VOLUME

I KELLEY’S
Textbook of
Rheumatology
EIGHTH EDITION

Gary S. Firestein, MD Professor of Medicine

Chief, Division of Rheumatology, Allergy,
and Immunology
Dean, Translational Medicine
University of California, San Diego, School of Medicine
La Jolla, California

Ralph C. Budd, MD Professor of Medicine

Director, Immunobiology Program
University of Vermont College of Medicine
Burlington, Vermont

Edward D. Harris, Jr., MD George DeForest Barnett Professor of Medicine, Emeritus

Stanford University School of Medicine
Academic Secretary to Stanford University, Emeritus
Stanford University
Stanford, California

Iain B. McInnes, PhD, FRCP Professor of Experimental Medicine

Honorary Consultant Rheumatologist
Centre for Rheumatic Diseases, Faculty of Medicine
University of Glasgow
Glasgow, United Kingdom

Shaun Ruddy, MD Professor Emeritus, Department of Internal Medicine,

Division of Rheumatology, Allergy, and Immunology
Virginia Commonwealth University School of Medicine
at the Medical College of Virginia
Richmond, Virginia

John S. Sergent, MD Professor of Medicine

Vice Chair for Education and Residency Program Director
Vanderbilt University School of Medicine
Nashville, Tennessee
1600 John F. Kennedy Blvd.
Ste 1800
Philadelphia, PA 19103-2899

ISBN: 978-1-4160-3285-4 (Expert Consult)

KELLEY’S TEXTBOOK OF RHEUMATOLOGY 978-1-4160-4842-8 (Expert Consult Premium Ed.)
Copyright © 2009, 2005, 2001, 1997, 1993, 1989, 1985, 1981 by Saunders, an imprint of Elsevier Inc.

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Notice

Knowledge and best practice in this field are constantly changing. As new research and experience broaden
our knowledge, changes in practice, treatment, and drug therapy may become necessary or appropriate.
Readers are advised to check the most current information provided (i) on procedures featured or (ii) by
the manufacturer of each product to be administered, to verify the recommended dose or formula, the
method and duration of administration, and contraindications. It is the responsibility of the practitioner,
relying on experience and knowledge of the patient, to make diagnoses, to determine dosages and the best
treatment for each individual patient, and to take all appropriate safety precautions. To the fullest extent of
the law, neither the Publisher nor the Editors assume any liability for any injury and/or damage to persons
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The Publisher

Library of Congress Cataloging-in-Publication Data

Kelley’s textbook of rheumatology / [edited by] Gary S. Firestein ... [et al.]. -- 8th ed.
p. ; cm.
Includes bibliographical references and index.
ISBN 978-1-4160-3285-4
1. Rheumatology. 2. Rheumatism. 3. Arthritis. I. Firestein, Gary S. II. Kelley, William N., 1939-
III. Title: Textbook of rheumatology.
[DNLM: 1. Rheumatic Diseases. 2. Arthritis. WE 544 K29 2009]
RC927.T49 2009
616.7'23--dc22
2007048387

Acquisitions Editor: Kimberly Murphy

Developmental Manager: Cathy Carroll
Developmental Editor: Angela Norton
Publishing Services Manager: Linda Van Pelt
Project Manager: Francisco Morales
Design Direction: Ellen Zanolle
Cover Design: Ellen Zanolle

Printed in Canada

Last digit is the print number: 9 8 7 6 5 4 3 2 1

Sincerest thanks to my wonderful wife, Linda, and our children,
David and Cathy, for their patience and support.
Also, the editorial help of our two Cavalier King Charles puppies,
Winston and Humphrey, was invaluable.
Gary S. Firestein

Sincere thanks for the kind mentoring

from Edward D. Harris Jr.,
H. Robson MacDonald, and C. Garrison Fathman,
as well as for the support of my wife, Lenore,
and my children, Graham and Laura.
Ralph C. Budd

Many thanks to my mentor, Steve Krane,

and for the support of the Harris boys,
Ned, Tom, and Chandler, and Eileen . . .
and for the happy smiles of the grandkids—
Andrew, Eliza, Maeve, and Liam.
Ted Harris

To my wife, Karin, for her patience,

understanding, and love and to our wonderful girls,
Megan and Rebecca, who continue to enlighten me.
Iain B. McInnes

To my wife, Millie; our children, Christi and Candace;

and our grandchildren, Kevin, Matthew, and Katharine.
Shaun Ruddy

To Carole and our children, Ellen and Katie,

and to our grandchildren, Kathryn, Henry,
Emmaline, and Romy.
John S. Sergent
 CONTRIBUTORS

Steven B. Abramson, MD Stanley P. Ballou, MD

Professor of Medicine and Pathology Associate Professor of Medicine
New York University School of Medicine Case Western Reserve University School of Medicine
New York, New York Director of Rheumatology
  Neutrophils and Eosinophils; Pathogenesis of MetroHealth Medical Center
Osteoarthritis Cleveland, Ohio
  Acute-Phase Reactants and the Concept of
Leyla Alparslan, MD Inflammation
Instructor in Orthopaedic Radiology
Uppsala University Faculty of Medicine Walter G. Barr, MD
Staff Radiologist, Akademiska Hospital Professor of Medicine
Uppsala, Sweden Northwestern University Feinberg School of Medicine
  Imaging Modalities in Rheumatic Disease Chicago, Illinois
  Mycobacterial Infections of Bones and Joints; Fungal
Thomas P. Andriacchi, PhD Infections of the Bones and Joints
Professor
Department of Mechanical Engineering Dorcas Eleanor Beaton, BScOT, MSc, PhD
Stanford University School of Engineering Assistant Professor, Department of Health Policy,
Department of Orthopaedics Management and Evaluation
Stanford University School of Medicine University of Toronto Faculty of Medicine
Stanford Scientist and Director, Mobility Program Clinical Research
VA Palo Alto Research & Development, Bone and Joint Unit
Research Center St. Michael’s Hospital
Palo Alto, California Toronto, Ontario, Canada
 Joint Biomechanics: The Role of Mechanics in Joint   Assessment of Health Outcomes
Pathology
Robert M. Bennett, MD, FRCP, MACR
John P. Atkinson, MD Professor of Medicine and Nursing Research
Samuel B. Grant Professor of Medicine and Professor of Oregon Health & Science University School of Medicine
Molecular Microbiology and School of Nursing
Washington University in St. Louis School of Medicine Portland, Oregon
Physician, Barnes-Jewish Hospital   Overlap Syndromes
St. Louis, Missouri
  Complement System Francis Berenbaum, MD, PhD
Professor of Rheumatology
Stefan Bachmann, MD Pierre and Marie Curie University (UPMC—Paris
FMH Specialist in Internal Medicine and Rheumatology Universitas) Faculty of Medicine
FMH Specialist in Physical Medicine and Rehabilitation Hospital Saint-Antoine
Leitender Artz/Chefarzt-Stellvertreter Paris, France
Klinik für Rheumatologie und Rehabilitation des   Clinical Features of Osteoarthritis
Bewegungsapparates
Valens, Switzerland Johannes W.J. Bijlsma, MD, PhD
  Introduction to Physical Medicine and Rehabilitation Professor and Chair, Department of Rheumatology  
& Clinical Immunology
Leslie R. Ballou, PhD University Medical Center Utrecht
Professor of Medicine and Molecular Sciences, Department Utrecht, The Netherlands
of Rheumatology   Glucocorticoid Therapy
University of Tennessee College of Medicine
Research Chemist, VA Medical Center and UT Health Linda K. Bockenstedt, MD
Science Center Harold W. Jockers Professor of Medicine
Memphis, Tennessee Department of Internal Medicine, Section of Rheumatology
  Nonsteroidal Anti-inflammatory Drugs Yale University School of Medicine
New Haven, Connecticut
  Lyme Disease

vi
 CONTRIBUTORS vii

Maarten Boers, MSc, MD, PhD Leonard H. Calabrese, DO

Professor of Clinical Epidemiology Professor of Medicine and R.J. Fasenmyer Chair of Clinical
Department of Clinical Epidemiology and Biostatistics Immunology
VU University Amsterdam Faculty of Medicine Cleveland Clinic Lerner College of Medicine
Amsterdam, The Netherlands Vice Chairman, Department of Rheumatic and
  Assessment of Health Outcomes Immunologic Diseases
Cleveland Clinic Foundation
Robert Alan Bonakdar, MD Cleveland, Ohio
Assistant Clinical Professor, Department of Family and   Antineutrophil Cytoplasmic Antibody–Associated
Preventive Medicine Vasculitis
UC San Diego School of Medicine
Director of Pain Management Amy C. Cannella, MD
Scripps Center for Integrative Medicine Assistant Professor, Department of Medicine, Section of
La Jolla, California Rheumatology and Immunology
  Integrative Medicine in Rheumatology:   University of Nebraska College of Medicine
An Evidence-Based Approach Omaha, Nebraska
  Methotrexate, Leflunomide, Sulfasalazine,
Dimitrios T. Boumpas, MD, FACP Hydroxychloroquine, and Combination Therapies
Professor and Chairman, Department of Internal Medicine,
Division of Rheumatology, Clinical Immunology, and Eugene J. Carragee, MD
Allergy Professor of Orthopaedic Surgery
University of Crete Medical School Stanford University School of Medicine
Chief of Medicine Director, Spine Surgery Section
Heraklion University General Hospital Stanford University Hospital and Clinics
Crete, Greece Stanford, California
  Clinical Features and Treatment of Systemic Lupus   Low Back Pain
Erythematosus
Steven Carsons, MD
Barry Bresnihan, MD Professor of Medicine
Professor of Rheumatology State University of New York at Stony Brook School  
University College Dublin School of Medicine   of Medicine
and Medical Science Stony Brook
National University of Ireland Chief, Division of Rheumatology, Allergy  
Consultant Rheumatologist and Immunology
St. Vincent’s University Hospital Winthrop University Hospital
Prinicipal Investigator Mineola, New York
Conway Institute of Biomedical Research   Sjögren’s Syndrome
Dublin, Ireland
  Synovium James T. Cassidy, MD
Professor, Department of Child Health
Doreen B. Brettler, MD University of Missouri–Columbia School of Medicine
Professor of Medicine Chief of Pediatric Rheumatology
University of Massachusetts Medical School University of Missouri Health Sciences Center
Director, New England Hemophilia Center Columbia, Missouri
University of Massachusetts Memorial Healthcare   Systemic Lupus Erythematosus, Juvenile
Worcester, Massachusetts Dermatomyositis, Scleroderma, and Vasculitis
  Hemophilic Arthropathy
Eliza F. Chakravarty, MD, MS
Paul L. Briant, PhD, MS Assistant Professor, Department of Medicine, Division  
VA Palo Alto Research & Development of Immunology and Rheumatology
Bone and Joint Research Center Stanford University School of Medicine
Palo Alto, CA Stanford, California
  Joint Biomechanics: The Role of Mechanics in Joint   Musculoskeletal Syndromes in Malignancy
Pathology
Christopher Chang, MD, PhD
Ralph C. Budd, MD Associate Clinical Professor, Department of Internal
Professor of Medicine Medicine, Division of Rheumatology/Allergy/Clinical
Director, Immunobiology Program Immunology
University of Vermont College of Medicine UC Davis School of Medicine
Burlington, Vermont Sacramento
  T Lymphocytes Staff, UC Davis Genome and Biomedical Services  
Facility
Davis, California
  Osteonecrosis
 viii CONTRIBUTORS

Joseph S. Cheng, MD, MS Jeroen DeGroot, MD

Assistant Professor of Neurological Surgery Operations Manager, Inflammatory and Degenerative
Vanderbilt University School of Medicine Diseases
Director, Neurosurgery Spine Program BioSciences Division
Vanderbilt University Medical Center TNO Quality of Life
Nashville, Tennessee Leiden, The Netherlands
  Neck Pain   Biologic Markers
Christopher P. Chiodo, MD Christopher P. Denton, PhD, FRCP
Instructor in Orthopaedic Surgery, Department   Professor of Experimental Rheumatology
of Orthopedic Surgery Royal Free and University College Medical School
Harvard Medical School Honorary Consultant Rheumatologist, Centre for
Chief, Foot and Ankle Division Rheumatology
Brigham and Women’s Hospital London, United Kingdom
Boston, Massachusetts   Systemic Sclerosis and the Scleroderma-Spectrum
  Foot and Ankle Pain Disorders
Paul P. Cook, MD Clinton Devin, MD
Associate Professor of Medicine Orthopedic Surgeon, Department of Orthopaedics  
Division of Infectious Diseases and Rehabilitation
Department of Infectious Diseases Vanderbilt Sports Medicine Center
Department of Internal Medicine Nashville, Tennessee
Brody School of Medicine at East Carolina University   Neck Pain
Greenville, North Carolina
  Bacterial Arthritis Betty Diamond, MD
Professor, Department of Microbiology & Immunology  
Joseph E. Craft, MD and Department of Medicine (Rheumatology)
Professor of Medicine and Immunobiology Albert Einstein College of Medicine
Chief, Section of Rheumatology, and Director, Bronx
Investigative Medicine Head and Investigator, Center for Autoimmune  
Yale University School of Medicine and Musculoskeletal Diseases
Chief of Rheumatology and Attending Physician The Feinstein Institute for Medical Research
Yale–New Haven Hospital Manhasset, New York
New Haven, Connecticut   B Cells
  Antinuclear Antibodies
Federico Díaz-González, MD
Gaye Cunnane, MD, MB, PhD, FRCPI Associate Professor of Rheumatology
Senior Lecturer in Medicine Universidad de La Laguna Faculty of Medicine
Trinity College Dublin Faculty of Health Sciences School Staff Rheumatologist
of Medicine Hospital Universitario de Canarias
Consultant in Rheumatology and Internal Medicine La Laguna, Spain
St. James’ Hospital   Platelets and Rheumatic Diseases
Dublin, Ireland
  Hemochromatosis Paul E. Di Cesare, MD, FACS
Professor and Michael W. Chapman Chair, Department  
Jody A. Dantzig, BS, PhD of Orthopaedic Surgery
Medical Student UC Davis School of Medicine
University of Pennsylvania School of Medicine Sacramento, California
Philadelphia, Pennsylvania   Pathogenesis of Osteoarthritis
  Muscle: Anatomy, Physiology, and Biochemistry
Joost P.H. Drenth, MD, PhD
John M. Davis III, MD Professor of Molecular Gastroenterology and Hepatology
Assistant Professor of Medicine Department of Gastroenterology and Hepatology
Division of Rheumatology Radboud University Nijmegen Medical Centre Faculty of
Mayo Clinic Medical Sciences
Rochester, Minnesota Nijmegen, The Netherlands
  History and Physical Examination of the   Familial Auto-inflammatory Syndromes
Musculoskeletal System
George F. Duna, MD, FACP
Associate Professor of Medicine
Baylor College of Medicine
Houston, Texas
  Antineutrophil Cytoplasmic Antibody–Associated
Vasculitis
 CONTRIBUTORS ix

Michael L. Dustin, PhD John P. Flaherty, MD

Irene Diamond Professor of Immunology and Associate Professor of Medicine
Professor of Pathology Associate Chief and Director of Clinical Services, Division
Department of Molecular Pathogenesis of Infectious Diseases
The Helen L. and Martin S. Kimmel Center for Biology Northwestern University Feinberg School of Medicine
and Medicine, Skirball Institute of Biomolecular Chicago, Illinois
Medicine   Mycobacterial Infections of Bones and Joints; Fungal
New York University School of Medicine Infections of the Bones and Joints
New York, New York
  Adaptive Immunity Including Organization of Adrienne M. Flanagan, MD, PhD
Lymphoid Tissues Professor
Institute of Orthopaedics and Musculoskeletal Science
Hani S. El-Gabalawy, MD, FRCPC University College London
Professor of Medicine and Immunology and Head, Division London
of Rheumatology Royal National Orthopaedic Hospital
University of Manitoba Faculty of Medicine Stanmore
Rheumatologist Department of Histopathology, University College
Winnipeg Health Sciences Centre Hospital
Winnipeg, Manitoba, Canada London, United Kingdom
  Synovial Fluid Analysis, Synovial Biopsy, and Synovial   Synovium
Pathology
Karen A. Fortner, PhD
Keith B. Elkon, MD Research Assistant Professor Immunobiology Program
Professor of Medicine and Immunology and Head, Division Department of Medicine
of Rheumatology University of Vermont College of Medicine
Department of Medicine Burlington, Vermont
University of Washington School of Medicine   T Lymphocytes
Seattle, Washington
  Cell Survival and Death in Rheumatic Diseases Howard A. Fuchs, MD
Associate Professor of Medicine
Doruk Erkan, MD Division of Rheumatology
Assistant Professor of Medicine Vanderbilt University
Weill Medical College of Cornell University Tennessee Valley Healthcare System
Associate Physician-Scientist and Assistant Attending Department of Veterans Affairs Medical Center
Physician Nashville, TN
Barbara Volcker Center for Women and Rheumatic   Polyarticular Arthritis
Diseases
Hospital for Special Surgery Steffen Gay, MD
New York, New York Professor
  Antiphospholipid Syndrome Department of Rheumatology
University Hospital
Gary S. Firestein, MD Zurich, Switzerland
Professor of Medicine   Fibroblasts and Fibroblast-like Synoviocytes
Chief, Division of Rheumatology, Allergy,  
and Immunology Mark C. Genovese, MD
Dean, Translational Medicine Professor of Medicine
University of California, San Diego, School of Medicine Stanford University School of Medicine
La Jolla, California Co-Chief, Division of Immunology and Rheumatology
  Etiology and Pathogenesis of Rheumatoid Arthritis; Stanford University Medical Center
Clinical Features of Rheumatoid Arthritis Stanford, California
  Treatment of Rheumatoid Arthritis
Oliver FitzGerald, MD, FRCPI, FRCP(UK)
Newman Clinical Research Professor M. Eric Gershwin, MD
University College Dublin School of Medicine   Distinguished Professor of Medicine
and Medical Science UC Davis School of Medicine
National University of Ireland Sacramento, California
Consultant Rheumatologist   Osteonecrosis
St. Vincent’s University Hospital
Dublin, Ireland
  Psoriatic Arthritis
 x CONTRIBUTORS

Allan Gibofsky, MD, JD, FACP, FCLM Carl S. Goodyear, PhD

Professor of Medicine and Public Health Lecturer and Arthritis Research Campaign
Weill Medical College of Cornell University University of Glasgow Faculty of Medicine
Adjunct Professor of Law NCCD Fellow, Division of Clinical Neurosciences
Fordham University School of Law Glasgow Biomedical Research Centre
Attending Rheumatologist Glasgow, United Kingdom
Hospital for Special Surgery   Rheumatoid Factors and Other Autoantibodies in
New York, New York Rheumatoid Arthritis
  Poststreptoccocal Arthritis and Rheumatic Fever
Siamon Gordon, MBChB, PhD, FRS, FMedSci
Mark H. Ginsberg, MD Professor Emeritus
Professor, Department of Medicine, Rheumatology Section Sir William Dunn School of Pathology
UC San Diego School of Medicine University of Oxford
La Jolla, California Oxford, United Kingdom
  Platelets and Rheumatic Diseases   Mononuclear Phagocytes in Rheumatic Diseases
Joseph Golbus, MD Adam Greenspan, MD, FACR
Associate Professor of Medicine Professor Emeritus of Radiology
Northwestern University Feinberg School of Medicine Department of Radiology, Section of Musculoskeletal
Senior Attending Physician, Division of Rheumatology Imaging
President UC Davis School of Medicine
Evanston Northwestern Healthcare Medical Group Sacramento, California
Chicago, Illinois Osteonecrosis
  Monarticular Arthritis
Peter K. Gregersen, MD
Yale E. Goldman, MD, PhD Professor of Medicine and Pathology
Professor, Department of Physiology New York University School of Medicine
University of Pennyslvania School of Medicine New York, New York
Director, Pennsylvania Muscle Institute   Genetics of Rheumatic Diseases
Philadelphia, Pennsylvania
  Muscle: Anatomy, Physiology, and Biochemistry Christine Grimaldi, PhD
Assistant Professor, Department of Microbiology &
Mary B. Goldring, PhD Immunology
Weill Medical College of Cornell University Albert Einstein College of Medicine
Senior Scientist Bronx
Hospital for Special Surgery Assistant Investigator, Center for Autoimmune and
New York, New York Musculoskeletal Disease
  Biology of the Normal Joint; Cartilage and The Feinstein Institute for Medical Research
Chondrocytes Manhasset, New York
  B Cells
Steven R. Goldring, MD
Weill Medical College of Cornell University Bevra Hannahs Hahn, MD, FACR, MACR
Chief Scientific Officer Professor and Vice Chair, Department of Medicine
Hospital for Special Surgery David Geffen School of Medicine at UCLA
New York, New York Chief, Rheumatology and Arthritis
  Biology of the Normal Joint UCLA Medical Center
Los Angeles, California
Stuart B. Goodman, MD, PhD, FRCSC, FACS, FBSE   Pathogenesis of Systemic Lupus Erythematosus
Robert L. and Mary Ellenburg Professor of Surgery,
Department of Orthopaedic Surgery J. Timothy Harrington, MD
Stanford University School of Medicine Associate Professor, Department of Medicine
Attending Orthopaedic Surgeon, Stanford University University of Wisconsin School of Medicine and Public
Medical Center Health
Consultant Orthopaedic Surgeon, Lucile Salter Packard Madison, Wisconsin
Children’s Hospital at Stanford   Mycobacterial Infections of Bones and Joints; Fungal
Stanford Infections of the Bones and Joints
Consultant Orthopaedic Surgeon, Palo Alto Veterans
Administration Hospital Edward D. Harris, Jr., MD, MACR
Palo Alto, California George DeForest Barnett Professor of Medicine, Emeritus
  Hip and Knee Pain Stanford University School of Medicine
Academic Secretary to Stanford University, Emeritus
Stanford University
Stanford, California
  Clinical Features of Rheumatoid Arthritis
 CONTRIBUTORS xi

David B. Hellmann, MD Arthur Kavanaugh, MD

Aliki Perroti Professor of Medicine Professor of Medicine
Johns Hopkins University School of Medicine Center for Innovative Therapy, Division of Rheumatology,
Vice Dean and Chairman, Department of Medicine Allergy and Immunology
Johns Hopkins Bayview Medical Center UC San Diego School of Medicine
Baltimore, Maryland La Jolla, California
  Giant Cell Arteritis, Polymyalgia Rheumatica,     Anticytokine Therapies
and Takayasu’s Arteritis
Alisa E. Koch, MD
George Ho, Jr., MD Frederick G.L. Huetwell and William D. Robinson, MD
Professor of Medicine Professor of Rheumatology
Brody School of Medicine at East Carolina University University of Michigan Medical School
Greenville, North Carolina Ann Arbor, Michigan
  Bacterial Arthritis   Cell Recruitment and Angiogenesis
James I. Huddleston, MD Deborah Krakow, MD
Assistant Professor, Department of Orthopaedic Surgery Associate Professor of Obstetrics and Gynecology
Stanford University School of Medicine David Geffen School of Medicine at UCLA
Stanford, California Attending Physician, Department of Obstetrics  
  Hip and Knee Pain and Gynecology
Cedars-Sinai Medical Center
Gene G. Hunder, MD Los Angeles, California
Professor Emeritus   Heritable Diseases of Connective Tissue
Mayo Clinic College of Medical Sciences
Emeritus Member Joel M. Kremer, MD
Department of Internal Medicine, Division of Pfaff Family Professor of Medicine
Rheumatology Albany Medical College
Mayo Clinic Director of Research
Rochester, Minnesota The Center for Rheumatology
  History and Physical Examination of the Albany, New York
Musculoskeletal System   Nutrition and Rheumatic Diseases
Johannes W.G. Jacobs, MD, PhD Hollis E. Krug, BS, MD
Associate Professor, Department of Rheumatology   Associate Clinical Professor of Medicine
and Clinical Immunology University of Minnesota Medical School
Rheumatologist and Senior Researcher Staff Rheumatologist
University Medical Center Utrecht VA Medical Center
Utrecht, The Netherlands Minneapolis, Minnesota
  Glucocorticoid Therapy   Management of Chronic Pain
Joanne M. Jordan, MD, MPH Irving Kushner, MD
Associate Professor of Medicine and Orthopaedics Professor of Medicine
Chief, Division of Rheumatology, Allergy, and Case Western Reserve University School of Medicine
Immunology Staff Rheumatologist
University of North Carolina at Chapel Hill School   MetroHealth Medical Center
of Medicine Cleveland, Ohio
Director, Thurston Arthritis Research Center   Acute-Phase Reactants and the Concept of
Chapel Hill, North Carolina Inflammation
  Principles of Epidemiology in Rheumatic Disease
Robert B.M. Landewé, MD
Joseph L. Jorizzo, MD Professor of Rheumatology, Department of Internal
Professor and Former (Founding) Chair, Department   Medicine, Division of Rheumatology
of Dermatology Maastricht University Faculty of Medicine
Wake Forest University School of Medicine Staff Rheumatologist
Winston-Salem, North Carolina Atrium Medical Center Heerlen
  Behçet’s Disease Maastricht, The Netherlands
  Clinical Trial Design and Analysis
Kenneth C. Kalunian, MD
Professor of Medicine and Director of Rheumatology, Nancy E. Lane, MD
Allergy and Immunology Professor of Medicine and Rheumatology
UC San Diego School of Medicine UC Davis School of Medicine
La Jolla, California Director, Center for Healthy Aging
  Rheumatic Manifestations of Hemoglobinopathies Sacramento, California
  Metabolic Bone Disease
 xii CONTRIBUTORS

Daniel J. Laskin, DDS, MS B. Asher Louden, MD

Professor and Chairman Emeritus, Department of Oral   Resident in Dermatology
and Maxillofacial Surgery Wake Forest University Baptist Medical Center
School of Dentistry and School of Medicine Winston-Salem, North Carolina
Virginia Commonwealth University   Behçet’s Disease
Richmond, Virginia
  Temporomandibular Joint Pain Carlos J. Lozada, MD, FACP, FACR
Associate Professor of Medicine
David M. Lee, MD University of Miami Miller School of Medicine
Assistant Professor of Medicine Director, Rheumatology Fellowship Program  
Harvard Medical School and Rheumatology Clinical Services
Associate Physician Jackson Memorial Hospital
Brigham and Women’s Hospital Miami, Florida
Boston, Massachusetts   Management of Osteoarthritis
  Mast Cells
Ingrid E. Lundberg, MD, PhD
Lela A. Lee, MD Professor of Medicine and Head, Rheumatology Unit,
Professor of Dermatology and Medicine Department of Medicine
University of Colorado School of   Karolinska Institute/Karolinska University Hospital
Medicine Stockholm, Sweden
Director of Dermatology   Inflammatory Diseases of Muscle and Other
Denver Health Medical Center Myopathies
Denver, Colorado
  The Skin and Rheumatic Diseases Reuven Mader, MD
Senior Clinical Lecturer
Marjatta Leirisalo-Repo, MD, PhD B. Rappaport Faculty of Medicine, Technion Israel
Professor of Rheumatology, Department of Medicine, Institute of Technology
Division of Rheumatology Head, Rheumatic Diseases Unit
Helsinki University Faculty of Medicine Ha’Emek Medical Center
Staff Rheumatologist, Helsinki University Central Haifa, Israel
Hospital   Proliferative Bone Diseases
Helsinki, Finland
  Undifferentiated Spondyloarthritis and Reactive Rashmi M. Maganti, MD
Arthritis Fellow, Division of Rheumatology and Clinical
Immunogenetics
David C. Leopold, MD, DABFM The University of Texas Health Science Center at Houston
Faculty Physician and Director, Integrative Medical Houston, Texas
Education   Rheumatic Manifestations of Human
Scripps Center for Integrative Medicine Immunodeficiency Virus Infection
La Jolla, California
  Integrative Medicine in Rheumatology:   Maren Lawson Mahowald, MD
An Evidence-Based Approach Professor of Medicine
University of Minnesota Medical School
Peter E. Lipsky, MD Rheumatology Section Chief
Chief, Autoimmunity Branch Minneapolis VA Medical Center
National Institute of Arthritis and Musculoskeletal   Minneapolis, Minnesota
and Skin Diseases   Management of Chronic Pain
National Institutes of Health
Bethesda, Maryland Walter P. Maksymowych, MBChB, FRCPC, FACP,
  Autoimmunity FRCP(UK)
Professor of Medicine
Michael D. Lockshin, MD, MACR University of Alberta Faculty of Medicine
Professor of Medicine and Obstetrics-Gynecology Senior Scientist
Weill Medical College of Cornell University Alberta Heritage Foundation for Medical Research
New York, New York Edmonton, Alberta, Canada
  Antiphospholipid Syndrome   Ankylosing Spondylitis
Kate R. Lorig, RN, DrPH Scott David Martin, MD
Professor, Department of Medicine, Division of Assistant Professor of Orthopedics
Immunology and Rheumatology Harvard Medical School
Stanford University School of Medicine Attending Staff Physician, Department of Orthopedics
Director, Patient Education Research Center Brigham and Women’s Hospital
Stanford, California Boston, Massachusetts
  Arthritis Self-Management   Shoulder Pain
 CONTRIBUTORS xiii

Helena Marzo-Ortega, MD, MRCP Lee S. Newman, MD, MA

Consultant Rheumatologist and Honorary Senior Lecturer Professor, Department of Medicine, Division of Allergy
Academic Section of Musculoskeletal Disease and Clinical Immunology and Division of Pulmonary
Leeds Institute of Molecular Medicine University of Leeds Sciences and Critical Care Medicine
and Chapel Allerton Hospital Professor of Epidemiology, Department of Preventive
Leeds, United Kingdom Medicine and Biometrics
  Undifferentiated Spondyloarthritis and Reactive University of Colorado School of Medicine
Arthritis Denver, Colorado
  Sarcoidosis
Dennis McGonagle, PhD, FRCPI
Professor of Investigative Rheumatology Peter A. Nigrovic, MD
The University of Leeds Instructor in Medicine
Leeds, United Kingdom Harvard Medical School
  Undifferentiated Spondyloarthritis and Reactive Staff Rheumatologist
Arthritis Department of Rheumatology, Immunology, and Allergy,
Brigham & Women’s Hospital
Iain B. McInnes, PhD, FRCP Division of Immunology, Children’s Hospital Boston
Professor of Experimental Medicine Boston, Massachusetts
Honorary Consultant Rheumatologist   Mast Cells
Centre for Rheumatic Diseases, Faculty of Medicine
University of Glasgow Kiran Nistala, MD, MRCP, MSc
Glasgow, United Kingdom Clinical Research Fellow in Paediatric Rheumatology
  Cytokines; Atherosclerosis in Rheumatic Disease; Institute of Child Health
Rheumatoid Factors and Other Autoantibodies in University College London
Rheumatoid Arthritis London, United Kingdome
  Juvenile Idiopathic Arthritis
Kevin G. Moder, MD
Consultant and Associate Professor James R. O’Dell, MD
Division of Rheumatology and Department of Internal Larson Professor and Vice-Chairman, Department of
Medicine Internal Medicine
Mayo Clinic University of Nebraska College of Medicine
Rochester, Minnesota Chief of Rheumatology and Residency Program Director,
  History and Physical Examination of the Department of Internal Medicine
Musculoskeletal System University of Nebraska Medical Center
Omaha, Nebraska
Eamonn S. Molloy, MD, MRCPI   Methotrexate, Leflunomide, Sulfasalazine,
Associate Staff Hydroxychloroquine, and Combination Therapies
Department of Rheumatic and Immunologic Disease
Cleveland Clinic Foundation Peter R. Oesch, MSc, Dipl PT
Cleveland, Ohio Head, Department of Ergonomics and Clinical Research
  Antineutrophil Cytoplasmic Antibody–Associated Valens Rehabilitation Clinic
Vasculitis Valens, Switzerland
  Introduction to Physical Medicine and Rehabilitation
Kanneboyina Nagaraju, PhD, DVM
Associate Professor of Pediatrics Yasunori Okada, MD, PhD
George Washington University School of Medicine   Professor and Chairman, Department of Pathology
and Health Sciences Keio University School of Medicine
Director, Murine Drug Testing Facility Tokyo, Japan
Center for Genetic Medicine Research   Proteinases and Matrix Degradation
Children’s Research Institute
Children’s National Medical Center Eugenia C. Pacheco-Pinedo, MD, MSc
Washington, DC Postdoctoral Researcher
  Inflammatory Diseases of Muscle and Other Department of Medicine
Myopathies Molecular Cardiology Research Center
Postdoctoral Researcher
Stanley J. Naides, MD Department of Physiology
Medical Director, Immunology R&D Pennsylvania Muscle Institute
Quest Diagnostics Nichols Institute Philadelphia, Pennsylvania
San Juan Capistrano, California   Muscle: Anatomy, Physiology, and Biochemistry
  Viral Arthritis
 xiv CONTRIBUTORS

Richard S. Panush, MD Steven A. Porcelli, MD

Professor of Medicine Weinstock Professor of Microbiology & Immunology  
Mount Sinai School of Medicine and Professor of Medicine
Chair, Department of Medicine Albert Einstein College of Medicine
Saint Barnabas Medical Center Bronx, New York
New York, New York   Innate Immunity
  Occupational and Recreational Musculoskeletal  
Disorders Mark D. Price, MD, PhD
Chief Resident in Orthopedic Surgery
Thomas Pap, MD Harvard Combined Orthopedic Surgery Program
Professor of Experimental Medicine Massachusetts General Hospital
Head, Division of Molecular Medicine of Musculoskeletal Boston, Massachusetts
Tissue   Foot and Ankle Pain
University of Münster
Institute of Experimental Musculoskeletal Medicine Johannes J. Rasker, MD, PhD
Director Professor Emeritus of Rheumatology
University Hospital Münster Faculty of Behavioral Sciences, Department of Psychology
Münster, Germany and Communication of Health and Risk
  Fibroblasts and Fibroblast-like Synoviocytes University of Twente
Enschede, The Netherlands
Stanford L. Peng, MD, PhD   Fibromyalgia
Senior Director, Translational Medicine Leader
Clinical Research and Exploratory Development John D. Reveille, MD
Roche Palo Alto Professor of Internal Medicine
Palo Alto Director, Division of Rheumatology and Clinical
Assistant Clinical Professor, Department of Medicine, Immunogenetics
Division of Rheumatology–Arthritis Department of Internal Medicine
University of California, San Francisco, School of University of Texas Medical School at Houston
Medicine Houston, Texas
San Francisco, California   Rheumatic Manifestations of Human
  Antinuclear Antibodies Immunodeficiency Virus Infection

Harris Perlman, PhD W. Neal Roberts, Jr., MD

Associate Professor, Department of Molecular Rheumatology Fellowship Program Director
Microbiology & Immunology Chas. W. Thomas Professor of Medicine
Saint Louis University School of Medicine Virginia Commonwealth University School of Medicine,
St. Louis, Missouri Medical College of Virginia Campus
  Signal Transduction Richmond, Virginia
  Psychosocial Management of Rheumatic Diseases
Jean-Charles Piette, MD
Department of Internal Medicine James T. Rosenbaum, MD
Groupe Hospitalier Pitié-Salpêtrière Edward E. Rosenbaum Professor of Inflammation Research
Paris, France and Professor of Ophthalmology, Medicine, and Cell
  Relapsing Polychondritis Biology
Chair, Division of Arthritis and Rheumatic Diseases
Michael H. Pillinger, MD Vice-Chair, Department of Ophthalmology
Chief of Rheumatology Oregon Health & Science University School of Medicine
New York Hospital for Joint Diseases Portland, Oregon
New York, New York   The Eye and Rheumatic Diseases
  Neutrophils and Eosinophils
Andrew E. Rosenberg, MD
Robert S. Pinals, MD Associate Professor
Acting Chief, Division of Rheumatology & Connective Harvard Medical School
Tissue Research Director of Surgical Pathology
Professor, Department of Medicine Massachusetts General Hospital
UMDNJ–Robert Wood Johnson Medical School Boston, Massachusetts
New Brunswick, New Jersey   Tumors and Tumor-like Lesions of Joints and Related  
  Felty’s Syndrome Structures
 CONTRIBUTORS xv

Clinton T. Rubin, PhD David C. Seldin, MD, PhD

Distinguished Professor and Chair Professor of Medicine and Microbiology
Department of Biomedical Engineering Boston University School of Medicine
State University of New York Stony Brook University Director, Amyloid Treatment and Research Program
School of Medicine Boston Medical Center
Stony Brook, New York Boston, Massachusetts
  Biology, Physiology, and Morphology of Bone   Amyloidosis
Janet E. Rubin, MD Jérémie Sellam, MD, PhD
Professor of Medicine and Pharmacology Assistant Professor of Rheumatology
University of North Carolina at Chapel Hill School   Paris Universitas – Pierre & Marie Curie Paris VI
of Medicine Assistant Professor of Rheumatology
Chapel Hill, North Carolina Saint-Antoine Hospital
  Biology, Physiology, and Morphology of Bone Paris, France
  Clinical Features of Osteoarthritis
Holly M. Sackett, MSPH
Senior Professional Research Assistant John S. Sergent, MD, MACR
University of Colorado Denver Professor of Medicine
Colorado School of Public Health Vice Chair for Education and Residency Program Director
Denver, Colorado Vanderbilt University School of Medicine
  Sarcoidosis Nashville, Tennessee
  Polyarticular Arthritis; Polyarteritis and Related
Jane E. Salmon, MD Disorders; Isolated Angiitis of the Central Nervous
Professor of Medicine System; Arthritis Accompanying Endocrine  
Professor of Obstetrics and Gynecology and Metabolic Disorders
Weill Medical College of Cornell University
Attending Physician Richard M. Siegel, MD, PhD
Hospital for Special Surgery Investigator, Autoimmunity Branch
New York Presbyterian Hospital National Institute of Arthritis and Musculoskeletal  
New York, New York and Skin Diseases
  Antiphospholipid Syndrome National Institutes of Health
Bethesda, Maryland
Jonathan Samuels, MD   Autoimmunity
Instructor in Medicine (Rheumatology)
NYU School of Medicine Karl Sillay, MD
Director, Clinical Immunulogy Laboratory Assistant Professor and Director, Functional Surgery,
NYU Langone Medical Center Department of Neurological Surgery
  Pathogenesis of Osteoarthritis University of Wisconsin School of Medicine and Public
Health
Naveed Sattar, MBChB, PhD, MRCPath Neurosurgeon, University of Wisconsin Hospital and
Professor of Metabolic Medicine Clinics, William S. Middleton Memorial Veterans
British Heart Foundation Glasgow Cardiovascular Hospital, St. Mary’s Hospital Medical Center, and
Research Centre Meriter Hospital
University of Glasgow Madison, Wisconsin
Honorary Consultant Endocrinologist   Neck Pain
Glasgow Royal Infirmary
Glasgow, United Kingdom Anna Simon, MD, PhD
  Atherosclerosis in Rheumatic Disease Clinical Investigator, Department of General Internal
Medicine
John C. Scatizzi, PhD Radboud University Nijmegen Medical Centre Faculty  
Post-Doctoral Fellow, Division of Rheumatology, Allergy, of Medical Sciences
& Immunology Nijmegen, The Netherlands
UC San Diego School of Medicine   Familial Auto-inflammatory Syndromes
La Jolla, California
  Signal Transduction Dawd S. Siraj, MD, MPH, TM
Assistant Professor of Medicine
Jose U. Scher, MD Brody School of Medicine at East Carolina University
Teaching Assistant Director
Department of Medicine, Division of Rheumatology ECU Physicians International Travel Clinic, Section  
New York University School of Medicine of Infectious Diseases
New York, New York Greenville, North Carolina
  Neutrophils and Eosinophils   Bacterial Arthritis
 xvi CONTRIBUTORS

Martha Skinner, MD Ioannis O. Tassiulas, MD

Professor of Medicine Senior Investigator, Department of Medicine, Division  
Boston University School of Medicine of Rheumatology
Boston, Massachusetts University of Crete Medical School
  Amyloidosis Heraklion, Greece
  Clinical Features and Treatment of Systemic Lupus
Kathleen A. Sluka, PT, PhD Erythematosus
Professor of Physical Therapy
Graduate Programs in Physical Therapy and Rehabilitation H. Guy Taylor, MBChB, MRCP(UK), FRACP,
Science, in Pain Research, and in Neuroscience Dipl MSM(Otago)
University of Iowa Carver College of Medicine Consultant Rheumatologist
Iowa City, Iowa Wanganui Hospital
  Neurological Regulation of Inflammation Wanganui, New Zealand
  Immunoregulatory Drugs
C. Michael Stein, MBChB, MRCP
Dan May Professor of Medicine and Professor   Peter C. Taylor, MA, PhD, FRCP
of Pharmacology Professor of Experimental Rheumatology
Vanderbilt University School of Medicine Head, Clinical Trials
Nashville, Tennessee Kennedy Institute of Rheumatology Division
  Immunoregulatory Drugs Faculty of Medicine
Imperial College London
John H. Stone, MD, MPH London, United Kingdom
Clinical Director of Rheumatology   Cell-Targeted Biologics and Emerging Targets:
Massachusetts General Hospital Rituximab, Abatacept, and Other Biologics
Boston, Massachusetts
  The Classification and Epidemiology of Systemic Robert Terkeltaub, MD
Vasculitis; Immune Complex–Mediated Small Vessel Professor of Medicine
Vasculitis Rheumatology Training Program Director and Associate
Division Director for Rheumatology–Allergy/
Bob Sun, MD Immunology
Instructor, Department of Medicine, Division of UC San Diego School of Medicine
Rheumatology La Jolla
Northwestern University Feinberg School of Medicine Section Chief, Rheumatology–Allergy
Attending Rheumatologist VA Medical Center San Diego
Evanston Northwestern Healthcare San Diego, California
Evanston, Illinois   Diseases Associated with Articular Deposition of
  Rheumatic Manifestations of Hemoglobinopathies Calcium Pyrophosphate Dihydrate and Basic Calcium
Carrie R. Swigart, MD Phosphate Crystals
Assistant Professor, Department of Orthopaedics and Thomas S. Thornhill, MD
Rehabilitation, Hand and Upper Extremity Section Professor of Orthopedics
Yale University School of Medicine Harvard Medical School
New Haven, Connecticut
Chief of Orthopedics
  Hand and Wrist Pain Brigham and Women’s Hospital
Zoltán Szekanecz, MD, PhD, DSc Boston, Massachusetts
Professor, Department of Medicine, Division of   Shoulder Pain
Rheumatology and Immunology Helen Tighe, BSc, PhD
Institute for Internal Medicine, Rheumatology Division
Associate Adjunct Professor, Department of Medicine,
University of Debrecen Medical and Health Science
Division of Rheumatology, Allergy, and Immunology
Center UC San Diego School of Medicine
Debrecen, Hungary La Jolla, California
  Cell Recruitment and Angiogenesis   Rheumatoid Factors and Other Autoantibodies in
Paul P. Tak, MD, PhD Rheumatoid Arthritis
Professor of Medicine and Director, Division of Clinical Betty P. Tsao, PhD
Immunology & Rheumatology Professor of Medicine, Department of Medicine, Division
Academic Medical Center/University of Amsterdam of Rheumatology
Faculty of Medicine David Geffen School of Medicine at UCLA
Amsterdam, The Netherlands Los Angeles, California
  Biologic Markers   Pathogenesis of Systemic Lupus Erythematosus
 CONTRIBUTORS xvii

Peter Tugwell, MSc, MD, FRCPC John Varga, MD

Professor of Medicine, Department of Medicine Hughes Distinguished Professor of Medicine
Ottawa Health Research Institute Department of Medicine, Division of Rheumatology
University of Ottawa Faculty of Medicine Northwestern University Feinberg School of Medicine
Ottawa, Ontario, Canada Chicago, Illinois
  Assessment of Health Outcomes   Systemic Sclerosis and the Scleroderma-Spectrum
Disorders
Zuhre Tutuncu, MD
Associate Professor, Department of Rheumatology, Allergy, Philippe Vinceneux, MD
and Immunology Medecine Interne 2
UC San Diego School of Medicine Hospital Pitié-Salpêtrière
La Jolla, California Paris, France
  Anticytokine Therapies   Relapsing Polychondritis
Katherine S. Upchurch, MD Benjamin W.E. Wang, MD, FRCPC
Associate Professor of Medicine Associate Professor, Department of Medicine, Division  
University of Massachusetts Medical School of Rheumatology
Clinical Chief, Division of Rheumatology University of Tennessee College of Medicine
UMass Memorial Medical Center Memphis, Tennessee
Worcester, Massachusetts   Nonsteroidal Anti-inflammatory Drugs
  Hemophilic Arthropathy
Lucy R. Wedderburn, MD
Wim B. Van den Berg, PhD Reader in Paediatric Rheumatology
Professor of Experimental Rheumatology, Rheumatology Rheumatology Unit, Institute of Child Health–University
Research, and Advanced Therapeutics College London
Radboud University Nijmegen Medical Centre Faculty of London, United Kingdom
Medical Sciences   Juvenile Idiopathic Arthritis
Nijmegen, The Netherlands
  Animal Models of Inflammatory Arthritis Barbara N. Weissman, MD
Professor of Radiology
Filip Van den Bosch, MD, PhD Harvard Medical School
Rheumatologist Director, Radiology Residency Program
University Hospital Gent—Department of Rheumatology Vice Chair, Department of Radiology
Ghent, Belgium Brigham & Women’s Hospital
  Undifferentiated Spondyloarthritis and Reactive Boston, Massachusetts
Arthritis   Imaging Modalities in Rheumatic Disease
Désirée M. F. M. Van der Heijde, MD, PhD Victoria P. Werth, MD
Professor of Rheumatology, Department of   Professor of Dermatology and Medicine
Rheumatology University of Pennsylvania School of Medicine
Leiden University Faculty of Medicine Chief of Dermatology
Leiden, The Netherlands Philadelphia VA Medical Center
  Clinical Trial Design and Analysis; Ankylosing   Philadelphia, Pennsylvania
Spondylitis   The Skin and Rheumatic Diseases
Sjef M. van der Linden, MD Karin N. Westlund-High, PhD
Professor of Rheumatology, Department of Medicine Professor, Department of Physiology
University of Maastricht Faculty of Health, Medicine,   University of Kentucky College of Medicine
and Life Sciences Lexington, Kentucky
CAPHRI Research Institute   Neurological Regulation of Inflammation
Head, Division of Rheumatology, Department of  
Medicine Michael S. Wildstein, MD
University Hospital of Maastricht President
Maastricht, The Netherlands Wildstein Spine Center
  Ankylosing Spondylitis Charleston, South Carolina
  Low Back Pain
Jos W.M. van der Meer, MD, PhD, FRCP
Department of General Internal Medicine
Radboud University Nijmegen Medical Centre
Nijmegen, The Netherlands
  Familial Auto-inflammatory Syndromes
xviii CONTRIBUTORS

Christopher M. Wise, MD Robert L. Wortmann, MD

W. Robert Irby Professor of Medicine Professor of Medicine
Department of Medicine, Division of Rheumatology, Dartmouth-Hitchcock Medical Center
Allergy, and Immunology Lebanon, New Hampshire
Virginia Commonwealth University School of Medicine,   Gout and Hyperuricemia
Medical College of Virginia Campus
Richmond, Virginia David Tak Yan Yu, MD
  Arthrocentesis and Injection of Joints and Soft Tissue Professor of Medicine
David Geffen School of Medicine at UCLA
Frederick Wolfe, MD Los Angeles, California
Clinical Professor of Medicine   Undifferentiated Spondyloarthritis and Reactive
University of Kansas School of Medicine Arthritis
Director, National Data Bank for Rheumatic  
Diseases John B. Zabriskie, MD
Wichita, Kansas Professor Emeritus
  Fibromyalgia Rockefeller University
New York, New York
Frank A. Wollheim, MD, PhD, FRCP   Poststreptoccocal Arthritis and Rheumatic Fever
Emeritus Professor, Department of Rheumatology
Lund University Faculty of Medicine/Lund University Robert B. Zurier, MD
Hospital Professor, Department of Medicine, Division  
Lund, Sweden of Rheumatology
  Enteropathic Arthritis University of Massachusetts Medical School
Worcester, Massachusetts
Patricia Woo, MBBS, BSc, PhD, CBE, FRCP, FMedSci   Prostaglandins, Leukotrienes, and Related Compounds
Professor of Paediatric Rheumatology
Faculty of Medicine Anne-Marie Zuurmond, PhD
University College London Biosciences Division
London, United Kingdom TNO Quality of Life
  Juvenile Idiopathic Arthritis Leiden, The Netherlands
  Biologic Markers
Anthony D. Woolf, BSc, MBBS, FRCP
Professor of Rheumatology
Peninsula College of Medicine and Dentistry, Universities
of Exeter & Plymouth
Exeter
Consultant Rheumatologist
Duke of Cornwall Rheumatology Unit, Royal Cornwall
Hospital
Truro, United Kingdom
  Economic Burden of Rheumatic Diseases
 PREFACE
“Plus ça change, plus c’est la même chose” –Jean-Baptiste
Alphonse Karr
As we, the editors, worked on the 8th edition of Kelley’s
Textbook of Rheumatology, we were struck by Monsieur
Karr’s quote from 160 years ago. This textbook continues to
change and evolve. The incomparable Ted Harris, who was
editor-in-chief of the 7th edition, has stepped down, leaving
Gary Firestein to fill his shoes. For the first time, a European
editor, Iain McInnes, joined the group and helped create a
truly international edition. Full color was introduced, new
chapter formats were designed to assure a consistent look
and feel for each topic, and algorithms for diagnosis and
treatment as well as key point boxes were included. In addi-
tion, new authors were added to the list of luminaries that
already contribute to the book, and there was a major effort
to provide greater availability through electronic versions of
reference material and on-line access.
While change has been in the air regarding many aspects
of the book, some things never vary. The book was initially
designed decades ago to provide scholarly rigor to the field
of rheumatology and to offer definitive reviews of scientific
advances as they apply to clinical medicine. This remained
the touchstone of our enterprise for the past 4 years, just
as it was for the previous seven editions. The painstaking
job of identifying the premier authors to present definitive
i­nformation on each topic required months of work, culmi-
nating in an editors’ meeting in Costa Rica to finalize the
chapters (well, it wasn’t all work!). The arduous process of
guiding, reviewing, and editing the outstanding contribu-
tions of our authors was time consuming but paled com-
pared with the efforts that the authors put into creating
their chapters. The thread connecting the past to the pres-
ent was also evident in the continued effort of our valued
co-editors John Sergent, Ralph Budd, and Shaun Ruddy.
Our trusted colleagues at Elsevier, including Cathy Carroll
and Kimberly Murphy, were always available and suffered
with us in Costa Rica as well.
The present looks very encouraging indeed. Kelley’s Text-
book of Rheumatology, 8th edition, is a beautiful tome that
is designed to carry on the tradition of being the definitive
rheumatology resource. Looking to the future, we expect
that this work will continue to evolve and change. New edi-
tors, new science, new authors, and new technology will be
the rule rather than the exception.
As you begin to use this edition, please know that it is
truly a labor of love. We have enjoyed the experience and
hope that it is as valuable to you as the previous editions.
The Editors
xix
 Part Structure and Function of Bone,
1 Joints, and Connective Tissue
1 Biology of the Normal
KEY POINTS
Condensation of mesenchymal cells, which differentiate into
chondrocytes, results in formation of the cartilage anlagen,
which provides the template for the developing skeleton.
During development of the synovial joint, growth differentia-
tion factor-5 regulates interzone formation, and interference
with movement of the embryo during development impairs
joint cavitation.
Members of the bone morphogenetic protein/transforming
growth factor-β, fibroblast growth factor, and Wnt families and
the parathyroid hormone–related peptide/Indian hedgehog axis
are essential for joint development and growth plate formation.
The synovial lining of diarthrodial joints is a thin layer of cells
lacking a basement membrane and consisting of two principal
cell types—macrophages and fibroblasts.
The articular cartilage receives its nutritional requirements
via diffusion from the synovial fluid, and interaction of the
cartilage with components of the synovial fluid contributes
to the unique low-friction surface properties of the articular
cartilage.
The normal joint is a specialized, integrated structure
consisting of multiple connective tissue elements, includ-
ing muscles, tendons, ligaments, synovium and capsule,
cartilage, and bone, organized in a manner that permits
stability and movement of the human skeleton. The joint
structures are positioned to distribute normal mechanical
stresses optimally and are organized for low-friction load
bearing. Deviations from normal structure and physiology
of joint tissues have been implicated in the pathogenesis of
various forms of arthritis. The differentiation of articular
tissues during embryonic development dictates their capac-
ity to respond to insults in later life. Physiologic cellular
processes involved in normal joint development, such as
differentiation, angiogenesis, macrophage recruitment, and
fibroblast proliferation, may reappear in the mature joint
and contribute to the pathogenesis of joint disease. Knowl-
edge of the development, structure, and function of normal
joint tissues is essential for understanding the underlying
mechanisms involved in the pathogenesis of human joint
diseases.
Joint
Steven R. Goldring  • 
Mary B. Goldring
CLASSIFICATION OF JOINTS
Human joints provide the structures by which bones join
with one another and may be classified according to the
histologic features of the union and the range of joint
motion. There are three classes of joint design: (1) syno-
vial or ­diarthrodial joints (Fig. 1-1), which articulate with
free movement, have a synovial membrane lining the joint
cavity, and contain synovial fluid; (2) amphiarthroses, in
which adjacent bones are separated by articular cartilage or
a fibrocartilage disk and are bound by firm ligaments permit-
ting limited motion (e.g., pubic symphysis, intervertebral
disks of vertebral bodies, distal tibiofibular articulation, and
­sacroiliac joint articulation with pelvic bones); and (3) syn-
arthroses, which are found only in the skull (suture lines)
where thin, fibrous tissue separates adjoining cranial plates
that interlock to prevent detectable motion before the end
of normal growth, yet permit growth in childhood and ado-
lescence.1
Joints also can be classified according to the connective
tissues present. Symphyses have a fibrocartilaginous disk
separating bone ends that are joined by firm ligaments (e.g.,
symphysis pubis and intervertebral joints). In synchondroses,
the bone ends are covered with articular cartilage, but there
is no synovium or significant joint cavity (e.g., sternomanu-
brial joint). In syndesmoses, the bones are joined directly
by fibrous ligaments without a cartilaginous interface (the
distal tibiofibular articulation is the only joint of this type
outside the cranial vault). In synostoses, bone bridges are
formed between bones, producing ankylosis.
The synovial joints are classified further according to
their shapes, which include ball-and-socket (hip), hinge
(interphalangeal), saddle (first carpometacarpal), and plane
(patellofemoral) joints. These configurations reflect the
varying functions, as the shapes and sizes of the opposing
surfaces determine the direction and extent of motion. The
various designs permit flexion, extension, abduction, adduc-
tion, or rotation. Certain joints can act in one (humeroul-
nar), two (wrist), or three (shoulder) axes of motion.
This chapter concentrates on the developmental biology
and relationship between structure and function of a “proto-
typic,” “normal” human diarthrodial joint—the joint most
likely to develop arthritis. Most research that has been done

  GOLDRING  |  Biology of the Normal Joint
Cartilage
Bone Tide mark
Periosteum
Capsule
Synovium
Figure 1-1  A normal human interphalangeal joint, in sagittal section,
as an example of a synovial, or diarthrodial, joint. The tidemark repre-
sents the calcified cartilage that bonds articular cartilage to the subchon-
dral bone plate. (From Sokoloff L, Bland JH: The Musculoskeletal System.
Baltimore, Williams & Wilkins, 1975. © 1975, the Williams & Wilkins Co,
Baltimore.)
concerns the knee because of its accessibility, but other
joints are described when appropriate.
DEVELOPMENTAL BIOLOGY
OF THE DIARTHRODIAL JOINT
Skeletal development is initiated by the differentiation of
mesenchymal cells that arise from three sources: (1) neural
crest cells of the neural ectoderm that gives rise to craniofa-
cial bones; (2) the sclerotome of the paraxial mesoderm, or
somite compartment, which forms the axial skeleton; and
(3) the somatopleure of the lateral plate mesoderm, which
yields the skeleton of the limbs.2 The appendicular skeleton
develops in the human embryo from limb buds, which are
first visible at around 4 weeks of gestation. Structures resem-
bling adult joints are generated at approximately 4 to 7
weeks of gestation.3 Many other crucial phases of muscu-
loskeletal development follow, including vascularization of
epiphyseal cartilage (8 to 12 weeks), appearance of villous
folds in synovium (10 to 12 weeks), evolution of bursae
(3 to 4 months), and appearance of periarticular fat pads
(4 to 5 months).
The upper limbs develop approximately 24 hours earlier
than the analogous portions of the lower limbs. Proximal
structures, such as the glenohumeral joint, develop before
more distal ones, such as the wrist and hand. As a conse-
quence, insults to embryonic development during limb for-
mation affect a more distal portion of the upper limb than
of the lower limb. Long bones form as a result of replace-
ment of the cartilage template by endochondral ossifica-
tion. The stages of limb development are well described
by O’Rahilly and Gardner3,4 and are shown in Figure 1-2.
The ­developmental sequence of the events occurring during
Homogeneous
interzone 3-Layered
Mesenchyme Perichondrium Synovial interzone
Blastema Cartilage mesenchyme
A B C
D E
Cavities Articular Articular Synovial
capsule cavity tissue and fold
Figure 1-2  The development of a synovial joint. A, Condensation.
Joints develop from the blastema, not the surrounding mesenchyme. 
B, Chondrification and formation of the interzone. The interzone remains
avascular and highly cellular. C, Formation of synovial mesenchyme.
Synovial mesenchyme forms from the periphery of the interzone and is
invaded by blood vessels. D, Cavitation. Cavities are formed in the cen-
tral and peripheral interzone and merge to form the joint cavity. E, The 
mature joint. (From O’Rahilly R, Gardner E: The embryology of movable
joints. In Sokoloff L (ed): The Joints and Synovial Fluid, Vol 1. New York,
­Academic Press, 1978.)
synovial joint formation and some of the regulatory factors
and extracellular matrix components involved are summa-
rized in Figures 1-3 and 1-4.
INTERZONE FORMATION AND JOINT CAVITATION
The morphology of the developing synovial joint and the
process of joint cavitation have been described in many
classic studies done on the limbs of mammalian and avian
embryos.5 In the human embryo, cartilage condensations,
or chondrifications, can be detected at stage 17, when the
embryo is small, approximately 11.7 mm long.3,4 In the
region of the future joint, following formation of the homo-
geneous chondrogenic interzone at 6 weeks (stages 18 and
19), a three-layered interzone is formed at approximately
7 weeks (stage 21), which consists of two chondrogenic,
perichondrium-like layers that cover the opposing surfaces
of the cartilage anlagen and are separated by a narrow band
of densely packed cellular blastema that remains and forms
the interzone. Cavitation begins in the central interzone at
about 8 weeks (stage 23).
Although these cellular events associated with joint
formation have been recognized for many years, only more
recently have the genes regulating these processes been
elucidated. These genes include growth differentiation
factor (GDF)-5, Wnt-14, bone morphogenetic protein
(BMP)-2, BMP-4, BMP-6, BMP-7, and the GDF-BMP
antagonists.5-8 In addition, joint formation is ­accompanied
by the expression of several fibroblast growth factor (FGF)
family members, including FGF-2 and FGF-4.9 The balance
of signaling between BMP and FGF determines the rate of
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE 

Chondrocyte
Mesenchymal cell Chondrocyte Chondrocyte hypertrophy and
condensation differentiation proliferation vascular invasion Ossification
TGF-β Epiphyseal
Wnt-3A, 7A FGF-18/FGFR3 VEGF ossification
FGF-2, 4, 8,10 IGF-1 BMP-2, 7 FGF-2/FGF-R1 center
Sonic Hh FGF-2/FGFR2 PTHrP Bone Wnt14/ (secondary)
BMP-2, 4, 7 BMP-2, 4, 7, 14 Ihh/Ptc collar β-catenin
Diaphyseal
HoxA, HoxD Sox9, 5, 6 Stat1 Runx2 ossification
Sox9 Gli3, 2 Osterix center
Gli3 Runx2 TCF/Lef1 (primary)
Fra2/JunD Growth
plate

FGF-2 Periarticular
PTHrP
Gli
BMPs FGFR2 (resting)
Perichondrium

Proliferating
Collagen II, IX, XI
FGF-18 BMP-7 FGFR3
Prehypertrophic
Ihh
Ptc

Aggrecan
BMP-2 FGFR1 COMP
FGF-2
Hypertrophic Collagen X
Osteocalcin
BMP-6

Figure 1-3  The stages of diarthrodial joint formation, and the temporal pattern of expression of the genes involved in regulation at different stages.

Subperiosteal Diaphyseal Epiphyseal

ring ossification ossification
center center

Synovial
capsule
TGF-β Sox9,5,6 Wnt14
FGF-2,4,8,10 IGF-1 GDF-5
Wnt3A,7A FGF-2,18 BMP-2,4 Hyaluronan C-1-1
Shh BMP-2,4,7,14 FGF-2 CD44
BMP-2,4,7 Runx2 Articular
PTHrP
Gli3 Cux1 cartilage
Ihh
HoxA, D Erg5
r-Fng
Lmx1b
RA
Mesenchymal Interzone formation Joint initiation Interzone Cavitation Joint
condensation and chondrocyte and ossification formation maturation
differentiation
Figure 1-4  Development of long bones from cartilage anlagen.

proliferation, adjusting the pace of the differentiation.10
Two transcription factors, Cux-1, a homeobox factor, and
the ETS factor ERG/C-1-1, are expressed concurrently with
GDF-5 and Wnt-14 at the onset of joint formation.11,12
Hartmann and Tabin13 have proposed two major roles for
Wnt-14. First, it acts at the onset of joint formation as
a negative regulator of chondrogenesis. Second, it facili-
tates interzone formation and cavitation by inducing the
expression of GDF-5 (also known as cartilage-derived mor-
phogenetic protein-1 [CDMP-1]), Wnt-4, chordin, and the
hyaluronan receptor, CD44.13-15 Paradoxically, application
of GDF-5 to developing joints in mouse embryo limbs in
organ culture causes joint fusion,16 suggesting that tem-
porospatial interactions among distinct cell populations
are important for the correct response. The current view
is that GDF-5 is required at the early stages of condensa-
tions, where it stimulates recruitment and differentiation
of chondrogenic cells, and later, when its expression is
restricted to the interzone.
The distribution of collagen types and keratan sulfate
in developing avian and rodent joints has been character-
ized by immunohistochemistry.17-21 Collagen types I and
III characterize the matrix produced by mesenchymal cells,
which switch to the production of types II, IX, and XI col-
lagens that typify the cartilaginous matrix at the time of
condensation.22 The messenger RNAs encoding the small
proteoglycans, biglycan and decorin, may be expressed at
this time, but the proteins do not appear until after cavi­
tation in the regions destined to become articular carti-
lage.23 The interzone regions are marked by the expression
  GOLDRING  |  Biology of the Normal Joint

A B

C D

C
C

E F
Figure 1-5  In situ hybridization of a 13-day-old (stage 39) chicken embryo middle digit, proximal interphalangeal joint, midfrontal sections. A, Bright-
field image showing developing joint and capsule (C). B, Equivalent paraffin section of opposite limb of same animal, showing onset of cavitation
laterally (arrow). C, Expression of type IIA collagen mRNA in articular surface cells, perichondrium, and capsule. D, Type IIB collagen mRNA is expressed
only in chondrocytes of the anlagen. E, Type XI collagen mRNA is expressed in the surface cells, perichondrium, and capsule, with lower levels in chon-
drocytes. F, Type I collagen mRNA is present in cells of the interzone and capsule. C through F images are dark field. Calibration bar = 1 μm. (From Nalin
AM, Greenlee TK Jr, Sandell LJ: Collagen gene expression during development of avian synovial joints: Transient expression of types II and XI collagen genes in
the joint capsule. Develop Dyn 203:352-362, 1995.)

of type IIA ­ collagen by chondrocyte progenitors in the
perichondrial layers, type IIB and XI collagens by overt­
chondrocytes in the cartilage anlagen, and type I collagen
in the interzone and in the developing capsule and peri-
chondrium (Fig. 1-5).24
The interzone region contains cells in two outer layers that
are destined to differentiate into chondrocytes and become
incorporated into the epiphyses, and in a thin intermedi-
ate zone that are programmed to undergo joint cavitation
and may remain as articular chondrocytes.25 Fluid and mac-
romolecules accumulate in this space and create a nascent
synovial cavity. Blood vessels appear in the surrounding
capsulosynovial blastemal mesenchyme before separation
of the adjacent articulating surfaces.26 Although it was first
assumed that these interzone cells should undergo necrosis
or programmed cell death (apoptosis),27 some investigators
have found no evidence of DNA ­fragmentation preceding
cavitation.24,25,28,29 There also is no evidence that metal-
loproteinases are involved in loss of tissue strength in the
region undergoing cavitation.30 Instead, the actual joint cav-
ity seems to be formed by mechanospatial changes induced
by the synthesis of hyaluronan via uridine diphosphoglucose
dehydrogenase (UDPGD) and hyaluronan synthase. Inter-
action of hyaluronan with its cell surface receptor, CD44,
modulates cell migration, but it is thought that the accu-
mulation of hyaluronan and the associated mechanical
influences play the major role in forcing the cells apart and
inducing rupture of the intervening extracellular matrix by
tensile forces.20,31 This mechanism accounts for the obser-
vation that joint cavitation is incomplete in the absence
of movement.32,33 Equivalent data from human embryonic
joints are difficult to obtain. In all large joints in humans,
complete joint cavities are apparent at the beginning of the
fetal period.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
CARTILAGE FORMATION AND
ENDOCHONDRAL OSSIFICATION
The skeleton develops from the primitive, avascular, densely
packed cellular mesenchyme, termed the skeletal blastema.
Common precursor mesenchymal cells divide into chon-
drogenic, myogenic, and osteogenic lineages that determine
the differentiation of cartilage centrally, muscle peripherally,
and bone. The surrounding tissues, particularly epithelium,
influence the differentiation of mesenchymal progenitor
cells to chondrocytes in cartilage anlagen. The cartilaginous
nodules appear in the middle of the blastema, and simultane-
ously cells at the periphery become flattened and elongated
to form the perichondrium. In the vertebral column, carti-
lage disks arise from portions of the somites surrounding the
notochord, and nasal and auricular cartilage and the embry-
onic epiphysis form from the perichondrium. In the limb,
the cartilage remains as a resting zone that later becomes
the articular cartilage, or undergoes terminal hypertrophic
differentiation to become calcified (growth plate formation)
and is replaced by bone (endochondral ossification). The
latter process requires extracellular matrix remodeling and
vascularization (angiogenesis). These events are controlled
exquisitely by cellular interactions with the surrounding
matrix, growth and differentiation factors, and other envi-
ronmental factors that initiate or suppress cellular signaling
pathways and transcription of specific genes in a temporo-
spatial manner.
Condensation and Limb-Bud Formation
Formation of the cartilage anlage occurs in four stages:
(1) cell migration, (2) aggregation regulated by mesenchy-
mal-epithelial cell interactions, (3) condensation, and (4)
overt chondrocyte differentiation, or chondrification.3,4,34
Interactions with the epithelium determine mesenchymal
cell recruitment and migration, proliferation, and condensa-
tion.34-36 The aggregation of chondroprogenitor mesenchy-
mal cells into precartilage condensations was first described
by Fell37 and depends on signals initiated by cell-cell and
cell-matrix interactions, the formation of gap junctions,
and changes in the cytoskeletal architecture. Before con-
densation, the prechondrocytic mesenchymal cells produce
extracellular matrix that is rich in hyaluronan and type I
collagen and type IIA collagen, which contains the exon-
2-encoded aminopropeptide found in noncartilage colla-
gens.38 The initiation of condensation is associated with
increased hyaluronidase activity and the appearance of the
cell adhesion molecules, neural cadherin (N-cadherin) and
neural cell adhesion molecule (N-CAM), which facilitate
cell-cell interactions.
Before chondrocyte differentiation, the cell-matrix inter-
actions are facilitated by fibronectin binding to syndecan,
downregulating N-CAM and setting the condensation
boundaries. Increased cell proliferation and extracellular
matrix remodeling, with the disappearance of type I col-
lagen, fibronectin, and N-cadherin, and the appearance of
tenascins, matrilins, and thrombospondins, including carti-
lage oligomeric protein, initiate the transition from chon-
droprogenitor cells to a fully committed chondrocyte.2,36,39,40
N-cadherin and N-CAM disappear in differentiating chon-
drocytes and are detectable later only in perichondrial cells.

The differentiated chondrocytes can proliferate and undergo
the complex process of hypertrophic maturation or remain
within cartilage elements in articular joints.
Zwilling41 proposed that positional information for orga-
nization of the limb bud was imparted by diffusible agents
generated at the tip of the limb bud and along its poste-
rior margin, promoting the development of a cartilaginous
anlage along proximal-distal and anterior-posterior axes.
Limb buds develop from the lateral plate mesoderm.42
The patterning of limb mesenchyme is due to interactions
between the mesenchyme and the overlying epithelium.35
The embryonic limb possesses two signaling centers, the api-
cal ectodermal ridge (AER) and the zone of polarizing activ-
ity (ZPA), which produce signals responsible for directing
the proximal-distal outgrowth (AER)and anterior-­posterior
patterning (ZPA).2,39
Much of the current understanding of limb development
is based on early studies in chickens and more recently in
mice. The regulatory events are controlled by interacting
patterning systems involving FGF, hedgehog, BMP, and Wnt
pathways, each of which functions sequentially over time
(see Fig. 1-3).42 Wnt signaling via β-catenin is required to
induce FGFs, such as FGF-10 and FGF-8, which act in posi-
tive feedback loops.42,43 FGF-2, FGF-4, and FGF-8 (induced
by Wnt-3A44), from the specialized epithelial cells in the
AER that are covering the limb-bud tip, control proximal-
distal (shoulder/finger) outgrowth.45 The homeobox (Hox)
transcription factors encoded by the HoxA and HoxD gene
clusters, which are crucial for the early events of limb pat-
terning in the undifferentiated mesenchyme, are required
for the expression of FGF-8 and Sonic hedgehog (Shh),46
and they modulate the proliferation of cells within the con-
densations.34 Among the Hox genes, Hoxa13 and Hoxd13
enhance and Hoxa11 and Hoxd11 suppress early events in
the formation of the cartilage anlagen.
Wnt7a is expressed early during limb bud development
where it acts to maintain Shh expression.42 Shh, produced
by a small group of cells in the posterior zone of the ZPA (in
response to retinoic acid in the mesoderm47 and FGF-4 in
the AER48), plays a key role in directing anterior-posterior
(e.g., little finger/thumb) patterning47,49 and stimulating
expression of BMP-2, BMP-4, BMP-7, and Hox genes.50-52
Shh signaling, which is required for early limb patterning,
but not for limb formation, is mediated by the Shh recep-
tor Patched (Ptc1), which activates another transmembrane
protein, Smoothened (Smo), and inhibits processing of the
Gli3 transcription factor to a transcriptional repressor.43,53
Dorsal-ventral (e.g., knuckles/palm) patterning depends on
the secretion of Wnt-7A54 and expression of the follow-
ing transcription factors: radical fringe (r-Fng) by the dor-
sal ectoderm, and engrailed (En-1) and Lmx1b (which is
induced by Wnt-7A) by the ventral endoderm.43,55
BMP-2, BMP-4, and BMP-7 coordinately regulate
the patterning of limb elements within the condensa-
tions depending on the temporal and spatial expression of
BMP receptors, involving SMAD-dependent and SMAD-
­independent signaling and BMP antagonists, such as nog-
gin and chordin.42,56-58 In vitro and in vivo studies have
shown that BMP signaling is required for the formation of
precartilaginous condensations and for the differentiation
of precursors into chondrocytes.59 Growth of the condensa-
tion ceases when noggin inhibits BMP signaling and permits
  GOLDRING  |  Biology of the Normal Joint
overt ­differentiation to chondrocytes, which are often des-
ignated as “chondroblasts.” The cartilage formed serves as a
template for formation of cartilage elements in the vertebra,
sternum, and rib, and for limb elongation or endochondral
bone formation.
Molecular Signals in Cartilage Morphogenesis
and Growth Plate Development
The cartilage anlagen grow by cell division and deposition
of the extracellular matrix and by apposition of proliferat-
ing cells from the inner chondrogenic layer of the perichon-
drium. The nuclear transcription factor, Sox9, is one of
earliest markers expressed in cells undergoing condensation
and is required for the subsequent stage of chondrogenesis
characterized by the deposition of matrix containing colla-
gens II, IX, and XI and aggrecan in the cartilage anlagen.60,61
Two additional Sox family members, L-Sox5 and Sox6,
which are not present in early mesenchymal condensations,
but are coexpressed with Sox9 during chondrocyte differen-
tiation,62 have a high degree of sequence identity with each
other, but have no sequence homology with Sox9 except
in the HMG box. They can form homodimers or heterodi-
mers, which bind more efficiently to pairs of HMG box sites
than to single sites, and in contrast to Sox9, they contain
no transcriptional activation domain. The expression of
SOX proteins depends on BMP signaling via BMPR1A and
BMPR1B, which are functionally redundant and active in
chondrocyte condensations, but not in the perichondrium.59
L-Sox5 and Sox6 are required for the expression of Col9a1,
aggrecan, link protein, and Col2a1 during overt chondro-
cyte differentiation.63 The runt-domain transcription factor,
Runx2 (also known as core binding factor, Cbfa1), also is
expressed in all condensations, including those that are des-
tined to form bone.64-66
Throughout chondrogenesis, the balance of signaling by
BMPs and FGFs determines the rate of proliferation, adjust-
ing the pace of the differentiation.10 In the long bones,
long after condensation, BMP-2, BMP-3, BMP-4, BMP-5,
and BMP-7 are expressed primarily in the perichondrium,
and only BMP-7 is expressed in the proliferating chondro-
cytes.10 BMP-6 is found later exclusively in hypertrophic
chondrocytes along with BMP-2. More than 23 FGFs have
been identified so far.67 The specific ligands that activate
each FGF receptor (R) during chondrogenesis in vivo have
been difficult to identify because the signaling depends on
the temporal and spatial location of not only the ligands,
but also the receptors.68 FGFR2 is upregulated early in con-
densing mesenchyme and is present later in the periphery
of the condensation along with FGFR1, which is expressed
in surrounding loose mesenchyme. FGFR3 is associated
with proliferation of chondrocytes in the central core of the
mesenchymal condensation and may overlap with FGFR2.
Proliferation of chondrocytes in the embryonic and postna-
tal growth plate is regulated by multiple mitogenic stimuli,
including FGFs, which converge on the cyclin D1 gene.69
In the growth plate, FGFR3 serves as a master inhibi-
tor of chondrocyte proliferation via phosphorylation of
the Stat1 transcription factor, which increases the expres-
sion of the cell cycle inhibitor p21.70 More recent stud-
ies suggest that FGF-18 is the preferred ligand of FGFR3
because Fgf18-deficient mice have an expanded zone of
proliferating chondrocytes similar to that in Fgfr3-deficient
mice, and that FGF-18 can inhibit Indian hedgehog (Ihh)
expression.71 FGF18 and FGF9 are expressed in the peri-
chondrium and periosteum and form a functional gradient
from the proximal proliferating zone, where FGF18 acts via
FGFR3 to downregulate proliferation and subsequent matu-
ration.71,72 FGF18 and FGF9 interact with FGFR1 in the
prehypertrophic and hypertrophic zones, where more recent
evidence indicates that they regulate vascular invasion by
inducing the expression of vascular endothelial growth fac-
tor (VEGF) and VEGFR1. As the epiphyseal growth plate
develops, FGFR3 disappears, and FGFR1 expression is
upregulated in prehypertrophic and hypertrophic chondro-
cytes, suggesting a role for FGFR1 in the regulation of cell
survival and differentiation and possibly cell death.68
The proliferation of chondrocytes in the lower prolifera-
tive and the prehypertrophic zones is under the control of
a local negative feedback loop involving signaling by para-
thyroid hormone related protein (PTHrP) and Ihh.73 Ihh
expression is restricted to the prehypertrophic zone, and the
PTHrP receptor is expressed in the distal zone of periarticu-
lar chondrocytes. The adjacent, surrounding perichondrial
cells express the Hedgehog receptor patched (ptc), which
on Ihh binding, similar to Shh in the mesenchymal con-
densations, activates Smo and induces Gli transcription
factors, which can feedback regulate Ihh target genes in a
positive (Gli1 and Gli2) or negative (Gli3) manner.74 Ihh
induces expression of PTHrP in the perichondrium,75 and
PTHrP signaling stimulates cell proliferation via its receptor
expressed in the periarticular chondrocytes.76 These interac-
tions are modulated by a balance of BMP and FGF signaling
that adjusts the pace of chondrocyte terminal differentia-
tion to the proliferation rate.10 FGF-18 or FGFR3 signaling
can inhibit Ihh expression,71 and BMP signaling upregulates
the expression of Ihh in cells that are beyond the range of
the PTHrP-induced signal.10 Evidence indicates that Ihh
acts independently of PTHrP on periarticular chondrocytes
to stimulate differentiation of columnar chondrocytes in
the proliferative zone, whereas PTHrP acts by preventing
premature differentiation into prehypertrophic and hyper-
trophic chondrocytes, suppressing premature expression of
Ihh.77 Ihh and PTHrP, by transiently inducing proliferation
markers and repressing differentiation markers, function in
a temporospatial manner to determine the number of cells
that remain in the chondrogenic lineage versus the number
that enter the endochondral ossification pathway.73,78
Endochondral Ossification
The development of long bones from the cartilage anlagen
occurs by a process termed endochondral ossification, which
involves terminal differentiation of chondrocytes to the
hypertrophic phenotype, cartilage matrix calcification, vas-
cular invasion, and ossification (see Fig. 1-4).28,78-80 This
process is initiated when the cells in the central region of
the anlage begin to hypertrophy, increasing cellular fluid
volume by almost 20 times. Ihh plays a pivotal role in reg-
ulating endochondral bone formation by synchronizing
perichondrial maturation with chondrocyte hypertrophy,
which is essential for initiating the process of vascular inva-
sion.81 Ihh is expressed in prehypertrophic chondrocytes as
they exit the proliferative phase and enter the hypertrophic
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
phase, at which time they begin to express the hypertrophic
chondrocyte marker, type X collagen (Col10a1) and alkaline
phosphatase. These cells are responsible for laying down the
cartilage matrix that subsequently undergoes mineralization.
Runx2, which serves as a positive regulatory factor in
chondrocyte maturation to hypertrophy,82 is expressed in the
adjacent perichondrium and in prehypertrophic chondro-
cytes, but less in late hypertrophic chondrocytes,83,84 over-
lapping with Ihh, Col10a1, and BMP-6.78,85 BMP-induced
Smad1 interacts with Runx2, and Runx2 and Smad1 are
important for chondrocyte hypertrophy.82,86,87 An essential
role for Runx2 in the process of chondrocyte hypertrophy is
supported by the observation that the late stages of chondro-
cyte hypertrophy are blocked in Runx2-deficient mice.65,88
Interactions with components of the extracellular matrix
also contribute to regulation of the process of chondrocyte
hypertrophy. Matrix metalloproteinase (MMP)-13, a down-
stream target of Runx2, is expressed by terminal hypertro-
phic chondrocytes,89-92 and MMP-13 deficiency results in
significant interstitial collagen accumulation leading to the
delay of endochondral ossification in the growth plate with
increased length of the hypertrophic zone.93,94
In contrast, Col10a1 knockout mice and transgenic mice
with a dominant interference Col10a1 mutation have subtle
growth plate phenotypes with compressed proliferative and
hypertrophic zones and altered mineral deposition.95 Muta-
tions in the COL10A1 gene are associated with the dwarf-
ism observed in human chondrodysplasias. These mutations
affect regions of the growth plate that are under great
mechanical stress, and it has been suggested that the defect
in skeletal growth may be due partly to alteration of the
mechanical integrity of the pericellular matrix in the hyper-
trophic zone, although a role for defective vascularization
also has been proposed.96 The extracellular matrix remodel-
ing that accompanies chondrocyte terminal differentiation is
thought to induce an alteration in the environmental stress
experienced by hypertrophic chondrocytes, which eventu-
ally undergo apoptosis.78,97,98 Together these studies indicate
that the composition and remodeling of the extracellular
matrix play an important role in processes associated with
chondrocyte hypertrophy, vascular invasion, and, as dis-
cussed subsequently, osteoblast recruitment and subsequent
bone formation.91
Vascular invasion of the hypertrophic zone is required
for the replacement of calcified cartilage by bone.85,92 The
angiogenic factor, VEGF, promotes vascular invasion by
specifically activating localized receptors, including Flk
expressed in endothelial cells in the perichondrium or sur-
rounding soft tissues, neuropilin 1 (Npn1) expressed in late
hypertrophic chondrocytes, or Npn2 expressed exclusively
in the perichondrium.28 VEGF is expressed as three differ-
ent isoforms: VEGF188, a matrix-bound form, is essential
for metaphyseal vascularization, whereas the soluble form,
VEGF120 (VEGFA), regulates chondrocyte survival and
epiphyseal cartilage angiogenesis.99-101 VEGF164 can be
either soluble or matrix bound and may act directly on
chondrocytes via Npn2. VEGF is released from the extra-
cellular matrix by MMPs, including MMP-9, membrane-
type (MT)1-MMP (MMP-14), and MMP-13. MMP-9 is
expressed by endothelial cells that migrate into the central
region of the hypertrophic cartilage.91 MMP-14, which has
a broader range of expression than MMP-9, is essential for

chondrocyte proliferation and secondary ossification,102
whereas MMP-13 is found exclusively in late hypertrophic
chondrocytes.83 These events of cartilage matrix remodel-
ing and vascular invasion are required for the migration and
differentiation of osteoclasts and osteoblasts, which remove
the mineralized cartilage matrix and replace it with bone.
DEVELOPMENT OF THE JOINT CAPSULE
AND SYNOVIUM
The interzone and the contiguous perichondrial envelope, of
which the interzone is a part, contain the mesenchymal cell
precursors that give rise to other joint components, includ-
ing the joint capsule, synovial lining, menisci, intracapsular
ligaments, and tendons.3,4,103,104 The external mesenchymal
tissue condenses as a fibrous capsule. The peripheral mes-
enchyme becomes vascularized and is incorporated as the
synovial mesenchyme, which differentiates into a pseudo-
membrane at about the same time as cavitation begins in
the central interzone (stage 23, approximately 8 weeks).
The menisci arise from the eccentric portions of the articu-
lar interzone. In common usage, the term synovium refers
to the true synovial lining and the subjacent vascular and
areolar tissue, up to—but excluding—the capsule. Synovial
lining cells can be distinguished as soon as the multiple cav-
ities within the interzone begin to coalesce. At first, these
are exclusively fibroblast-like (type B) cells.
As the joint cavity increases in size, synovial-lining cell
layers expand by proliferation of fibroblast-like cells and
recruitment of macrophage-like (type A) cells from the cir-
culation.105 The synovial lining cells express the hyaluronan
receptor, CD44, and UDPGD, the levels of which remain
elevated after cavitation. This increased activity likely con-
tributes to the high concentration of hyaluronan in joint flu-
ids.31,106 Further synovial expansion results in the appearance
of synovial villi at the end of the second month, early in the
fetal period, which greatly increases the surface area available
for exchange between the joint cavity and the vascular space.
The role of innervation in the developing joint is not
well understood. A dense capillary network develops in the
subsynovial tissue, with numerous capillary loops that pen-
etrate into the true synovial lining layer. The human syno-
vial microvasculature is already innervated by 8 weeks (stage
23) of gestation, around the time of joint cavitation,103 as is
shown by immunoreactivity for the neuronal “housekeeping”
enzymes.107 Evidence of neurotransmitter function is not
found until much later, however, with the appearance of the
sensory neuropeptide, substance P, at 11 weeks. The putative
sympathetic neurotransmitter, neuropeptide Y, appears at
13 weeks of gestation, along with the catecholamine-
­synthesizing enzyme tyrosine hydroxylase. The finding that
the Slit2 gene, which functions for the guidance of neuronal
axons and neurons, is expressed in the mesenchyme adjacent
to the AER (stages 20 to 22) and in peripheral mesenchyme
of the limb bud (stages 23 to 28) suggests that innervation is
an integral part of synovial joint development.108
DEVELOPMENT OF NONARTICULAR JOINTS
In contrast to articular joints, the temporomandibular joint
develops slowly, with cavitation at a crown-rump length of
57 to 75 mm (i.e., well into the fetal stage).109 This slow
  GOLDRING  |  Biology of the Normal Joint
development may be because this joint develops in the
absence of a continuous blastema and involves the insertion
between bone ends of a fibrocartilaginous disk that arises
from muscular and mesenchymal derivatives of the first pha-
ryngeal arch.
The development of other types of joints, such as synar-
throses, is similar to that of diarthrodial joints except that
cavitation does not occur, and synovial mesenchyme is not
formed. In these respects, synarthroses and amphiarthroses
resemble the “fused” peripheral joints induced by paralyz-
ing chicken embryos,110 and they may develop as they do
because there is relatively little motion during their forma-
tion.
Human vertebrae and intervertebral disks develop as
units, each derived from a homogeneous blastema arising
from a somite. Each embryonic intervertebral disk serves as
a rostral and caudal chondrogenic zone for the two adjacent
evolving vertebral bodies. The periphery of the embryonic
“disk” is replaced by the anulus fibrosus.111 The interver-
tebral disk bears many similarities to the joint; the anulus
is the joint capsule, the nucleus pulposus is the joint cav-
ity, and the vertebral end plates are the cartilage-covered
bone ends composing the articulation. The proteoglycans
and collagens expressed during development of the inter-
vertebral disk have been mapped and reflect the complex
structure-function relationships that allow flexibility and
resistance to compression in the spine.112-115
DEVELOPMENT OF ARTICULAR CARTILAGE
In the vertebrate skeleton, cartilage is the product of cells
from three distinct embryonic lineages. Craniofacial carti-
lage is formed from cranial neural crest cells, the cartilage
of the axial skeleton (intervertebral disks, ribs, and ster-
num) forms from paraxial mesoderm (somites), and the
articular cartilage of the limbs is derived from the lateral
plate mesoderm.2 In the developing limb bud, mesenchymal
condensations, followed by chondrocyte differentiation and
maturation, occur in digital zones, whereas undifferentiated
mesenchymal cells in the interdigital web zones undergo
cell death.116 Embryonic cartilage is destined for one of sev-
eral fates: It can remain as permanent cartilage, as on the
articular surfaces of bones, or it can provide a template for
the formation of bones by endochondral ossification. Dur-
ing development, chondrocyte maturation expands from
the central site of the original condensation, which forms
the cartilage anlage resembling the shape of the future bone,
toward the ends of the forming bones. During joint cavita-
tion, the peripheral interzone is absorbed into each adjacent
cartilaginous zone, evolving into the articular surface. The
articular surface is destined to become a specialized carti-
laginous structure that does not normally undergo vascular-
ization and ossification.
More recent evidence indicates that postnatal maturation
of the articular cartilage involves an appositional growth
mechanism originating from progenitor cells at the articu-
lar surface, rather than by an interstitial mechanism.113 The
chondrocytes of mature articular cartilage are terminally dif-
ferentiated cells that continue to express cartilage-specific
matrix molecules, such as type II collagen and aggrecan (see
following section).19,21,24 Through the processes described
previously, the articular joint spaces are developed and lined
on all surfaces either by cartilage or by synovial lining cells.
These two different tissues merge at the enthesis, the region
at the periphery of the joint where the cartilage melds into
bone, and where ligaments and the capsule are attached.117
In the postnatal growth plate, the differentiation of the peri-
chondrium also is linked to the differentiation of the chon-
drocytes in the epiphysis into different zones of the growth
plate, and contributes to longitudinal bone growth.28,78
ORGANIZATION AND PHYSIOLOGY
OF THE MATURE JOINT
The unique structural properties and biochemical compo-
nents of diarthrodial joints make them extraordinarily dura-
ble load-bearing devices.118 The mature diarthrodial joint
is a complex structure, influenced by its environment and
mechanical demands (see Chapter 6). There are structural
differences between joints determined by their different
functions. The shoulder joint, which demands an enormous
range of motion, is stabilized primarily by muscles, whereas
the hip, requiring motion and antigravity stability, has an
intrinsically stable ball-and-socket configuration. The com-
ponents of the “typical” synovial joint are the synovium,
muscles, tendons, ligaments, bursae, menisci, articular carti-
lage, and subchondral bone. The anatomy and physiology of
muscles are described in detail in Chapter 5.
SYNOVIUM
The synovium lines the joint cavity and is the sight of pro-
duction of synovial fluid that provides the nutrition for the
articular cartilage and lubricates the cartilage surfaces. It
is a thin membrane between the fibrous joint capsule and
fluid-filled synovial cavity that attaches to skeletal tissues at
the bone-cartilage interface and does not encroach on the
surface of the articular cartilage. It is divided into functional
compartments: the lining region (synovial intima), the sub-
intimal stroma, and the neurovasculature (Fig. 1-6). The
synovial intima, also termed synovial lining, is the superficial
layer of the normal synovium that is in contact with the
intra-articular cavity.106,119 The synovial lining is loosely
attached to the subintima, which contains blood vessels,
lymphatics, and nerves. Capillaries and arterioles generally
are located directly underneath the synovial intima, whereas
venules are located closer to the joint capsule.
A transition from loose to dense connective tissue occurs
from the joint cavity to the capsule. Most cells in the nor-
mal subintimal stroma are fibroblasts and macrophages,
although adipocytes and occasional mast cells are present.106
These compartments are not circumscribed by basement
membranes, but nonetheless have distinct functions; they
are separated from each other by chemical barriers, such as
membrane peptidases, which limit the diffusion of regula-
tory factors between compartments. Synovial compartments
are unevenly distributed within a single joint. Vascular-
ity is high at the enthesis where synovium, ligament, and
cartilage coalesce.120 Far from being a homogeneous tissue
in continuity with the synovial cavity, synovium is highly
heterogeneous, and synovial fluid may be poorly represen-
tative of the tissue-fluid composition of any synovial tissue
compartment. In rheumatoid arthritis, the synovial lining
of diarthrodial joints is the site of the initial inflammatory
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE 

Intimal Intimal
macrophage fibroblasts Synovial fluid

Subintimal
fibroblast
Subintimal
macrophage

Blood vessels

A B
Figure 1-6  A, Schematic representation of normal human synovium. The intima contains specialized fibroblasts expressing vascular cell adhe-
sion molecule-1 (VCAM-1) and uridine diphosphoglucose (UDPG) and specialized macrophages expressing FcγRIIIa. The deeper subintima contains
­unspecialized counterparts. B, Microvascular endothelium in human synovium contains receptors for the vasodilator/growth factor substance P. Silver
grains represent specific binding of [125I]Bolton Hunter–labeled substance P to synovial microvessels (arrows). Arrowheads indicate the synovial surface.
Emulsion-dipped in vitro receptor autoradiography preparations with hematoxylin and eosin counterstain. Calibration bar = 1 μm. (A from Edwards JCW:
Fibroblast biology: Development and differentiation of synovial fibroblasts in arthritis. Arthritis Res 2:344-347, 2000.)

process.121,122 This lesion is characterized by proliferation
of the synovial lining cells, increased vascularization, and
infiltration of the tissue by inflammatory cells, including
lymphocytes, plasma cells, and activated macrophages (see
Chapter 65).123-125
Synovial Lining
The synovial lining, a specialized condensation of mesen-
chymal cells and extracellular matrix, is located between the
synovial cavity and stroma. In normal synovium, the lining
layer is two to three cells deep, although intra-articular fat
pads usually are covered by only a single layer of synovial
cells, and ligaments and tendons are covered by synovial
cells that are widely separated. At some sites, lining cells are
absent, and the extracellular connective tissue constitutes
the lining layer.126 Such “bare areas” become increasingly
frequent with advancing age.127 Although the synovial lin-
ing is often referred to as the synovial membrane, the term
membrane is more correctly reserved for epithelia that have
basement membranes, tight intercellular junctions, and
desmosomes. Instead, synovial lining cells lie loosely in a
bed of hyaluronate interspersed with collagen fibrils. This is
the macromolecular sieve that imparts the semipermeable
nature of the synovium. The absence of any true epithelial
tissue, including basement membrane, is a major determi-
nant of joint physiology.
Early electron microscopic studies characterized lin-
ing cells as macrophage-derived type A synoviocytes and
fibroblast-derived type B synoviocytes.128 High UDPGD
activity and CD55 are used to distinguish type B synovial
cells, whereas nonspecific esterase and CD68 typify type
A cells.129,130 Normal synovium is lined predominantly by
fibroblast-like cells, whereas macrophage-like cells compose
only 10% to 20% of lining cells (see Fig. 1-6).
Type A, macrophage-like synovial cells contain vacu-
oles, a prominent Golgi apparatus, and filopodia, but they
have little rough endoplasmic reticulum. These cells express
numerous cell surface markers of the monocyte-macrophage
lineage, including CD11b, CD68, CD14, CD163, and the
IgG Fc receptor, FcγRIIIa.106 Synovial intimal macrophages
are phagocytic and may provide a mechanism by which par-
ticulate matter can be cleared from the normal joint cavity.
Similar to other tissue macrophages, these cells have little
capacity to proliferate and are likely localized to the joint
during development. The op/op osteopetrotic mouse that is
deficient in macrophages because of an absence of macro-
phage colony-stimulating factor also lacks synovial macro-
phages.131 This finding provides further evidence that type
A synovial cells are of a common lineage with other tissue
macrophages. Although they represent only a small percent-
age of the cells in the normal synovium, the macrophages
are recruited from the circulation during synovial inflamma-
tion, partly from subchondral bone marrow through vascu-
lar channels near the enthesis.
The type B, fibroblast-like synovial cell contains fewer
vacuoles and filopodia than type A cells and has abundant
protein-synthetic organelles. Similar to other fibroblasts,
lining cells express the collagen synthesis enzyme prolyl
hydroxylase and synthesize extracellular matrix compo-
nents, including collagens, sulfated proteoglycans, fibronec-
tin, fibrillin-1, and tenascin.106,132 They have the potential
to proliferate, although proliferation markers are rarely seen
in normal synovium.133 In contrast to stromal fibroblasts,
synovial intimal fibroblasts express UDPGD and synthesize
hyaluronan, an important constituent of synovial fluid.106
They also synthesize lubricin, which, together with hyaluro-
nan, is necessary for the low-friction interaction of cartilage
surfaces in the diarthrodial joint. Despite not being a true
epithelium, synovial lining cells bear abundant membrane
peptidases on their surface, capable of degrading a wide
range of regulatory peptides, such as substance P and angio-
tensin II.134 These enzymes may be important in limiting
the diffusion of these potent peptide mediators away from
the immediate vicinity of their site of release and action.
Normal synovial lining cells also express a rich array of
adhesion molecules, including CD44, the principal receptor
for hyaluronan; vascular cell adhesion molecule (VCAM)-1;
and intercellular adhesion molecule (ICAM)-1.106,135-137
These are probably essential for cellular attachment to
 10 GOLDRING  |  Biology of the Normal Joint
specific matrix components in the synovial lining region,
preventing loss into the synovial cavity of cells subjected
to deformation and shear stresses during joint movement.
Adhesion molecules such as VCAM-1 and ICAM-1 poten-
tially also are involved in the recruitment of inflammatory
cells during the evolution of arthritis. Cadherins mediate
cell-cell adhesion between adjacent cells of the same type.
The identification of cadherin-11 as a key adhesion mol-
ecule that regulates the formation of the synovial lining dur-
ing development and the synoviocyte function postnatally
has provided the opportunity to examine its role in inflam-
matory joint disease.138 Cadherin-11 deficiency or treatment
with cadherin-11 antibody or a cadherin-11 fusion protein
reduced synovial inflammation and reduced cartilage ero-
sion in an animal model of arthritis.139
Synovial Vasculature
The subintimal synovium contains blood vessels, providing
the blood flow that is required for solute and gas exchange
in the synovium itself and for the generation of synovial
fluid.120 The avascular articular cartilage also depends on
nutrition in the synovial fluid, derived from the synovial
vasculature. The vascularized synovium behaves similar to
an endocrine organ, generating factors that regulate syn-
oviocyte function and serving as a selective gateway that
recruits cells from the circulation during stress and inflam-
mation.140 Finally, synovial blood flow plays an important
role in regulating intra-articular temperature.
The synovial vasculature can be divided, on morpho-
logic and functional grounds, into arterioles, capillaries, and
venules. In addition, lymphatics accompany arterioles and
larger venules.106,120 Arterial and venous networks of the
joint are complex and are characterized by arteriovenous
anastomoses that communicate freely with blood vessels in
periosteum and periarticular bone. As large synovial arteries
enter the deep layers of the synovium near the capsule, they
give off branches, which bifurcate again to form “microvas-
cular units” in the subsynovial layers. The synovial lining
region, the surfaces of intra-articular ligaments, and the
entheses (in the angle of ligamentous insertions into bone)
are particularly well vascularized.120
The distribution of synovial vessels, which were formed
largely as a result of vasculogenesis during development
of the joint, displays considerable plasticity. Vasculogen-
esis is a dynamic process that depends on the cellular
interactions with regulatory factors and the extracellular
matrix that are also important in angiogenesis. In inflam-
matory arthritis, the density of blood vessels decreases
relative to the growing synovial mass, creating a hypoxic
and acidotic environment.141,142 Angiogenic factors such
as VEGF, acting via VEGF receptor 1 and 2 (Flt-1 and
Flk-1), and basic FGF promote proliferation and migra-
tion of endothelial cells, a process that is facilitated by
matrix-degrading enzymes and adhesion molecules such
as integrin αvβ3 and E-selectin, expressed by activated
endothelial cells.143-145 Vessel maturation is facilitated by
angiopoietin-1 acting via the Tie-2 receptor. The angio-
genic molecules are restricted to the capillary epithe-
lium in normal synovium, but their levels are elevated in
inflamed synovium in perivascular sites and areas remote
from vessels.146,147
Regulation of Synovial Blood Flow
Synovial blood flow is regulated by intrinsic (autocrine
and paracrine) and extrinsic (neural and humoral) systems.
Locally generated factors, such as the peptide vasoconstrictors
angiotensin II and endothelin-1, act on adjacent arteriolar
smooth muscle to regulate regional vascular tone.120 Nor-
mal synovial arterioles are richly innervated by sympathetic
nerves containing vasoconstrictors, such as norepinephrine
and neuropeptide Y, and by “sensory” nerves that also play
an efferent vasodilatory role by releasing neuropeptides, such
as substance P and calcitonin gene–related peptide.148,149
Arterioles regulate regional blood flow. Capillaries and post-
capillary venules are sites of fluid and cellular exchange. Cor-
respondingly, regulatory systems are differentially distributed
along the vascular axis. Angiotensin-converting enzyme,
which generates angiotensin II, is localized predominantly
in arteriolar and capillary endothelia and decreases during
inflammation.150 Specific receptors for angiotensin II and
for substance P are abundant on synovial capillaries, with
lower densities on adjacent arterioles. Dipeptidyl peptidase
IV, a peptide-degrading enzyme, is specifically localized to
the cell membranes of venular endothelium. The synovial
vasculature is not only functionally compartmentalized from
the surrounding stroma, but also highly specialized along its
arteriovenous axis. Other unique characteristics of the nor-
mal synovial vasculature include the presence of inducible
nitric oxidase synthase–independent 3-nitrotyrosine, a reac-
tion product of peroxynitrite,151 and the localization of the
synoviocyte-derived CXCL12 chemokine on heparan sulfate
receptors on endothelial cells,152 suggesting physiologic roles
for these molecules in normal vascular function.
JOINT INNERVATION
Dissection studies have shown that each joint has a dual
nerve supply, consisting of specific articular nerves that
penetrate the capsule as independent branches of adjacent
peripheral nerves and articular branches that arise from
related muscle nerves. The definition of joint position and
the detection of joint motion are monitored separately and
by a combination of multiple inputs from different receptors
in varied systems. Nerve endings in muscle and skin and
in the joint capsule mediate sensation of joint position and
movement.153,154 Normal joints have afferent (sensory)
and efferent (motor) innervations. Fast-conducting, myelin-
ated A fibers innervating the joint capsule are important
for proprioception and detection of joint movement; slow-
­conducting, unmyelinated C fibers transmit diffuse pain
sensation and regulate synovial microvascular function.
Normal synovium is richly innervated by fine, unmyelin-
ated nerve fibers that follow the courses of blood vessels and
extend into the synovial lining layers.148 These nerve fibers
do not have specialized endings and are slow-conducting
fibers; they may transmit diffuse, burning, or aching pain
sensation. Sympathetic nerve fibers surround blood ves-
sels, particularly in the deeper regions of normal synovium.
They contain and release classic neurotransmitters, such as
norepinephrine, and neuropeptides that constrict synovial
blood vessels. Neuropeptides that are markers of sensory
nerves include substance P, calcitonin gene–related peptide,
neuropeptide Y, and vasoactive intestinal peptide.148,155-157
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
Afferent nerves containing substance P also have an
efferent role in the synovium. Substance P is released from
peripheral nerve terminals into the joint, and specific, G
protein–coupled receptors for substance P are localized to
microvascular endothelium in normal synovium. Abnor-
malities of articular innervation that are associated with
inflammatory arthritis may contribute to the failure of syno-
vial inflammation to resolve.148,158 Excessive local neuro-
peptide release may result in the loss of nerve fibers owing to
neuropeptide depletion. Synovial tissue proliferation with-
out concomitant growth of new nerve fibers may lead to
an apparent partial denervation of synovium.148,158 Studies
in patients suggest that free nerve endings containing sub-
stance P may modulate inflammation and the pain pathway
in osteoarthritis.159 Afferent nerve fibers from the joint play
an important role in the reflex inhibition of muscle contrac-
tion. Trophic factors generated by motoneurons, such as the
neuropeptide calcitonin gene–related peptide, are impor-
tant in maintaining muscle bulk and a functional neuromus-
cular junction.160 Decreases in motoneuron trophic support
during articular inflammation probably contribute to muscle
wasting.
Mechanisms of joint pain have been reviewed in de-
tail.161,162 In a noninflamed joint, most sensory nerve fibers
do not respond to movement within the normal range;
these are referred to as silent nociceptors. In an acutely
inflamed joint, however, these nerve fibers become sensi-
tized by mediators, such as bradykinin, neurokinin 1, and
prostaglandins (peripheral sensitization), such that normal
movements induce pain. Pain sensation is upregulated or
downregulated further in the central nervous system, at the
level of the spinal cord and in the brain, by central sensitiza-
tion and “gating” of nociceptive input. Although the nor-
mal joint may respond predictably to painful stimuli, there
is often a poor correlation between apparent joint disease
and perceived pain in chronic arthritis. Pain associated with
joint movements within the normal range is a characteristic
symptom described by patients with chronically inflamed
joints caused by rheumatoid arthritis. Chronically inflamed
joints may not be painful at rest, however, unless acutely
inflamed.163
TENDONS
Tendons are functional and anatomic bridges between mus-
cle and bone.164,165 They focus the force of a large mass of
muscle into a localized area on bone and, by splitting to form
numerous insertions, may distribute the force of a single
muscle to different bones. Tendons are formed of longitu-
dinally arranged collagen fibrils embedded in an organized,
hydrated proteoglycan matrix with blood vessels, lymphat-
ics, and fibroblasts.166 Cross-links between adjacent colla-
gen chains or molecules contribute to the tensile strength
of the tendon. 167,168 Tendon collagen fibrillogenesis is initi-
ated during early development by a highly ordered process
of alignment involving the actin cytoskeleton and cad-
herin-11.169,170 Many tendons, particularly tendons with a
large range of motion, run through vascularized, discontinu-
ous sheaths of collagen lined with mesenchymal cells resem-
bling synovium. Gliding of tendons through their sheaths is
enhanced by hyaluronic acid produced by the lining cells.
Tendon movement is essential for the embryogenesis and
11
maintenance of tendons and their sheaths. Degenerative
changes appear in tendons, and fibrous adhesions form
between tendons and sheaths when inflammation or surgi-
cal incision is followed by long periods of immobilization.171
At the myotendinous junction, recesses between muscle
cell processes are filled with collagen fibrils, which blend
into the tendon. At its other end, collagen fibers of the
tendon typically blend into fibrocartilage, mineralize, and
merge into bone through a fibrocartilaginous transition zone
termed the enthesis, or insertion site.172
Tendon fibroblasts synthesize and secrete collagens, pro-
teoglycans, and other matrix components, such as fibronec-
tin and tenascin C, and MMPs and their inhibitors, which
can contribute to the breakdown and repair of tendon com-
ponents.166,173-176 Collagen fibrils in tendon are composed
primarily of type I collagen with some type III collagen, but
there are regional differences in the distribution of other
matrix components. The compressed region contains the
small proteoglycans, biglycan, decorin, fibromodulin, and
lumican, and the large proteoglycan versican.177,178 The
major components in the tensile region of the tendon are
decorin, microfibrillar type VI collagen, fibromodulin, and
proline and arginine-rich end leucine-rich repeat protein.
The presence of cartilage oligomeric matrix protein, aggre-
can, and biglycan and collagen types II, IX, and XI is indica-
tive of fibrocartilage.179,180 The collagen fiber orientation at
the tendon-to-bone enthesis is important for maintaining
microarchitecture, by reducing the stress concentrations and
shielding the outward splay of the insertion from the highest
stresses.181 Understanding the structure has implications for
tendon repair because motion between a tendon graft and
bone tunnel may impair early graft incorporation and lead
to tunnel widening secondary to bone resorption.182
Failure of the muscle-tendon apparatus is rare, but when
it does occur, it is secondary to enormous, quickly generated
forces across a joint and usually occurs near the tendon inser-
tion into bone.183,184 Factors that may predispose to tendon
failure are aging processes, including loss of extracellular
water and an increase in intermolecular cross-links of colla-
gen; tendon ischemia; iatrogenic factors, including injection
of glucocorticoids; and deposition of calcium hydroxyapatite
crystals within the collagen bundles. Alterations in collagen
fibril composition and structure are associated with tendon
degeneration during aging and may predispose to osteoar-
thritis.185,186 Evidence indicates that BMPs promote tendon
repair if osteogenic signaling is impaired.187
LIGAMENTS
Ligaments provide a stabilizing bridge between bones, per-
mitting a limited range of movement.188 The ligaments
often are recognized only as hypertrophied components
of the fibrous joint capsule and are structurally similar to
tendons.189 Although the fibers are oriented parallel to the
longitudinal axis of both tissues,164 the collagen fibrils in
ligaments are nonparallel and arranged in fibers that are ori-
ented roughly along the long axis in a wavy, undulating pat-
tern, or “crimp,” which can straighten in response to load.
Some ligaments have a higher ratio of elastin to collagen
(1:4) than tendons (1:50), which permits a greater degree
of stretch. Ligaments also have larger amounts of reducible
cross-links, more type III collagen, slightly less total ­collagen,
 12 GOLDRING  |  Biology of the Normal Joint
and more glycosaminoglycans compared with tendons. The
cells in ligaments seem to be more metabolically active than
the cells in tendons because they have more plump cellular
nuclei and higher DNA content.
During postnatal growth, the development of ligament
attachment zones involves changes in the ratios and distri-
bution of collagen types I, III, and V and the synthesis of
type II collagen and proteoglycans by fibrochondrocytes that
develop from ligament cells at the attachment zone.190,191
Attachment zones are believed to permit gradual transmis-
sion of the tensile force between ligament and bone.
Ligaments play a major role in the passive stabilization
of joints, aided by the capsule and, when present, menisci.
In the knee, the collateral and cruciate ligaments provide
stability when there is little or no load on the joint. As com-
pressive load increases, there is an increasing contribution
to stability from the joint surfaces themselves and the sur-
rounding musculature. Injured ligaments generally heal, and
structural integrity is restored by contracture of the heal-
ing ligament so that it can act again as a stabilizer of the
joint.192
BURSAE
The many bursae in the human body facilitate gliding of
one tissue over another, much as a tendon sheath facili-
tates movement of its tendon. Bursae are closed sacs, lined
sparsely with mesenchymal cells similar to synovial cells,
but they are generally less well vascularized than synovium.
Most bursae differentiate concurrently with synovial joints
during embryogenesis. During life, however, trauma or
inflammation may lead to the development of new bursae,
hypertrophy of previously existing ones, or communication
between deep bursae and joints. In patients with rheuma-
toid arthritis, communications may exist between the sub-
acromial bursae and the glenohumeral joint, between the
gastrocnemius or semimembranosus bursae and the knee
joint, and between the iliopsoas bursa and the hip joint. It
is unusual, however, for subcutaneous bursae, such as the
prepatellar bursa or olecranon bursa, to develop communi-
cation with the underlying joint.193
MENISCI
The meniscus, a fibrocartilaginous, wedge-shaped structure,
is best developed in the knee, but also is found in the acro-
mioclavicular and sternoclavicular joints, the ulnocarpal
joint, and the temporomandibular joint.194,195 Until more
recently, menisci were thought to have little function and a
quiescent metabolism with no capability of repair, although
early observations indicated that removal of menisci from
the knee may lead to premature arthritic changes in the
joint.196 Evidence from an arthroscopic study of patients
with anterior cruciate ligament insufficiency indicates that
the pathology of the medial meniscus correlates with that of
the medial femoral cartilage.197 The meniscus is now con-
sidered to be an integral component of the knee joint that
has important functions in joint stability, load distribution,
shock absorption, and lubrication.194,195
The microanatomy of the meniscus is complex and
age dependent.198 The characteristic shape of the lat-
eral and the medial menisci is achieved early in prenatal
d­ evelopment. At that time, the menisci are cellular and
highly vascularized; with maturation, vascularity decreases
progressively from the central margin to the peripheral
margin. After skeletal maturity, the peripheral 10% to 30%
of the meniscus remains highly vascularized by a circumfer-
ential capillary plexus and is well innervated.199 Tears in
this vascularized peripheral zone may undergo repair and
remodeling.200 The central portion of the mature meniscus
is an avascular fibrocartilage, however, without nerves or
lymphatics, consisting of cells surrounded by an abundant
extracellular matrix of collagens, chondroitin sulfates, der-
matan sulfates, and hyaluronic acid. Tears in this central
zone heal poorly, if at all.
Collagen constitutes 60% to 70% of the dry weight of the
meniscus and is mostly type I collagen, with lesser amounts
of types III, V, and VI. A small quantity of cartilage-specific
type II collagen is localized to the inner, avascular portion of
the meniscus. Collagen fibers in the periphery are mostly cir-
cumferentially oriented, with radial fibers extending toward
the central portion.201-204 Elastin content is around 0.6%,
and proteoglycan content is around 2% to 3% dry weight.
Aggrecan and decorin are the major proteoglycans in the
adult meniscus.205,206 Decorin is the predominant proteo-
glycan synthesized in the meniscus from young individu-
als, whereas the relative proportion of aggrecan synthesis
increases with age. Although the capacity of the meniscus
to synthesize sulfated proteoglycans decreases after the teen-
age years, the age-related increases in expression of decorin
and aggrecan mRNA suggest that the resident cells are able
to respond quickly to alterations in the biomechanical envi-
ronment.207
The meniscus was defined originally as a fibrocartilage,
based on the rounded or oval shape of most of the cells
and the fibrous microscopic appearance of the extracellular
matrix.208 Based on molecular and spatial criteria, three dis-
tinct populations of cells are recognized in the meniscus of
the knee joint202:
1. The fibrochondrocyte is the most abundant cell in
the middle and inner meniscus, synthesizing primarily
type I collagen and relatively small amounts of type II
and III collagens. It is round or oval in shape and has
a pericellular filamentous matrix containing type VI
collagen.
2. The fibroblast-like cells lack a pericellular matrix
and are located in the outer portion of the menis-
cus. They are distinguished by long, thin, branching
cytoplasmic projections that stain for vimentin. They
make contact with other cells in different regions via
connexin 43–containing gap junctions. The pres-
ence of two centrosomes, one associated with a pri-
mary celium, suggests a sensory, rather than motile,
function that could enable the cells to respond to cir-
cumferential tensile loads, rather than compressive
loads.209
3. The superficial zone cells have a characteristic fusi-
form shape with no cytoplasmic projections. The
occasional staining of these cells in the uninjured
meniscus with α-actin and their migration into sur-
rounding wound sites suggest that they are specialized
progenitor cells that may participate in a remod­
eling response in the meniscus and surrounding
tissues.210,211
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE 13

b­ isphosphonates,228 and estrogen.229 Although receptor

MATURE ARTICULAR CARTILAGE activator of nuclear factor ĸB (NFĸB) ligand (RANKL),
Articular cartilage is a specialized connective tissue that cov- which mediates osteoclast differentiation and activity, and
ers the weight-bearing surfaces of diarthrodial joints.118,212,213 its receptor RANK, a member of the tumor necrosis fac-
The principal functions of cartilage layers covering bone ends tor receptor family, are expressed in adult articular chon-
are to permit low-friction, high-velocity movement between drocytes, exogenous RANKL does not activate NFĸB or
bones, to absorb the transmitted forces associated with loco- stimulate the production of collagenase or nitric oxide.230
motion, and to contribute to joint stability. Lubrication by Inhibition of RANKL expression does not block cartilage
synovial fluid provides frictionless movement of the articu- destruction in ­ inflammatory models,231 although RANKL
lating cartilage surfaces. Chondrocytes (see Chapter 3) are may have indirect effects on cartilage by its protective effect
the single cellular component of adult hyaline articular car- on bone.232
tilage and are responsible for synthesizing and maintaining
the highly specialized cartilage matrix macromolecules. The Table 1-1  Extracellular Matrix Components  
cartilage extracellular matrix is composed of an extensive of Articular Cartilage*
network of collagen fibrils, which confers tensile strength, Collagens
and an interlocking mesh of proteoglycans, which provides
Type II
compressive stiffness through the ability to absorb and Type IX
extrude water. Numerous other noncollagenous proteins also Type XI
contribute to the unique properties of cartilage (Table 1-1). Type VI
Histologically, the tissue appears to be fairly homogeneous Types XII, XIV
Type X (hypertrophic chondrocyte)
and clearly distinguished from the calcified cartilage and
underlying subchondral bone (Fig. 1-7). The organization of Proteoglycans
articular cartilage and structure-function relationships of car- Aggrecan
tilage matrix components are described in Chapter 3. Versican
Link protein
Biglycan (DS-PGI)
SUBCHONDRAL BONE INTERACTIONS Decorin (DS-PGII)
WITH ARTICULAR CARTILAGE Epiphycan (DS-PGIII)
Fibromodulin
The subchondral bone plate beneath the calcified base of Lumican
Proline/arginine-rich and leucine-rich repeat protein (PRELP)
articular cartilage may have many effects on the cartilage Chondroadherin
above it. Its stiffness modifies the compressive forces to Perlecan
which articular cartilage is subjected, its blood supply may Lubricin (SZP)
be important in cartilage nutrition (see following section), Other Noncollagenous Proteins (Structural)
and its cells may produce peptides that regulate chondrocyte
Cartilage oligomeric matrix protein (COMP) or thrombospondin-5
function. Several studies have suggested that the responses Thrombospondin-1 and thrombospondin-3
of the subchondral bone to mechanical stimulation may Cartilage matrix protein (matrilin-1) and matrilin-3
transmit signals into the articular cartilage.214 Tidemark Fibronectin
advancement with thickening of the calcified cartilage and Tenascin-C
Cartilage intermediate layer protein (CILP)
thinning of articular cartilage is associated with fibrillation Fibrillin
of the cartilage surface during aging.215 Elastin
An increase in subchondral bone density is an early fea-
Other Noncollagenous Proteins (Regulatory)
ture of osteoarthritis.216 Radin and Rose217 proposed that
the initiation of fibrillation is caused by an increase in sub- Glycoprotein (gp)-39, YKL-40
Matrix Gla protein (MGP)
chondral bone stiffness. These changes in subchondral bone Chondromodulin-I (SCGP) and chondromodulin-II
stiffness may be secondary to cartilage deterioration, but Cartilage-derived retinoic acid–sensitive protein (CD-RAP)
are necessary for progression of osteoarthritic lesions, and Growth factors
involve bone and calcified cartilage close to the joint.218-220 Cell Membrane–Associated Proteins
Also, at the junction of the articular hyaline cartilage and Integrins (α1β1, α2β1, α3β1, α5β1, α6β1, α10β1, αvβ3, αvβ5)
adjacent subchondral bone, there is evidence of vascular Anchorin CII (annexin V)
invasion and advancement in the zone of calcified cartilage Cell determinant 44 (CD44)
in the region of the so-called tidemark that contributes fur- Syndecan-3
ther to a decrease in articular cartilage thickness.221 A more Discoidin domain receptor 2
recent study showed that angiogenesis in the osteochondral *The collagens, proteoglycans, and other noncollagenous proteins in the
junction is independent of synovial angiogenesis and synovi- cartilage matrix are synthesized by chondrocytes at different stages during
development and growth of cartilage. In mature articular cartilage, proteo-
tis, but is associated with cartilage changes and clinical dis- glycans and other noncollagen proteins are turned over slowly, whereas
ease activity.222 These structural alterations in the articular the collagen network is stable unless exposed to proteolytic cleavage. Pro-
cartilage and periarticular bone may lead to modification of teins that are associated with chondrocyte cell membranes also are listed
the contours of the adjacent articulating surfaces.217,223-225 because they permit specific interactions with extracellular matrix proteins.
Because increased trabecular bone volume with tra- The specific structure-function relationships are discussed in Chapter 3 and
described in Table 3-1.
becular sclerosis and increased bone turnover are fea- DS-PG, dermatan sulfate proteoglycan; SCGP, small cartilage–derived gly-
tures of osteoarthritis pathogenesis, therapies that target coprotein; SZP, superficial zone protein; YKL-40, 40KD chitinase 3-like gly-
bone have been proposed. Examples are calcitonin,226,227 coprotein.
 14 GOLDRING  |  Biology of the Normal Joint
Figure 1-7  A and B, Representative sec-
tions of normal human adult articular car-
tilage, showing nearly the same field in
plain (A) and polarized (B) light. Note the
clear demarcation of the articular cartilage
from the calcified cartilage below the tide-
mark and the underlying subchondral bone. 
(Hematoxylin-eosin stain; original magnifi-
cation ×60.) (Courtesy of Edward F. DiCarlo,
MD, Pathology Department, Hospital for Spe- A B
cial Surgery, New York, NY.)
SYNOVIAL FLUID AND NUTRITION
OF JOINT STRUCTURES
The volume and composition of synovial fluid are determined
by the properties of the synovium and its vasculature. Fluid
in normal joints is present in small quantities (2.5 mL in the
normal knee) sufficient to coat the synovial surface, but not
to separate one surface from the other. Tendon sheath fluid
and synovial fluid are biochemically similar. Both are essen-
tial for the nutrition and lubrication of adjacent avascular
structures, including tendon and articular cartilage, and for
limiting adhesion formation, maintaining movement. Char-
acterization and measurement of synovial fluid constituents
have proved useful for the identification of locally generated
regulatory factors, markers of cartilage turnover, and the met-
abolic status of the joint, and for the assessment of the effects
of therapy on cartilage homeostasis. Interpretation of such
data requires, however, an understanding of the generation
and clearance of synovial fluid and its various components.
GENERATION AND CLEARANCE
OF SYNOVIAL FLUID
Synovial fluid concentrations of a protein represent the net
contributions of synovial blood flow, plasma concentration,
microvascular permeability, and lymphatic removal and its
production and consumption within the joint space. Syno-
vial fluid is a mixture of a protein-rich ultrafiltrate of plasma
and hyaluronan synthesized by synoviocytes. Generation
of this ultrafiltrate depends on the difference between
intracapillary and intra-articular hydrostatic pressures and
between colloid osmotic pressures of capillary plasma and
synovial tissue fluid. Fenestrations, small pores covered by a
thin membrane, in the synovial capillaries and the macro-
molecular sieve of hyaluronic acid facilitate rapid exchange
of small molecules, such as glucose and lactate, assisted—in
the case of glucose—by an active transport system.233 Pro-
teins are present in synovial fluid at concentrations inversely
proportional to molecular size, with synovial fluid albumin
concentrations being about 45% of those in plasma (Fig.
1-8).234 Concentrations of electrolytes and small molecules
are equivalent to those in plasma.235
Synovial fluid is cleared through lymphatics in the
synovium, assisted by joint movement. In contrast to ultra-
filtration, lymphatic clearance of solutes is independent of
molecular size. In addition, constituents of synovial fluid,
such as regulatory peptides, may be degraded locally by
enzymes, and low-molecular-weight metabolites may diffuse
along concentration gradients into plasma. The kinetics of
delivery and removal of a protein must be determined (e.g.,
using albumin as a reference solute) to assess the significance
of its concentration in the joint.236
Hyaluronic acid is synthesized by fibroblast-like syno-
vial lining cells, and it appears in high concentrations in
synovial fluid at around 3 g/L, compared with a plasma con-
centration of 30 μg/L. Lubricin, a glycoprotein that assists
articular lubrication, is another constituent of synovial fluid
that is generated by the lining cells.363 It is now believed
that hyaluronan functions in fluid-film lubrication, whereas
lubricin is the true boundary lubricant in synovial fluid (see
following). Because the volume of synovial fluid is deter-
mined by the amount of hyaluronan, water retention seems
to be the major function of this large molecule.233,237
Despite the absence of a basement membrane, synovial
fluid does not mix freely with extracellular synovial tissue
fluid. Hyaluronan may trap molecules within the synovial
cavity by acting as a filtration screen on the surface of the
synovial lining, resisting the movement of synovial fluid
out from the joint space.237 Synovial fluid and its constitu-
ent proteins have a rapid turnover time (around 1 hour
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
1.0
Probably RA
Gout
Osteoarthritis
Classic RA
Ratio
Normal
SF
.10
S
Conc.
α2 Macro-
Oroso- Trans- Cerulo-
mucoid ferrin plasmin globulin
44 74 160 820
.01
1 100 1000
Molecular Weight (×103)
Figure 1-8  Ratio of the concentration of proteins in synovial fluid to
that found in serum, plotted as a function of molecular weight. Larger
proteins are selectively excluded from normal synovial fluid, but this
macromolecular sieve is less effective in diseased synovium. Conc., con-
centration; RA, rheumatoid arthritis; S, serum; SF, synovial fluid. (From
Kushner I, Somerville JA: Permeability of human synovial membrane to
­plasma proteins. Arthritis Rheum 14:560, 1971. Reprinted with permission of
the American College of Rheumatology.)
in normal knees), and equilibrium is not usually reached
among all parts of the joint. Tissue fluid around fenestrated
endothelium reflects plasma ultrafiltrate most closely, with
a low content of hyaluronate compared with synovial fluid.
Alternatively, locally generated or released peptides, such as
endothelin and substance P, may attain much higher peri-
vascular concentrations than those measured in synovial
fluid. The turnover time for hyaluronan in the normal joint
(13 hours) is an order of magnitude slower, however, than
that of small solutes and proteins. Association with hyaluro-
nan may result in trapping of solutes within synovial fluid.238
In normal joints, intra-articular pressures are slightly sub-
atmospheric at rest (0 to −5 mm Hg).239 During exercise,
hydrostatic pressure in the normal joint may decrease fur-
ther. Resting intra-articular pressures in rheumatoid joints
are around 20 mm Hg, whereas during isometric exercise,
they may increase to greater than 100 mm Hg, well above
capillary perfusion pressure and, at times, above arterial
pressure. Repeated mechanical stresses can interrupt syno-
vial perfusion during joint movement, particularly in the
presence of a synovial effusion.
SYNOVIAL FLUID AS AN INDICATOR
OF JOINT FUNCTION
In the absence of a basement membrane separating
synovium or cartilage from synovial fluid, measurements
made on synovial fluid may reflect the activity of these
structures. A wide range of regulatory factors and products
15
of ­synoviocyte metabolism and cartilage breakdown may be
generated locally within the joint, resulting in marked differ-
ences between the composition of synovial fluid and plasma
ultrafiltrate. Because there is little capacity for the selective
concentration of solutes in synovial fluid, solutes present
at higher concentrations than in plasma are probably syn-
thesized locally. It is necessary to know the local clearance
rate, however, to determine whether the solutes present in
synovial fluid at lower concentrations than in plasma are
generated locally.235 Although microvascular permeability
to protein in highly inflamed rheumatoid joints is more than
twice that in osteoarthritic joints, synovial fluid protein
concentrations vary little between the two joint diseases240
because the enhanced entry of proteins through the micro-
vasculature is largely offset by the increased lymphatic clear-
ance.241 Because clearance rates from synovial fluid may be
slower than those from plasma, however, synovial fluid lev-
els of drugs or urate may remain elevated after plasma levels
have declined.233
Comparisons of synovial fluid constituents between
disease groups are often limited by the sparseness of data
on normal synovial fluid as a result of difficulties in its col-
lection. Extrapolation from synovial fluid concentrations
to local synthetic rates is complicated further because of
variations in clearance rates and in synovial fluid volume.
Plasma proteins are less effectively filtered in inflamed
synovium, perhaps because of increased size of endothelial
cell fenestrations or because interstitial hyaluronate-protein
complexes are fragmented by enzymes associated with the
inflammatory process.234 Concentrations of proteins, such
as α2-macroglobulin (the principal proteinase inhibitor of
plasma), fibrinogen, and IgM, are elevated in inflammatory
synovial fluids (see Fig. 1-8), as are associated protein-bound
cations. Membrane peptidases may limit the diffusion of
regulatory peptides from their sites of release into synovial
fluid. In inflammatory arthritis, fibrin deposits may retard
flow between the tissue and the liquid phase. The cautious
interpretation of synovial fluid analysis has important impli-
cations in understanding how to use data on biomarkers
of cartilage damage and repair in rheumatoid arthritis and
osteoarthritis (see Chapter 48).
LUBRICATION AND NUTRITION
OF THE ARTICULAR CARTILAGE
Lubrication
Synovial fluid serves as a lubricant for articular cartilage and
a source of nutrition for the chondrocytes within. Lubri-
cation is essential for protecting cartilage and other joint
structures from friction and shear stresses associated with
movement under loading. There are two basic categories of
joint lubrication. In fluid-film lubrication, cartilage surfaces
are separated by an incompressible fluid film; hyaluronan
functions as the lubricant. In boundary lubrication, special-
ized molecules attached to the cartilage surface permit sur-
face-to-surface contact, while decreasing the coefficient of
friction.
During loading, a noncompressible fluid film is trapped
between opposing cartilage surfaces and prevents the sur-
faces from touching. Irregularities in the cartilage surface
and its deformation during compression may augment this
 16 GOLDRING  |  Biology of the Normal Joint
trapping of fluid. This stable film is approximately 0.1 μm
thick in the normal human hip joint, but it can be much
thinner in the presence of inflammatory synovial fluids or
with increased cartilage porosity.242,243
Lubricin is the major boundary lubricant in the human
joint.244 It is a glycoprotein, also called superficial zone pro-
tein and proteoglycan 4, which is synthesized by synovial
cells and chondrocytes.245-248 It has a molecular weight of
225,000, is 200 nm in length, and is 1 to 2 nm in diam-
eter.249 Dipalmitoyl phosphatidylcholine, which constitutes
45% of the lipid in normal synovial fluid, acts together with
lubricin as a boundary lubricant.250 More recent work indi-
cates that lubricin functions as a phospholipid carrier via a
mechanism that is common to all tissues.251,252 Lipid com-
poses 1% to 2% of dry weight of cartilage,253 and experimen-
tal treatment of cartilage surfaces with fat solvents impairs
lubrication qualities.254
Nutrition
As observed by Hunter in 1743,255 normal adult articular
cartilage contains no blood vessels. Vascularization of car-
tilage would be expected to alter its mechanical properties.
Blood flow would be repeatedly occluded during weight
bearing and exercise, with reactive oxygen species generated
during reperfusion, resulting in repeated damage to cartilage
matrix and chondrocytes. Chondrocytes synthesize specific
inhibitors of angiogenesis that maintain articular cartilage as
an avascular tissue.256-258 As a result of the lack of adjacent
blood vessels, the chondrocyte normally lives in an hypoxic
and acidotic environment, with extracellular fluid pH val-
ues around 7.1 to 7.2,259 and it uses anaerobic glycolysis for
energy production.260 High lactate levels in normal synovial
fluid, compared with paired plasma measurements, partially
reflect this anaerobic metabolism.261 There are two sources
of nutrients for articular cartilage: (1) the synovial fluid and
(2) subchondral blood vessels.
The synovial fluid and, indirectly, the synovial lining,
through which synovial fluid is generated, are the major
sources of nutrients for articular cartilage. Nutrients may
enter cartilage from synovial fluid either by diffusion or
by mass transport of fluid during compression-relaxation
cycles.262 Molecules as large as hemoglobin (65 kD) can dif-
fuse through normal articular cartilage,263 and the solutes
needed for cellular metabolism are much smaller. Diffusion
of uncharged small solutes, such as glucose, is not impaired
in matrices containing large amounts of glycosaminogly-
cans, and diffusivity of small molecules through hyaluronate
is enhanced.264,265
Intermittent compression may serve as a pump mecha-
nism for solute exchange in cartilage. The concept has
arisen from observations that joint immobilization or dislo-
cation leads to degenerative changes. In contrast, exercise
increases solute penetration into cartilage in experimental
systems.263 During weight bearing, fluid escapes from the
load-bearing region by flow to other cartilage sites. When
the load is removed, cartilage re-expands and draws back
fluid, exchanging nutrients with waste materials.266
In a growing child, the deeper layers of cartilage are
vascularized, such that blood vessels penetrate between
columns of chondrocytes in the hypertrophic zone of the
growth plate. It is likely that nutrients diffuse from these
tiny end capillaries through the matrix to chondrocytes.
Diffusion from subchondral blood vessels is not consid-
ered a major route for the nutrition of normal adult articu-
lar cartilage because of the barrier provided by its densely
calcified lower layer, the “tidemark.” Nonetheless, partial
defects may normally exist in this barrier,267 and in arthritis,
neovascularization of the deeper layers of articular cartilage
may contribute to cartilage nutrition and to entry of inflam-
matory cells and cytokines.221,268 In aging and osteoarthri-
tis, tidemark “duplication” may indicate communication
between the bone and cartilage.218,269 Experimental stud-
ies have indicated that cartilage lesions of chondromalacia
may develop if the subchondral blood supply of the patella
is compromised.270
SUMMARY AND CONCLUSION
Normal human synovial joints are complex structures that
comprise interacting connective tissue elements that per-
mit constrained and low-friction movement of adjacent
bones. The development of synovial joints in the embryo
is a highly ordered process involving complex cell-cell and
cell-matrix interactions that lead to the formation of the
cartilage anlage and interzone and joint cavitation. Under-
standing of the cellular interactions and molecular factors
involved in cartilage morphogenesis and limb development
has provided clues to understanding the functions of the
synovium, articular cartilage, and associated structures in
the mature joint.
The synovial joint is uniquely adapted to responding to
environmental and mechanical demands. The synovial lin-
ing is composed of two to three cell layers, and there is no
basement membrane separating the lining cells from the
underlying connective tissue. The synovium produces syno-
vial fluid, which provides nutrition and lubrication to the
avascular articular cartilage. Normal articular cartilage con-
tains a single cell type, the articular chondrocyte, which is
responsible for maintaining the integrity of the extracellular
cartilage matrix. This matrix consists of a complex network of
collagens, proteoglycans, and other noncollagenous proteins,
which provide tensile strength and compressive resistance.
Proper distribution and relative composition of these pro-
teins is required for the function of cartilage in protecting the
subchondral bone from adverse environmental influences.
Maintenance of the unique composition and organiza-
tion of each joint tissue is crucial for normal joint function,
which is compromised in response to inflammation, biome-
chanical injury, and aging. Knowledge of the normal struc-
ture-function relationships within joint tissues is essential
for understanding the pathogenesis and consequences of
joint diseases.
REFERENCES
  1. Simkin PA: The muskuloskeletal system, A: Joints. In Klippel JH,
Crofford LJ, Stone JH, et al (eds): Primer on the Rheumatic Diseases.
Atlanta, Arthritis Foundation, 2001, pp 5-9.
  2. Olsen BR, Reginato AM, Wang W: Bone development. Annu Rev
Cell Dev Biol 16:191-220, 2000.
  3. O’Rahilly R, Gardner E: The timing and sequence of events in
the development of the limbs in the human embryo. Anat Embryol
(Berl) 148:1-23, 1975.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
  4. O’Rahilly R, Gardner E: The embryology of movable joints. In Sokol-  
off L (ed): The Joints and Synovial Fluid, Vol 1. New York, Academic
Press, 1978, p 49.
  5. Pacifici M, Koyama E, Iwamoto M: Mechanisms of synovial joint and  
articular cartilage formation: Recent advances, but many lingering
mysteries. Birth Defects Res C Embryo Today 75:237-248, 2005.
  6. Church VL, Francis-West P: Wnt signalling during limb develop-  
ment. Int J Dev Biol 46:927-936, 2002.
  7. Francis-West PH, Abdelfattah A, Chen P, et al: Mechanisms of GDF-
5 action during skeletal development. Development 126:1305-1315,
1999.  
  8. Edwards CJ, Francis-West PH: Bone morphogenetic proteins in the
development and healing of synovial joints. Semin Arthritis Rheum  
31:33-42, 2001.
  9. Xu X, Weinstein M, Li C, et al: Fibroblast growth factor recep-
tors (FGFRs) and their roles in limb development. Cell Tissue Res  
296:33-43, 1999.
10. Minina E, Kreschel C, Naski MC, et al: Interaction of FGF, Ihh/
Pthlh, and BMP signaling integrates chondrocyte proliferation and  
hypertrophic differentiation. Dev Cell 3:439-449, 2002.
11. Lizarraga G, Lichtler A, Upholt WB, et al: Studies on the role of
Cux1 in regulation of the onset of joint formation in the developing  
limb. Dev Biol 243:44-54, 2002.
12. Iwamoto M, Tamamura Y, Koyama E, et al: Transcription factor ERG  
and joint and articular cartilage formation during mouse limb and
spine skeletogenesis. Dev Biol 305:40-51, 2007.  
13. Hartmann C, Tabin CJ: Wnt-14 plays a pivotal role in inducing syno-
vial joint formation in the developing appendicular skeleton. Cell
104:341-351, 2001.
14. Tsumaki N, Tanaka K, Arikawa-Hirasawa E, et al: Role of CDMP-1 in  
skeletal morphogenesis: Promotion of mesenchymal cell recruitment
and chondrocyte differentiation. J Cell Biol 144:161-173, 1999.  
15. Archer CW, Dowthwaite GP, Francis-West P: Development of syno-
vial joints. Birth Defects Res C Embryo Today 69:144-155, 2003.  
16. Storm EE, Kingsley DM: GDF5 coordinates bone and joint formation  
during digit development. Dev Biol 209:11-27, 1999.
17. von der Mark H, von der Mark K, Gay S: Study of differential col-  
lagen synthesis during development of the chick embryo by immu-
nofluorescence, I: Preparation of collagen type I and type II specific  
antibodies and their application to early stages of the chick embryo.
Dev Biol 48:237-249, 1976.
18. Craig FM, Bentley G, Archer CW: The spatial and temporal pattern  
of collagens I and II and keratan sulphate in the developing chick
metatarsophalangeal joint. Development 99:383-391, 1987.  
19. Morrison EH, Ferguson MW, Bayliss MT, et al: The development of
articular cartilage, I: The spatial and temporal patterns of collagen
types. J Anat 189(Pt 1):9-22, 1996.  
20. Pitsillides AA, Archer CW, Prehm P, et al: Alterations in hyaluronan
synthesis during developing joint cavitation. J Histochem Cytochem  
43:263-273, 1995.
  21. Murphy JM, Heinegard R, McIntosh A, et al: Distribution of carti-
lage molecules in the developing mouse joint. Matrix Biol 18:487-  
497, 1999.
  22. Mundlos S, Olsen BR: Heritable diseases of the skeleton, Part II:
Molecular insights into skeletal development-matrix components  
and their homeostasis. FASEB J 11:227-233, 1997.  
  23. Kavanagh E, Ashhurst DE: Development and aging of the articular
cartilage of the rabbit knee joint: Distribution of biglycan, decorin,
and matrilin-1. J Histochem Cytochem 47:1603-1616, 1999.  
  24. Nalin AM, Greenlee TK Jr, Sandell LJ: Collagen gene expression
during development of avian synovial joints: Transient expression
of types II and XI collagen genes in the joint capsule. Dev Dyn
203:352-362, 1995.  
  25. Ito MM, Kida MY: Morphological and biochemical re-evaluation
of the process of cavitation in the rat knee joint: Cellular and
cell strata alterations in the interzone. J Anat 197(Pt 4):659-679,  
2000.
  26. Mitrovic D: Development of the diarthrodial joints in the rat embryo.
Am J Anat 151:475-485, 1978.  
  27. Kimura S, Shiota K: Sequential changes of programmed cell death in
developing fetal mouse limbs and its possible roles in limb morpho-  
genesis. J Morphol 229:337-346, 1996.
  28. Colnot CI, Helms JA: A molecular analysis of matrix remodeling and
angiogenesis during long bone development. Mech Dev 100:245-
250, 2001.
17
29. Roach HI, Clarke NM: Physiological cell death of chondrocytes in
vivo is not confined to apoptosis: New observations on the mamma-
lian growth plate. J Bone Joint Surg Br 82:601-613, 2000.
30. Edwards JCW, Wilkinson LS, Soothill P, et al: Matrix metallopro-
teinases in the formation of human synovial joint cavities. J Anat
188(Pt 2):355-360, 1996.
31. Edwards JCW, Wilkinson LS, Jones HM, et al: The formation of
human synovial joint cavities: A possible role for hyaluronan and
CD44 in altered interzone cohesion. J Anat 185(Pt 2):355-367,
1994.
32. Drachman DB, Sokoloff L: The role of movement in embryonic joint
development. Dev Biol 14:401, 1966.
33. Persson M: The role of movements in the development of sutural and
diarthrodial joints tested by long-term paralysis of chick embryos. J
Anat 137(Pt 3):591-599, 1983.
34. Hall BK, Miyake T: All for one and one for all: Condensations
and the initiation of skeletal development. Bioessays 22:138-147,
2000.
35. Capdevila J, Izpisua Belmonte JC: Patterning mechanisms control-
ling vertebrate limb development. Annu Rev Cell Dev Biol 17:
87-132, 2001.
36. Tuan RS: Biology of developmental and regenerative skeletogenesis.
Clin Orthop 427(Suppl):S105-S117, 2004.
37. Fell HB: The histogenesis of cartilage and bone in the long bones of
the embryonic fowl. J Morphol Physiol 40:417-459, 1925.
38. Sandell LJ, Nalin AM, Reife RA: Alternative splice form of type II
procollagen mRNA (IIA) is predominant in skeletal precursors and
non-cartilaginous tissues during early mouse development. Dev Dyn
199:129-140, 1994.
39. DeLise AM, Fischer L, Tuan RS: Cellular interactions and signaling
in cartilage development. Osteoarthritis Cartilage 8:309-334, 2000.
40. Eames BF, de la Fuente L, Helms JA: Molecular ontogeny of the skel-
eton. Birth Defects Res C Embryo Today 69:93-101, 2003.
41. Zwilling E: Limb morphogenesis. Dev Biol 28:12-17, 1972.
42. Tickle C: Patterning systems—from one end of the limb to the other.
Dev Cell 4:449-458, 2003.
43. Niswander L: Pattern formation: Old models out on a limb. Nat
Rev Genet 4:133-143, 2003.
44. Kengaku M, Capdevila J, Rodriguez-Esteban C, et al: Distinct WNT
pathways regulating AER formation and dorsoventral polarity in the
chick limb bud. Science 280:1274-1277, 1998.
45. Johnson RL, Riddle RD, Tabin CJ: Mechanisms of limb patterning.
Curr Opin Genet Dev 4:535-542, 1994.
46. Kmita M, Tarchini B, Zakany J, et al: Early developmental arrest
of mammalian limbs lacking HoxA/HoxD gene function. Nature
435:1113-1116, 2005.
47. Riddle RD, Johnson RL, Laufer E, et al: Sonic hedgehog mediates
the polarizing activity of the ZPA. Cell 75:1401-1416, 1993.
48. Yang Y, Niswander L: Interaction between the signaling molecules
WNT7a and SHH during vertebrate limb development: Dorsal sig-
nals regulate anteroposterior patterning. Cell 80:939-947, 1995.
49. Laufer E, Nelson CE, Johnson RL, et al: Sonic hedgehog and Fgf-4
act through a signaling cascade and feedback loop to integrate growth
and patterning of the developing limb bud. Cell 79:993-1003, 1994.
50. Riddle RD, Tabin C: How limbs develop. Sci Am 280:74-79, 1999.
51. Bitgood MJ, McMahon AP: Hedgehog and Bmp genes are coex-
pressed at many diverse sites of cell-cell interaction in the mouse
embryo. Dev Biol 172:126-138, 1995.
52. Roberts DJ, Johnson RL, Burke AC, et al: Sonic hedgehog is an
endodermal signal inducing Bmp-4 and Hox genes during induction
and regionalization of the chick hindgut. Development 121:3163-
3174, 1995.
53. Liu A, Wang B, Niswander LA: Mouse intraflagellar transport pro-
teins regulate both the activator and repressor functions of Gli tran-
scription factors. Development 132:3103-3111, 2005.
54. Parr BA, McMahon AP: Dorsalizing signal Wnt-7a required for nor-
mal polarity of D-V and A-P axes of mouse limb. Nature 374:350-
353, 1995.
55. Irvine KD, Vogt TF: Dorsal-ventral signalling in limb development.
Curr Opin Cell Biol 8:867-876, 1997.
56. Pizette S, Niswander L: BMPs are required at two steps of limb
chondrogenesis: Formation of prechondrogenic condensations and
their differentiation into chondrocytes. Dev Biol 219:237-249,
2000.
 18 GOLDRING  |  Biology of the Normal Joint
  57. Derynck R, Zhang YE: Smad-dependent and Smad-independent
pathways in TGF-β family signalling. Nature 425:577-584,
2003.
  58. Yoon BS, Lyons KM: Multiple functions of BMPs in chondrogenesis.
J Cell Biochem 93:93-103, 2004.
  59. Yoon BS, Ovchinnikov DA, Yoshii I, et al: Bmpr1a and Bmpr1b
have overlapping functions and are essential for chondrogenesis in
vivo. Proc Natl Acad Sci U S A 102:5062-5067, 2005.
  60. Ng LJ, Wheatley S, Muscat GE, et al: SOX9 binds DNA, activates
transcription, and coexpresses with type II collagen during chondro-
genesis in the mouse. Dev Biol 183:108-121, 1997.
  61. Lefebvre V, Huang W, Harley VR, et al: SOX9 is a potent activator
of the chondrocyte-specific enhancer of the pro α1(II) collagen gene.
Mol Cell Biol 17:2336-2346, 1997.
  62. Lefebvre V, Li P, de Crombrugghe B: A new long form of Sox5
(L-Sox5), Sox6 and Sox9 are coexpressed in chondrogenesis
and cooperatively activate the type II collagen gene. EMBO J 17:
5718-5733, 1998.
  63. Smits P, Li P, Mandel J, et al: The transcription factors L-Sox5 and
Sox6 are essential for cartilage formation. Dev Cell 1:277-290, 2001.
  64. Ducy P, Zhang R, Geoffroy V, et al: Osf2/Cbfa1: A transcriptional
activator of osteoblast differentiation. Cell 89:747-754, 1997.
  65. Otto F, Thornell AP, Crompton T, et al: Cbfa1, a candidate gene for
cleidocranial dysplasia syndrome, is essential for osteoblast differen-
tiation and bone development. Cell 89:765-771, 1997.
  66. Xu SC, Harris MA, Rubenstein JLR, et al: Bone morphogenetic
protein-2 (BMP-2) signaling to the Col2a1 gene in chondroblasts
requires the homeobox gene Dlx-2. DNA Cell Biol 20:359-365,
2001.
  67. Itoh N, Ornitz DM: Evolution of the Fgf and Fgfr gene families.
Trends Genet 20:563-569, 2004.
  68. Ornitz DM: FGF signaling in the developing endochondral skeleton.
Cytokine Growth Factor Rev 16:205-213, 2005.
  69. Beier F: Cell-cycle control and the cartilage growth plate. J Cell
Physiol 202:1-8, 2005.
  70. Sahni M, Ambrosetti DC, Mansukhani A, et al: FGF signaling
inhibits chondrocyte proliferation and regulates bone development
through the STAT-1 pathway. Genes Dev 13:1361-1366, 1999.
  71. Liu Z, Xu J, Colvin JS, et al: Coordination of chondrogenesis and
osteogenesis by fibroblast growth factor 18. Genes Dev 16:859-869,
2002.
  72. Ohbayashi N, Shibayama M, Kurotaki Y, et al: FGF18 is required for
normal cell proliferation and differentiation during osteogenesis and
chondrogenesis. Genes Dev 16:870-879, 2002.
  73. Kronenberg HM: PTHrP and skeletal development. Ann N Y
Acad Sci 1068:1-13, 2006.
  74. McMahon AP, Ingham PW, Tabin CJ: Developmental roles and clin-
ical significance of hedgehog signaling. Curr Top Dev Biol 53:1-114,
2003.
  75. Vortkamp A, Lee K, Lanske B, et al: Regulation of rate of cartilage
differentiation by Indian hedgehog and PTH-related protein. Sci-
ence 273:613-622, 1996.
  76. Lanske B, Karaplis AC, Lee K, et al: PTH/PTHrP receptor in early
development and Indian hedgehog-regulated bone growth. Science
273:663-666, 1996.
  77. Kobayashi T, Soegiarto DW, Yang Y, et al: Indian hedgehog stimu-
lates periarticular chondrocyte differentiation to regulate growth
plate length independently of PTHrP. J Clin Invest 115:1734-1742,
2005.
  78. Ferguson CM, Miclau T, Hu D, et al: Common molecular pathways
in skeletal morphogenesis and repair. Ann N Y Acad Sci 857:33-42,
1998.
  79. Ballock RT, O’Keefe RJ: The biology of the growth plate. J Bone
Joint Surg Am 85A:715-726, 2003.
  80. Provot S, Schipani E: Molecular mechanisms of endochondral bone
development. Biochem Biophys Res Commun 328:658-665, 2005.
  81. St-Jacques B, Hammerschmidt M, McMahon AP: Indian hedgehog
signaling regulates proliferation and differentiation of chondrocytes
and is essential for bone formation. Genes Dev 13:2072-2086, 1999.
  82. Enomoto H, Enomoto-Iwamoto M, Iwamoto M, et al: Cbfa1 is a
positive regulatory factor in chondrocyte maturation. J Biol Chem
275:8695-8702, 2000.
  83. Kim IS, Otto F, Zabel B, et al: Regulation of chondrocyte differentia-
tion by Cbfa1. Mech Dev 80:159-170, 1999.
  84. Takeda S, Bonnamy JP, Owen MJ, et al: Continuous expression
of Cbfa1 in nonhypertrophic chondrocytes uncovers its ability to
induce hypertrophic chondrocyte differentiation and partially res-
cues Cbfa1-deficient mice. Genes Dev 15:467-481, 2001.
  85. Colnot C: Cellular and molecular interactions regulating skeleto-
genesis. J Cell Biochem 95:688-697, 2005.
  86. Leboy P, Grasso-Knight G, D’Angelo M, et al: Smad-Runx inter-
actions during chondrocyte maturation. J Bone Joint Surg Am
83A(Suppl 1):S15-S22, 2001.
  87. Zheng Q, Zhou G, Morello R, et al: Type X collagen gene regulation
by Runx2 contributes directly to its hypertrophic chondrocyte-spe-
cific expression in vivo. J Cell Biol 162:833-842, 2003.
  88. Komori T, Yagi H, Nomura S, et al: Targeted disruption of Cbfa1
results in a complete lack of bone formation owing to maturational
arrest of osteoblasts. Cell 89:755-764, 1997.
  89. Jimenez MJ, Balbin M, Lopez JM, et al: Collagenase 3 is a target
of Cbfa1, a transcription factor of the runt gene family involved in
bone formation. Mol Cell Biol 19:4431-4442, 1999.
  90. Inada M, Yasui T, Nomura S, et al: Maturational disturbance of
chondrocytes in Cbfa1-deficient mice. Dev Dyn 214:279-290, 1999.
  91. Ortega N, Behonick DJ, Werb Z: Matrix remodeling during endo-
chondral ossification. Trends Cell Biol 14:86-93, 2004.
  92. Colnot C, Lu C, Hu D, et al: Distinguishing the contributions of
the perichondrium, cartilage, and vascular endothelium to skeletal
development. Dev Biol 269:55-69, 2004.
  93. Inada M, Wang Y, Byrne MH, et al: Critical roles for collagenase-
3 (Mmp13) in development of growth plate cartilage and in endo-
chondral ossification. Proc Natl Acad Sci U S A 101:17192-17197,
2004.
  94. Stickens D, Behonick DJ, Ortega N, et al: Altered endochondral
bone development in matrix metalloproteinase 13-deficient mice.
Development 131:5883-5895, 2004.
  95. Jacenko O, Chan D, Franklin A, et al: A dominant interference
collagen X mutation disrupts hypertrophic chondrocyte pericellu-
lar matrix and glycosaminoglycan and proteoglycan distribution in
transgenic mice. Am J Pathol 159:2257-2269, 2001.
  96. Gress CJ, Jacenko O: Growth plate compressions and altered hema-
topoiesis in collagen X null mice. J Cell Biol 149:983-993, 2000.
  97. Vu TH, Shipley JM, Bergers G, et al: MMP-9/gelatinase B is a key
regulator of growth plate angiogenesis and apoptosis of hypertrophic
chondrocytes. Cell 93:411-422, 1998.
  98. Gerber HP, Vu TH, Ryan AM, et al: VEGF couples hypertrophic car-
tilage remodeling, ossification and angiogenesis during endochondral
bone formation. Nat Med 5:623-628, 1999.
99. Maes C, Carmeliet P, Moermans K, et al: Impaired angiogenesis and
endochondral bone formation in mice lacking the vascular endo-
thelial growth factor isoforms VEGF164 and VEGF188. Mech Dev
111:61-73, 2002.
100. Maes C, Stockmans I, Moermans K, et al: Soluble VEGF isoforms are
essential for establishing epiphyseal vascularization and regulating
chondrocyte development and survival. J Clin Invest 113:188-199,
2004.
101. Zelzer E, Olsen BR: Multiple roles of vascular endothelial growth fac-
tor (VEGF) in skeletal development, growth, and repair. Curr Top
Dev Biol 65:169-187, 2005.
102. Zhou Z, Apte SS, Soininen R, et al: Impaired endochondral ossifi-
cation and angiogenesis in mice deficient in membrane-type matrix
metalloproteinase I. Proc Natl Acad Sci U S A 97:4052-4057, 2000.
103. Merida-Velasco JA, Sanchez-Montesinos I, Espin-Ferra J, et al:
Development of the human knee joint. Anat Rec 248:269-278,
1997.
104. Merida-Velasco JA, Sanchez-Montesinos I, Espin-Ferra J, et al:
Development of the human knee joint ligaments. Anat Rec 248:
259-268, 1997.
105. Izumi S, Takeya M, Takagi K, et al: Ontogenetic development of
synovial A cells in fetal and neonatal rat knee joints. Cell Tissue Res
262:1-8, 1990.
106. Edwards JCW: Fibroblast biology: Development and differentiation
of synovial fibroblasts in arthritis. Arthritis Res 2:344-347, 2000.
107. Hukkanen M, Konttinen YT, Rees RG, et al: Distribution of nerve
endings and sensory neuropeptides in rat synovium, meniscus and
bone. Int J Tissue React 14:1-10, 1992.
108. Holmes G, Niswander L: Expression of slit-2 and slit-3 during chick
development. Dev Dyn 222:301-307, 2001.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
109. Merida-Velasco JR, Rodriguez-Vazquez JF, Merida-Velasco JA, et al:
Development of the human temporomandibular joint. Anat Rec
255:20-33, 1999.
110. Bradley SJ: An analysis of self-differentiation of chick limb buds in
chorio-allantoic grafts. J Anat 107:479-490, 1970.
111. Roberts S, Evans H, Trivedi J, et al: Histology and pathology of the
human intervertebral disc. J Bone Joint Surg Am 88(Suppl 2):10-14,
2006.
112. Eyre DR, Matsui Y, Wu JJ: Collagen polymorphisms of the interver-
tebral disc. Biochem Soc Trans 30:844-848, 2001.
113. Hayes AJ, Benjamin M, Ralphs JR: Extracellular matrix in develop-
ment of the intervertebral disc. Matrix Biol 20:107-121, 2001.
114. McAlinden A, Zhu Y, Sandell LJ: Expression of type II procollagens
during development of the human intervertebral disc. Biochem Soc
Trans 30:831-838, 2001.
115. Zhu Y, McAlinden A, Sandell LJ: Type IIA procollagen in develop-
ment of the human intervertebral disc: Regulated expression of the
NH(2)-propeptide by enzymic processing reveals a unique develop-
mental pathway. Dev Dyn 220:350-362, 2001.
116. Pizette S, Niswander L: Early steps in limb patterning and chondro-
genesis. Novartis Found Symp 232:23-36; discussion 36-46, 2001.
117. Benjamin M, Ralphs JR: Tendons and ligaments—an overview. His-
tol Histopathol 12:1135-1144, 1997.
118. Poole AR: Cartilage in health and disease. In Koopman W (ed):
Arthritis and Allied Conditions: A Textbook of Rheumatology. Phil-
adelphia, Lippincott Williams & Wilkins, 2005, pp 223-269.
119. Henderson B, Pettipher ER: The synovial lining cell: Biology and
pathobiology. Semin Arthritis Rheum 15:1-32, 1985.
120. Haywood L, Walsh DA: Vasculature of the normal and arthritic
synovial joint. Histol Histopathol 16:277-284, 2001.
121. Tak PP, Bresnihan B: The pathogenesis and prevention of joint dam-
age in rheumatoid arthritis: Advances from synovial biopsy and tissue
analysis. Arthritis Rheum 43:2619-2633, 2000.
122. Firestein GS: Evolving concepts of rheumatoid arthritis. Nature
423:356-361, 2003.
123. Meyer LH, Franssen L, Pap T: The role of mesenchymal cells in the
pathophysiology of inflammatory arthritis. Best Pract Res Clin Rheu-
matol 20:969-981, 2006.
124. Knedla A, Neumann E, Muller-Ladner U: Developments in the syno-
vial biology field 2006. Arthritis Res Ther 9:209, 2007.
125. Szekanecz Z, Koch AE: Macrophages and their products in rheuma-
toid arthritis. Curr Opin Rheumatol 19:289-295, 2007.
126. Bassleer R, Lhosest-Gauthier M-P, Renard A-M, et al: Histological
structure and functions of synovium. In Franchimont P (ed): Articu-
lar Synovium. Basel, Karger, 1982, pp 1-26.
127. Pasquali-Ronchetti I, Frizziero L, Guerra D, et al: Aging of the
human synovium: An in vivo and ex vivo morphological study.
Semin Arthritis Rheum 21:400-414, 1992.
128. Barland P, Novikoff AB, Hamerman D: Electron microscopy of the
human synovial membrane. J Cell Biol 14:207-220, 1962.
129. Wilkinson LS, Pitsillides AA, Worrall JG, et al: Light microscopic
characterization of the fibroblast-like synovial intimal cell (synovio-
cyte). Arthritis Rheum 35:1179-1184, 1992.
130. Smith MD, Barg E, Weedon H, et al: Microarchitecture and protec-
tive mechanisms in synovial tissue from clinically and arthroscopi-
cally normal knee joints. Ann Rheum Dis 62:303-307, 2003.
131. Naito M, Umeda S, Takahashi K, et al: Macrophage differentia-
tion and granulomatous inflammation in osteopetrotic mice (op/op)
defective in the production of CSF-1. Mol Reprod Dev 46:85-91,
1997.
132. Smith SC, Folefac VA, Osei DK, et al: An immunocytochemical
study of the distribution of proline-4-hydroxylase in normal, osteo-
arthritic and rheumatoid arthritic synovium at both the light and
electron microscopic level. Br J Rheumatol 37:287-291, 1998.
133. Qu Z, Garcia CH, O’Rourke LM, et al: Local proliferation of fibro-
blast-like synoviocytes contributes to synovial hyperplasia: Results of
proliferating cell nuclear antigen/cyclin, c-myc, and nucleolar orga-
nizer region staining. Arthritis Rheum 37:212-220, 1994.
134. Walsh DA, Mapp PI, Wharton J, et al: Neuropeptide degrading
enzymes in normal and inflamed human synovium. Am J Pathol
142:1610-1621, 1993.
135. Morales-Ducret J, Wayner E, Elices MJ, et al: α4/β1 integrin (VLA-
4) ligands in arthritis: Vascular cell adhesion molecule-1 expression
in synovium and on fibroblast-like synoviocytes. J Immunol 149:
1424-1431, 1992.
19
136. Demaziere A, Athanasou NA: Adhesion receptors of intimal and
subintimal cells of the normal synovial membrane. J Pathol 168:
209-215, 1992.
137. Johnson BA, Haines GK, Harlow LA, et al: Adhesion molecule expres-
sion in human synovial tissue. Arthritis Rheum 36:137-146, 1993.
138. Valencia X, Higgins JM, Kiener HP, et al: Cadherin-11 provides
specific cellular adhesion between fibroblast-like synoviocytes. J Exp
Med 200:1673-1679, 2004.
139. Lee DM, Kiener HP, Agarwal SK, et al: Cadherin-11 in synovial lining
formation and pathology in arthritis. Science 315:1006-1010, 2007.
140. Simkin PA: Physiology of normal and abnormal synovium. Semin
Arthritis Rheum 21:179-183, 1991.
141. Firestein GS: Starving the synovium: Angiogenesis and inflamma-
tion in rheumatoid arthritis. J Clin Invest 103:3-4, 1999.
142. Walsh DA: Angiogenesis and arthritis. Rheumatology (Oxf) 38:
103-112, 1999.
143. Walsh DA, Wade M, Mapp PI, et al: Focally regulated endothe-
lial proliferation and cell death in human synovium. Am J Pathol
152:691-702, 1998.
144. Koch AE: Review: Angiogenesis: Implications for rheumatoid arthri-
tis. Arthritis Rheum 41:951-962, 1998.
145. Storgard CM, Stupack DG, Jonczyk A, et al: Decreased angiogen-
esis and arthritic disease in rabbits treated with an αvβ3 antagonist.
J Clin Invest 103:47-54, 1999.
146. Uchida T, Nakashima M, Hirota Y, et al: Immunohistochemical
localisation of protein tyrosine kinase receptors Tie-1 and Tie-2 in
synovial tissue of rheumatoid arthritis: Correlation with angiogenesis
and synovial proliferation. Ann Rheum Dis 59:607-614, 2000.
147. Gravallese EM, Pettit AR, Lee R, et al: Angiopoietin-1 is expressed
in the synovium of patients with rheumatoid arthritis and is induced
by tumour necrosis factor α. Ann Rheum Dis 62:100-107, 2003.
148. Mapp PI: Innervation of the synovium. Ann Rheum Dis 54:398-403,
1995.
149. Seegers HC, Hood VC, Kidd BL, et al: Enhancement of angiogenesis
by endogenous substance P release and neurokinin-1 receptors during
neurogenic inflammation. J Pharmacol Exp Ther 306:8-12, 2003.
150. Walsh DA, Catravas J, Wharton J: Angiotensin converting enzyme
in human synovium: Increased stromal [(125)I]351A binding in
rheumatoid arthritis. Ann Rheum Dis 59:125-131, 2000.
151. Mapp PI, Klocke R, Walsh DA, et al: Localization of 3-­nitrotyrosine
to rheumatoid and normal synovium. Arthritis Rheum 44:1534-
1539, 2001.
152. Pablos JL, Santiago B, Galindo M, et al: Synoviocyte-derived
CXCL12 is displayed on endothelium and induces angiogenesis in
rheumatoid arthritis. J Immunol 170:2147-2152, 2003.
153. Ferrell WR, Crighton A, Sturrock RD: Position sense at the proximal
interphalangeal joint is distorted in patients with rheumatoid arthri-
tis of finger joints. Exp Physiol 77:675-680, 1992.
154. Dee R: Structure and function of hip joint innervation. Ann R Coll
Surg Engl 45:357-374, 1969.
155. Buma P, Verschuren C, Versleyen D, et al: Calcitonin gene-related
peptide, substance P and GAP-43/B-50 immunoreactivity in the
normal and arthrotic knee joint of the mouse. Histochemistry 98:
327-339, 1992.
156. Hukkanen M, Platts LA, Corbett SA, et al: Reciprocal age-related
changes in GAP-43/B-50, substance P and calcitonin gene-related
peptide (CGRP) expression in rat primary sensory neurones and
their terminals in the dorsal horn of the spinal cord and subintima of
the knee synovium. Neurosci Res 42:251-260, 2002.
157. McDougall JJ, Watkins L, Li Z: Vasoactive intestinal peptide (VIP)
is a modulator of joint pain in a rat model of osteoarthritis. Pain
123:98-105, 2006.
158. Niissalo S, Hukkanen M, Imai S, et al: Neuropeptides in experimental
and degenerative arthritis. Ann N Y Acad Sci 966:384-399, 2002.
159. Saito T, Koshino T: Distribution of neuropeptides in synovium of the
knee with osteoarthritis. Clin Orthop 376:172-182, 2000.
160. New HV, Mudge AW: Calcitonin gene-related peptide regulates
muscle acetylcholine receptor synthesis. Nature 323:809-811, 1986.
161. Schaible HG, Ebersberger A, Von Banchet GS: Mechanisms of pain
in arthritis. Ann N Y Acad Sci 966:343-354, 2002.
162. McDougall JJ: Arthritis and pain: Neurogenic origin of joint pain.
Arthritis Res Ther 8:220, 2006.
163. Coggeshall RE, Hong KA, Langford LA, et al: Discharge characteris-
tics of fine medial articular afferents at rest and during passive move-
ments of inflamed knee joints. Brain Res 272:185-188, 1983.
 20 GOLDRING  |  Biology of the Normal Joint
164. Benjamin M, Ralphs JR: The cell and developmental biology of ten-
dons and ligaments. Int Rev Cytol 196:85-130, 2000.
165. Wang JH: Mechanobiology of tendon. J Biomech 39:1563-1582,
2006.
166. Vogel KG, Peters JA: Histochemistry defines a proteoglycan-rich
layer in bovine flexor tendon subjected to bending. J Musculoskelet
Neuronal Interact 5:64-69, 2005.
167. Knott L, Tarlton JF, Bailey AJ: Chemistry of collagen cross-linking:
Biochemical changes in collagen during the partial mineralization of
turkey leg tendon. Biochem J 322(Pt 2):535-542, 1997.
168. Ng GY, Oakes BW, Deacon OW, et al: Long-term study of the bio-
chemistry and biomechanics of anterior cruciate ligament-patellar
tendon autografts in goats. J Orthop Res 14:851-856, 1996.
169. Canty EG, Starborg T, Lu Y, et al: Actin filaments are required for
fibripositor-mediated collagen fibril alignment in tendon. J Biol
Chem 281:38592-38598, 2006.
170. Richardson SH, Starborg T, Lu Y, et al: Tendon development requires
regulation of cell condensation and cell shape via cadherin-11-
­mediated cell-cell junctions. Mol Cell Biol 27:6218-6228, 2007.
171. Kannus P, Jozsa L, Kvist M, et al: The effect of immobilization on
myotendinous junction: An ultrastructural, histochemical and immu-
nohistochemical study. Acta Physiol Scand 144:387-394, 1992.
172. Tan AL, Toumi H, Benjamin M, et al: Combined high-resolution
magnetic resonance imaging and histological examination to explore
the role of ligaments and tendons in the phenotypic expression of
early hand osteoarthritis. Ann Rheum Dis 65:1267-1272, 2006.
173. Dalton S, Cawston TE, Riley GP, et al: Human shoulder tendon
biopsy samples in organ culture produce procollagenase and tissue
inhibitor of metalloproteinases. Ann Rheum Dis 54:571-577, 1995.
174. Jain A, Nanchahal J, Troeberg L, et al: Production of cytokines, vas-
cular endothelial growth factor, matrix metalloproteinases, and tissue
inhibitor of metalloproteinases 1 by tenosynovium demonstrates its
potential for tendon destruction in rheumatoid arthritis. Arthritis
Rheum 44:1754-1760, 2001.
175. Tillander B, Franzen L, Norlin R: Fibronectin, MMP-1 and histologic
changes in rotator cuff disease. J Orthop Res 20:1358-1364, 2002.
176. Jarvinen TA, Jozsa L, Kannus P, et al: Mechanical loading regulates
the expression of tenascin-C in the myotendinous junction and ten-
don but does not induce de novo synthesis in the skeletal muscle.
J Cell Sci 116:857-866, 2003.
177. Berenson MC, Blevins FT, Plaas AH, et al: Proteoglycans of human
rotator cuff tendons. J Orthop Res 14:518-525, 1996.
178. Waggett AD, Ralphs JR, Kwan AP, et al: Characterization of col-
lagens and proteoglycans at the insertion of the human Achilles ten-
don. Matrix Biol 16:457-470, 1998.
179. Fukuta S, Oyama M, Kavalkovich K, et al: Identification of types II,
IX and X collagens at the insertion site of the bovine achilles tendon.
Matrix Biol 17:65-73, 1998.
180. Vogel KG, Meyers AB: Proteins in the tensile region of adult bovine
deep flexor tendon. Clin Orthop 367:S344-S355, 1999.
181. Thomopoulos S, Marquez JP, Weinberger B, et al: Collagen fiber ori-
entation at the tendon to bone insertion and its influence on stress
concentrations. J Biomech 39:1842-1851, 2006.
182. Rodeo SA, Kawamura S, Kim HJ, et al: Tendon healing in a bone
tunnel differs at the tunnel entrance versus the tunnel exit: An effect
of graft-tunnel motion? Am J Sports Med 34:1790-1800, 2006.
183. Sharma P, Maffulli N: Biology of tendon injury: Healing, model-
ing and remodeling. J Musculoskelet Neuronal Interact 6:181-190,
2006.
184. Rees JD, Wilson AM, Wolman RL: Current concepts in the manage-
ment of tendon disorders. Rheumatology (Oxf) 45:508-521, 2006.
185. Kumagai J, Sarkar K, Uhthoff HK: The collagen types in the attach-
ment zone of rotator cuff tendons in the elderly: An immunohisto-
chemical study. J Rheumatol 21:2096-2100, 1994.
186. Ameye L, Aria D, Jepsen K, et al: Abnormal collagen fibrils in ten-
dons of biglycan/fibromodulin-deficient mice lead to gait impair-
ment, ectopic ossification, and osteoarthritis. FASEB J 16:673-680,
2002.
187. Hoffmann A, Pelled G, Turgeman G, et al: Neotendon formation
induced by manipulation of the Smad8 signalling pathway in mesen-
chymal stem cells. J Clin Invest 116:940-952, 2006.
188. Woo SL, Abramowitch SD, Kilger R, et al: Biomechanics of knee
ligaments: Injury, healing, and repair. J Biomech 39:1-20, 2006.
189. Hoffmann A, Gross G: Tendon and ligament engineering: From cell
biology to in vivo application. Regen Med 1:563-574, 2006.
190. Bland YS, Ashhurst DE: Changes in the distribution of fibrillar colla-
gens in the collateral and cruciate ligaments of the rabbit knee joint
during fetal and postnatal development. Histochem J 28:325-334,
1996.
191. Nawata K, Minamizaki T, Yamashita Y, et al: Development of the
attachment zones in the rat anterior cruciate ligament: Changes in
the distributions of proliferating cells and fibrillar collagens during
postnatal growth. J Orthop Res 20:1339-1344, 2002.
192. Frank CB, Hart DA, Shrive NG: Molecular biology and biomechan-
ics of normal and healing ligaments—a review. Osteoarthritis Carti-
lage 7:130-140, 1999.
193. Kaufmann P, Bose P, Prescher A: New insights into the soft-tissue
anatomy anterior to the patella. Lancet 363:586, 2004.
194. Arnoczky SP, McDevitt CA: The meniscus: Structure, function
repair, and replacement. In Buckwalter JL, Einhorn TA, Simon SR
(eds): Orthopaedic Basic Science: Biology and Biomechanics of the
Musculoskeletal System. Park Ridge, Ill, American Academy of
Orthopaedic Surgeons, 2000, pp 531-546.
195. Sweigart MA, Athanasiou KA: Toward tissue engineering of the
knee meniscus. Tissue Eng 7:111-129, 2001.
196. Fairbank T: Knee joint changes after meniscectomy. J Bone Joint
Surg Br 30:664, 1948.
197. Murrell GA, Maddali S, Horovitz L, et al: The effects of time course
after anterior cruciate ligament injury in correlation with meniscal
and cartilage loss. Am J Sports Med 29:9-14, 2001.
198. Messner K, Gao J: The menisci of the knee joint: Anatomical and
functional characteristics, and a rationale for clinical treatment. J
Anat 193(Pt 2):161-178, 1998.
199. Mine T, Kimura M, Sakka A, et al: Innervation of nociceptors in
the menisci of the knee joint: An immunohistochemical study. Arch
Orthop Trauma Surg 120:201-204, 2000.
200. Arnoczky SP, Warren RF: The microvasculature of the meniscus and
its response to injury: An experimental study in the dog. Am J Sports
Med 11:131-141, 1983.
201. Eyre DR, Muir H: The distribution of different molecular species of
collagen in fibrous, elastic and hyaline cartilages of the pig. Biochem
J 151:595-602, 1975.
202. McDevitt CA, Mukherjee S, Kambic H, et al: Emerging concepts
of the cell biology of the meniscus. Curr Opin Orthop 13:345-350,
2002.
203. Petersen W, Tillmann B: Collagenous fibril texture of the human
knee joint menisci. Anat Embryol (Berl) 197:317-324, 1998.
204. Fithian DC, Kelly MA, Mow VC: Material properties and struc-
ture-function relationships in the menisci. Clin Orthop 252:19-31,
1990.
205. Roughley PJ, White RJ: The dermatan sulfate proteoglycans of the
adult human meniscus. J Orthop Res 10:631-637, 1992.
206. Bland YS, Ashhurst DE: Changes in the content of the fibrillar colla-
gens and the expression of their mRNAs in the menisci of the rabbit
knee joint during development and ageing. Histochem J 28:265-274,
1996.
207. McAlinden A, Dudhia J, Bolton MC, et al: Age-related changes
in the synthesis and mRNA expression of decorin and aggrecan in
human meniscus and articular cartilage. Osteoarthritis Cartilage
9:33-41, 2001.
208. Ghadially FN, Lalonde JM, Wedge JH: Ultrastructure of normal and
torn menisci of the human knee joint. J Anat 136(Pt 4):773-791,
1983.
209. Hellio Le Graverand MP, Ou Y, Schield-Yee T, et al: The cells
of the rabbit meniscus: Their arrangement, interrelationship, mor-
phological variations and cytoarchitecture. J Anat 198:525-535,
2001.
210. Kambic HE, Futani H, McDevitt CA: Cell, matrix changes and
α-smooth muscle actin expression in repair of the canine meniscus.
Wound Repair Regen 8:554-561, 2000.
211. Ahluwalia S, Fehm M, Murray MM, et al: Distribution of smooth
muscle actin-containing cells in the human meniscus. J Orthop Res
19:659-664, 2001.
212. Goldring MB: The muskuloskeletal system, B: Articular car-
tilage. In Klippel JH, Crofford LJ, Stone JH, et al (eds): Primer
on the Rheumatic Diseases. Atlanta, Arthritis Foundation, 2001,
pp 10-16.
213. Benedek TG: A history of the understanding of cartilage. Osteoar-
thritis Cartilage 14:203-209, 2006.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
214. Bailey AJ, Mansell JP, Sims TJ, et al: Biochemical and mechanical
properties of subchondral bone in osteoarthritis. Biorheology 41:
349-358, 2004.
215. Green WT Jr, Martin GN, Eanes ED, et al: Microradiographic study
of the calcified layer of articular cartilage. Arch Pathol 90:151-158,
1970.
216. Reimann I, Mankin HJ, Trahan C: Quantitative histologic analyses
of articular cartilage and subchondral bone from osteoarthritic and
normal human hips. Acta Orthop Scand 48:63-73, 1977.
217. Radin EL, Rose RM: Role of subchondral bone in the initiation
and progression of cartilage damage. Clin Orthop 213:34-40,
1986.
218. Burr DB: Anatomy and physiology of the mineralized tissues: Role in
the pathogenesis of osteoarthrosis. Osteoarthritis Cartilage 12(Suppl A):
S20-S30, 2004.
219. Buckland-Wright C: Subchondral bone changes in hand and knee
osteoarthritis detected by radiography. Osteoarthritis Cartilage
12(Suppl A):S10-S19, 2004.
220. Mrosek EH, Lahm A, Erggelet C, et al: Subchondral bone trauma
causes cartilage matrix degeneration: An immunohistochemical
analysis in a canine model. Osteoarthritis Cartilage 14:171-178,
2006.
221. Lane LB, Villacin A, Bullough PG: The vascularity and remodelling
of subchondrial bone and calcified cartilage in adult human femoral
and humeral heads: An age- and stress-related phenomenon. J Bone
Joint Surg Br 59:272-278, 1977.
222. Walsh DA, Bonnet CS, Turner EL, et al: Angiogenesis in the
synovium and at the osteochondral junction in osteoarthritis. Osteo-
arthritis Cartilage 15:743-751, 2007.
223. Bullough PG: The role of joint architecture in the etiology of arthri-
tis. Osteoarthritis Cartilage 12(Suppl A):S2-S9, 2004.
224. Messent EA, Ward RJ, Tonkin CJ, et al: Differences in trabecular
structure between knees with and without osteoarthritis quantified
by macro and standard radiography, respectively. Osteoarthritis Car-
tilage 14:1302-1305, 2006.
225. Coats AM, Zioupos P, Aspden RM: Material properties of subchon-
dral bone from patients with osteoporosis or osteoarthritis by micro-
indentation testing and electron probe microanalysis. Calcif Tissue
Int 73:66-71, 2003.
226. El Hajjaji H, Williams JM, Devogelaer JP, et al: Treatment with calci-
tonin prevents the net loss of collagen, hyaluronan and proteoglycan
aggregates from cartilage in the early stages of canine experimental
osteoarthritis. Osteoarthritis Cartilage 12:904-911, 2004.
227. Bagger YZ, Tanko LB, Alexandersen P, et al: Oral salmon calcito-
nin induced suppression of urinary collagen type II degradation in
postmenopausal women: A new potential treatment of osteoarthritis.
Bone 37:425-430, 2005.
228. Spector TD: Bisphosphonates: Potential therapeutic agents for dis-
ease modification in osteoarthritis. Aging Clin Exp Res 15:413-418,
2003.
229. Ham KD, Carlson CS: Effects of estrogen replacement therapy on
bone turnover in subchondral bone and epiphyseal metaphyseal can-
cellous bone of ovariectomized cynomolgus monkeys. J Bone Miner
Res 19:823-829, 2004.
230. Komuro H, Olee T, Kuhn K, et al: The osteoprotegerin/receptor
activator of nuclear factor ĸB/receptor activator of nuclear fac-
tor ĸB ligand system in cartilage. Arthritis Rheum 44:2768-2776,
2001.
231. Pettit AR, Ji H, von Stechow D, et al: TRANCE/RANKL knockout
mice are protected from bone erosion in a serum transfer model of
arthritis. Am J Pathol 159:1689-1699, 2001.
232. Zwerina J, Hayer S, Redlich K, et al: Activation of p38 MAPK is
a key step in tumor necrosis factor-mediated inflammatory bone
destruction. Arthritis Rheum 54:463-472, 2006.
233. Simkin PA, Bassett JE, Koh EM: Synovial perfusion in the human
knee: A methodologic analysis. Semin Arthritis Rheum 25:56-66,
1995.
234. Kushner I, Somerville JA: Permeability of human synovial membrane
to plasma proteins: Relationship to molecular size and inflammation.
Arthritis Rheum 14:560-570, 1971.
235. Simkin PA: Fluid dynamics of the joint space and trafficking of matrix
products. In Seibel MJ, Robins SP, Bilezekian JP (eds): Dynamics of
Bone and Cartilage Metabolism. New York, Academic Press, 2006,
pp 451-456.
21
236. Levick JR: A method for estimating macromolecular reflection by
human synovium, using measurements of intra-articular half lives.
Ann Rheum Dis 57:339-344, 1998.
237. Levick JR, McDonald JN: Fluid movement across synovium in
healthy joints: Role of synovial fluid macromolecules. Ann Rheum
Dis 54:417-423, 1995.
238. Myers SL, Brandt KD: Effects of synovial fluid hyaluronan concentra-
tion and molecular size on clearance of protein from the canine knee.
J Rheumatol 22:1732-1739, 1995.
239. Gaffney K, Williams RB, Jolliffe VA, et al: Intra-articular pressure
changes in rheumatoid and normal peripheral joints. Ann Rheum
Dis 54:670-673, 1995.
240. Wallis WJ, Simkin PA, Nelp WB: Protein traffic in human synovial
effusions. Arthritis Rheum 30:57-63, 1987.
241. Myers SL, O’Connor BL, Brandt KD: Accelerated clearance of albu-
min from the osteoarthritic knee: Implications for interpretation of
concentrations of “cartilage markers” in synovial fluid. J Rheumatol
23:1744-1748, 1996.
242. Hlavacek M: The role of synovial fluid filtration by cartilage in lubri-
cation of synovial joints, II: Squeeze-film lubrication: Homogeneous
filtration. J Biomech 26:1151-1160, 1993.
243. Jin ZM, Dowson D, Fisher J: The effect of porosity of articular carti-
lage on the lubrication of a normal human hip joint. Proc Inst Mech
Eng [H] 206:117-124, 1992.
244. Swann DA, Silver FH, Slayter HS, et al: The molecular structure
and lubricating activity of lubricin isolated from bovine and human
synovial fluids. Biochem J 225:195-201, 1985.
245. Jay GD, Tantravahi U, Britt DE, et al: Homology of lubricin and
superficial zone protein (SZP): Products of megakaryocyte stimulat-
ing factor (MSF) gene expression by human synovial fibroblasts and
articular chondrocytes localized to chromosome 1q25. J Orthop Res
19:677-687, 2001.
246. Flannery CR, Hughes CE, Schumacher BL, et al: Articular carti-
lage superficial zone protein (SZP) is homologous to megakaryocyte
stimulating factor precursor and is a multifunctional proteoglycan
with potential growth-promoting, cytoprotective, and lubricating
properties in cartilage metabolism. Biochem Biophys Res Commun
254:535-541, 1999.
247. Ikegawa S, Sano M, Koshizuka Y, et al: Isolation, characterization
and mapping of the mouse and human PRG4 (proteoglycan 4) genes.
Cytogenet Cell Genet 90:291-297, 2000.
248. Schmidt TA, Schumacher BL, Klein TJ, et al: Synthesis of proteo-
glycan 4 by chondrocyte subpopulations in cartilage explants, mono-
layer cultures, and resurfaced cartilage cultures. Arthritis Rheum
50:2849-2857, 2004.
249. Jay GD, Lane BP, Sokoloff L: Characterization of a bovine synovial
fluid lubricating factor, III: The interaction with hyaluronic acid.
Connect Tissue Res 28:245-255, 1992.
250. Williams PF 3rd, Powell GL, LaBerge M: Sliding friction analysis of
phosphatidylcholine as a boundary lubricant for articular cartilage.
Proc Inst Mech Eng [H] 207:59-66, 1993.
251. Hills BA: Boundary lubrication in vivo. Proc Inst Mech Eng 214:
83-94, 2000.
252. Jay GD, Harris DA, Cha CJ: Boundary lubrication by lubricin is
mediated by O-linked β(1-3)Gal-GalNAc oligosaccharides. Glyco-
conj J 18:807-815, 2001.
253. Stockwell RA: Lipid content of human costal and articular cartilage.
Ann Rheum Dis 26:481-486, 1967.
254. Pickard JE, Fisher J, Ingham E, et al: Investigation into the effects of
proteins and lipids on the frictional properties of articular cartilage.
Biomaterials 19:1807-1812, 1998.
255. Hunter W: On the structure and diseases of articulating cartilage.
Philos Trans R Soc Lond Biol 42:514, 1743.
256. Brem H, Folkman J: Inhibition of tumor angiogenesis mediated by
cartilage. J Exp Med 141:427-439, 1975.
257. Kuettner KE, Pauli BU: Inhibition of neovascularization by a carti-
lage factor. Ciba Found Symp 100:163-173, 1983.
258. Moses MA, Sudhalter J, Langer R: Identification of an inhibitor of
neovascularization from cartilage. Science 248:1408-1410, 1990.
259. Pita JC, Howell DS: Micro-biochemical studies of cartilage. In Sokol-
off L (ed): The Joints and Synovial Fluid. New York, Academic Press,
1978, p 273.
260. Bywaters E: The metabolism of joint tissues. J Pathol Bacteriol
44:247, 1937.
 22 GOLDRING  |  Biology of the Normal Joint
261. Naughton DP, Haywood R, Blake DR, et al: A comparative evalua-
tion of the metabolic profiles of normal and inflammatory knee-joint
synovial fluids by high resolution proton NMR spectroscopy. FEBS
Lett 332:221-225, 1993.
262. Strangeways T: The nutrition of the articular cartilage. BMJ 1:
661, 1920.
263. Maroudas A, Bullough P, Swanson SA, et al: The permeability of
articular cartilage. J Bone Joint Surg Br 50:166-177, 1968.
264. Hadler NM: Synovial fluids facilitate small solute diffusivity. Ann
Rheum Dis 39:580-585, 1980.
265. O’Hara BP, Urban JP, Maroudas A: Influence of cyclic loading on the
nutrition of articular cartilage. Ann Rheum Dis 49:536-539, 1990.
266. Lewis P, McCutchen CW: Experimental evidence for weeping lubri-
cation in mammalian joints. Nature 184:1285, 1959.
267. Mital MA, Millington PF: Osseous pathway of nutrition to articular
cartilage of the human femoral head. Lancet 1:842, 1970.
268. Bromley M, Bertfield H, Evanson JM, et al: Bidirectional erosion of
cartilage in the rheumatoid knee joint. Ann Rheum Dis 44:676-681,
1985.
269. Lane LB, Bullough PG: Age-related changes in the thickness of the
calcified zone and the number of tidemarks in adult human articular
cartilage. J Bone Joint Surg Br 62:372-375, 1980.
270. Neusel E, Graf J: The influence of subchondral vascularisation on
chondromalacia patellae. Arch Orthop Trauma Surg 115:313-315,
1996.
 2 Synovium
KEY POINTS
The synovium provides nutrients to cartilage and produces
lubricants for the joint.
The intimal lining of the synovium introduces macrophage-
like and fibroblast-like synoviocytes.
The sublining contains scattered immune cells, fibroblasts,
blood vessels, and fat cells.
Fibroblast-like synoviocytes in the intimal lining express
specialized proteins that synthesize proteoglycans such as
hyaluronic acid.
STRUCTURE
The synovium is a membranous structure that extends from
the margins of articular cartilage and lines the capsule of diar­
throdial joints, including the temporomandibular joint1 and
the facet joints of vertebral bodies (Fig. 2-1).2 The healthy
synovium covers intra-articular tendons and ­ ligaments,
and fat pads, but not articular cartilage or meniscal tissue.
Synovium also ensheathes tendons where they pass beneath
ligamentous bands. Normally, the synovial membrane has
two components—the intima, or lining cells, and the sub­
intima, otherwise referred to as the sublining or supportive
layer. The intima represents the interface between the cav­
ity containing synovial fluid and the subintimal layer. There
is no well-formed basement membrane to separate the
intima from the subintima. The subintima is composed of
fibrovascular connective tissue and merges with the densely
collagenous fibrous joint capsule.
SYNOVIAL LINING CELLS
The synovial intimal layer is composed of synovial lining
cells (SLCs), which have an epithelial-like arrangement
on the luminal aspect of the joint cavity. SLCs, termed
synoviocytes, are one to three cells deep, depending on the
anatomic location, and they extend 20 to 40 μm beneath
the lining layer surface. The major and minor axes of SLCs
measure 8 to 12 μm (major axis) and 6 to 8 μm (minor axis).
SLCs have poorly defined cell borders and elliptical nuclei
with generally a single small nucleolus.3
Ultrastructure of Synovial Lining Cells
Transmission electron microscopic analysis shows that the
intimal cells form a discontinuous layer, something not appre­
ciated under transmission light microscopy, so that the subin­
timal matrix is in direct contact with the synovial fluid (Fig.
2-2). The existence of two distinct cell types, type A and type B
BARRY BRESNIHAN  • 
ADRIENNE M. FLANAGAN
SLCs, originally was described by Barland and associates,4 and
several lines of evidence, including animal models, detailed
ultrastructural studies, and immunohistochemical analysis,
indicate that these cells represent macrophages (type A SLCs)
and fibroblasts (type B SLCs). Studies of the SLC populations
in a variety of species, including humans, have found that
macrophages make up approximately 20% and fibroblast-like
cells approximately 80% of the lining cell.5,6 The existence of
the two cell types has been substantiated by similar findings in
a wide variety of species, including hamsters, cats, dogs, guinea
pigs, rabbits, mice, rats, and horses.6-14
Distinguishing the different cell populations that form
the synovial lining is impossible by hematoxylin and eosin
staining under transmission light microscopy. At an ultra­
structural level, the type A cells are characterized by a con­
spicuous Golgi apparatus, large vacuoles, and small vesicles,
and contain little rough endoplasmic reticulum, giving
them a macrophage-like phenotype (Fig. 2-3A and B). The
plasma membrane of type A cells possesses numerous fine
extensions, termed filopodia, which are characteristic of mac­
rophages. These cells are located for the most part on the lin­
ing surface, where it is more than one cell thick. Type A cells
cluster at the tips of the synovial villi, and this uneven dis­
tribution at least partly explains early reports that suggested
type A cells were the predominant intimal cell type.4,8
Type B SLCs have prominent cytoplasmic extensions
that extend onto the surface of the synovial lining (Fig. 2-3C
and D).15 Frequent invaginations are seen along the plasma
membrane, and a large indented nucleus relative to the area
of the surrounding cytoplasm is also a feature. Type B cells
have abundant rough endoplasmic reticulum widely distrib­
uted in the cytoplasm, and the Golgi apparatus, vacuoles,
and vesicles are generally inconspicuous, although some
cells have small numbers of prominent vacuoles at their
apical aspect. Type B SLCs also are known to contain lon­
gitudinal bundles of different-sized filaments, supporting
their classification as fibroblasts. Desmosomes and gaplike
junctions have been described in rat, mouse, and rabbit
synovium, but the existence of these structures has never
been documented in human SLCs.
Cells exhibiting the ultrastructure of type A and type
B SLCs have been classified as intermediate, or type AB.
The existence of intermediate cells has been refuted on the
basis of detailed electron microscopic studies, and it is now
accepted that a proportion of type B cells have conspicu­
ous vacuoles, and that rough endoplasmic reticulum appears
in activated macrophages.16,17 The putative existence of an
intermediate SLC implies that type A and type B SLCs are
part of the same cell lineage. This concept is contrary to all
current evidence, which finds that type A and type B SLCs
are histogenetically and functionally distinct.
23
 24 bresnihan  |  Synovium
500 µm
Figure 2-1  The cartilage-synovium junction. Hyaline articular carti-
lage occupies the left half of this image, and fibrous capsule and syno-
vial membrane occupy the right half. A sparse intimal lining layer with
a fibrous subintima can be observed extending from the margin of the
cartilage across the capsular surface to assume a more cellular intimal
morphology with areolar subintima.
Immunohistochemical Profile of Synovial Intimal Cells
Synovial Intimal Macrophages. Synovial macrophages and
fibroblasts express lineage-specific molecules, which can be
detected by immunohistochemistry. Synovial macrophages
express common hematopoietic antigen CD45 (Fig. 2-4A);
monocyte/macrophage receptors CD163 and CD97; and ly­
sosomal enzymes CD68 (Fig. 2-4B), neuron-specific esterase,
and cathepsin B, L, and D. Cells expressing CD14, a mol­
ecule that acts as a coreceptor for the detection of bacterial
lipopolysaccharide, and expressed by circulating monocytes
and monocytes newly recruited to tissue, are rarely seen in
the healthy intimal layer, but small numbers are found close
to venules in the subintima.18-24
The Fcγ receptor, FcγRIII (CD16), expressed by Kupffer
cells of the liver and type II alveolar macrophages of the
lung, also is expressed on a subpopulation of synovial mac­
rophages.25-27 The synovial macrophage population also
expresses the major histocompatibility complex (MHC)
class II molecule which plays an important role in the
immune response. More recently, the macrophages, which
are responsible for the removal of debris, blood, and par­
ticulate material from the joint cavity and possess antigen
processing properties, have been found to express a new
complement-related protein, Z39Ig, a cell surface receptor
and immunoglobulin superfamily member, which is involved
in the induction of HLA-DR, and implicated in the reg­
ulation of phagocytosis and antigen-­mediated immune
responses.28-30
The expression of the β2 integrin chains, CD18, CD11a,
CD11b, and CD11c, varies; CD11a and CD11c may be
absent, or weakly expressed, on a few lining cells.31,32
Osteoclasts, which are tartrate-resistant, acid phosphatase–
positive, and express the αvβ3 vitronectin and calcitonin
receptors, do not appear in the normal synovium.
Synovial Intimal Fibroblasts. Synovial intimal and subin­
timal fibroblasts are indistinguishable by light microscopy.
They are generally considered to be closely related in terms
2 Microns
Figure 2-2  Transmission electron photomicrograph of synovial intimal
cells. The cell on the left exhibits the dendritic appearance of a synovial
intimal fibroblast (type B cell). Other overlying fibroblast dendrites can
be observed. The presence of intercellular gaps allows the synovial fluid
to be in direct contact with the synovial matrix.
of cell lineage, but because of their different microenviron­
ments, they do not always share the same phenotype. They
possess prominent synthetic capacity and produce the es­
sential joint lubricants hyaluronic acid (HA) and lubricin.33
Intimal fibroblasts express uridine diphosphoglucose dehy­
drogenase (UDPGD), an enzyme involved in HA synthesis,
which is recognized as a specific marker for this cell type.
UDPGD converts UDP-glucose to UDP-glucuronate, one
of the two substrates required by HA synthase for assembly
of the HA polymer.34 CD44 expression, the nonintegrin re­
ceptor for HA, is expressed by all SLCs.32,35,36
Synovial fibroblasts also synthesize normal matrix com­
ponents, including fibronectin, laminin, collagens, pro­
teoglycans, lubricin, and other identified and unidentified
proteins. They also have the capacity to produce large
amounts of metalloproteinases, metalloproteinase inhibi­
tors, prostaglandins, and cytokines. This capacity must
provide essential biologic advantages, but the complex
physiologic mechanisms relevant to normal function are
incompletely delineated. The expression of selected adhe­
sion molecules on synovial fibroblasts probably facilitates
the trafficking of some cell populations, such as neutrophils,
into the synovial fluid, and the retention of others, such
as mononuclear leukocytes, in the synovial tissue. Metal­
loproteinases, cytokines, adhesion molecules, and other cell
surface molecules are strikingly upregulated in inflamma­
tory states.
Specialized intimal fibroblasts also express many other
molecules that are not expressed by the intimal macrophage
population, including decay-accelerating factor (CD55),
previously identified by the antibody Mab67; vascular cell
adhesion molecule 1; intracellular adhesion molecule33,37-40;
and cadherin 11.41,42 PGP.95, a neuronal marker, is reported
as being specific for type B synoviocytes in horses.43 Decay-
accelerating factor, also expressed on the cells of other body
cavities and cells in bone marrow, interacts with CD97,
a glycoprotein that is present on the surface of most acti­
vated leukocytes, including intimal macrophages, and is
thought to be involved in the signaling processes early after
leukocyte activation.44,45 In contrast, FcγRIII is expressed
only by macrophages when they are in close contact with
decay-accelerating factor–positive fibroblasts, or decay-
accelerating factor–coated fibrillin-1 microfibrils in the
extracellular matrix.26
Cadherins are a class of tissue-restricted transmem­
brane proteins that play important roles in homophilic
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE 25

A 500 nm C 500 nm

100 nm
B D 500 nm

Figure 2-3  Transmission electron photomicrographs of synovial intimal macrophages (type A cells) and fibroblasts (type B cells). A, Low-powered
magnification showing the surface fine filopodia, characteristic of macrophages, and a smooth-surfaced nucleus. B, The boxed area in A is shown at a
higher magnification and reveals numerous vesicles characteristic of macrophages. The absence of rough endoplasmic reticulum also is noted. C, The
convoluted nucleus along with the prominent rough endoplasmic reticulum (boxed area) is characteristic of a synovial intimal fibroblast (type B cell). 
D, The rough endoplasmic reticulum is shown at greater magnification.

intercellular adhesion and are involved in maintaining
the integrity of tissue architecture. Cadherin 11, which
was cloned from rheumatoid arthritis synovial tissue, also
is expressed in normal synovial intimal fibroblasts, but not
in intimal macrophages. The finding that fibroblasts trans­
fected with cadherin 11 are induced to form a lining-like
structure in vitro implicates this molecule in the architec­
tural organization of the synovial lining.41,42,46 This sugges­
tion is supported by the observation that cadherin-deficient
mice have a hypoplastic synovial lining and are resistant to
inflammatory arthritis.47
β1 and β3 integrins are present on all SLCs, forming recep­
tors for laminin (CD49f and CD49b), collagen types I and
IV (CD49b), vitronectin (CD51), and fibronectin (CD49d
and CD49e). In contrast, the integrin collagen receptors,
CD49a, CD54 (a member of the immunoglobulin superfam­
ily), and CD4 and CD62 (selectin) present on lymphocytes,
and involved in their homing to high endothelial venules,
are not observed on these cells. CD31 (platelet–endothelial
cell adhesion molecule), a member of the immunoglobulin
superfamily that is expressed on endothelial cells, platelets,
and monocytes, is only weakly expressed on SLCs.32
 26 bresnihan  |  Synovium
A 20 µm
Figure 2-4  Transmitted light photomicrographs depicting synovial intimal macrophages by immunohistochemistry. A and B, Macrophages are deco-
rated with CD45 (arrow in A) and CD68 (B), markers that identify hematopoietic cells (CD45) and macrophages (CD68).
Turnover of Synovial Lining Cells
Proliferation of SLCs in humans is low, as shown when
normal human synovial explants, exposed to a pulse of 3H
thymidine, resulted in the SLCs having a labeling index of
approximately 0.05% to 0.3%48; this bears a striking con­
trast with labeling indices of approximately 50% for bowel
crypt epithelium. Similar evidence of low proliferation has
been found in the synovium of rats and rabbits. The advent
of immunohistochemistry saw this observation substanti­
ated when Revell and others reported that the proportion of
SLCs expressing the proliferation marker Ki67 was between
1 in 2800 and 1 in 30,000.49 It was subsequently shown
that the type B SLCs, the synovial fibroblasts, proliferated
in situ,22,50 a finding consistent with the concept that type
A synovial cells are macrophages. Mitotic activity of SLCs
also is low in inflammatory conditions, such as rheuma­
toid arthritis, a condition associated with SLC hyperplasia.
Coulton and coworkers51 reported “a few” mitotic figures in
only 1 of 600 cases of rheumatoid arthritis synovium sam­
ples analyzed.
Apart from the knowledge that synovial fibroblasts pro­
liferate slowly, little is known about their natural life span,
recruitment, or mode of death. Apoptosis likely is involved
in maintaining synovial homeostasis, but there is little in
the literature on this subject. The dearth of information is
likely to be explained by the lack of normal synovium avail­
able for analysis, in addition to the difficulty encountered
when quantifying this process on histological sections owing
to the rapid clearance of apoptotic bodies.52
Origin of Synovial Lining Cells
There is little doubt that the type A SLC population iden­
tified by Barland and associates4 is bone marrow derived
and represents cells of the mononuclear phagocyte system.
The studies conducted by Edwards53,54 proved informative
when they exploited the Beige (bg) mouse, which harbors
a homozygous mutation that confers the presence of giant
lysosomes in macrophages. It was shown that normal mice,
bone marrow depleted through irradiation, were rescued
B 20 µm
with bone marrow cells obtained from the bg mouse. Elec­
tron microscopic analysis of the synovium from the recipi­
ent animals revealed that type A SLCs contained the giant
lysosomes of the donor bg mouse, and that these structures
were never identified in type B cells. These findings pro­
vided powerful evidence that the type A SLCs represent
macrophages, that they are recruited from the bone mar­
row, and that they were unrelated histogenetically to type
B SLCs.
In addition to immunohistochemistry, several other
lines of evidence have added weight to the concept that
type A SLCs are recruited from the bone marrow: (1) The
op/op mouse, a spontaneously occurring mutant that fails to
produce macrophage colony-stimulating factor because of a
missense mutation in the csf-1 gene,55-57 has low numbers
of circulating and resident macrophage colony-­stimulating
factor–dependent macrophages, including those in the
synovium. (2) Type A cells in rat synovium do not occur
until after the development of synovial blood vessels.22
(3) Others have reported that type A SLCs were conspicu­
ous around vessels in the synovium in neonatal mice.6
(4) When synovial explants are placed in culture, the
reduction in the type A SLCs is partially explained by
their migration into the culture medium, an observation
that reflects the process of migration of macrophages into
the synovial fluid in vivo.1,58 (5) Macrophages are found
around venules in disease states and constitute 80% of the
intimal cells in inflammatory conditions, such as rheuma­
toid arthritis.
Type B intimal cells represent a resident fibroblast popu­
lation in the synovial lining, but little is known about the
cells from which they derive, and how their recruitment is
regulated. The existence of a mesenchymal stem cell in the
synovium is a prime candidate for the origin of the syno­
vial lining fibroblast, but this has not been substantiated. To
date, a transcription factor directing mesenchymal stem cell
differentiation into synovial fibroblast, similar to the factors
required for commitment by this multipotential population
into bone (cbfa-1), cartilage (Sox 9), and fat (peroxisome
proliferator-activated receptor γ [PPARγ]), has not been
identified.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE 27

There are three well-defined categories of subintima—

SUBINTIMAL LAYER
SLCs are not separated from the underlying subintima by a
well-formed basement membrane composed of the typical
trilaminar structure that is seen beneath epithelial mucosa
elsewhere. Nevertheless, most components of basement
membrane are present in the extracellular matrix surround­
ing SLCs. These components include tenascin X, perlecan
(a heparin sulfate proteoglycan), collagen type 4, laminin,
and fibrillin-1.59,60 Of note is the absence of laminin-5
and integrin α3β3γ2, which are components of epithelial
hemidesmosomes.61
The subintima is composed of loose connective tissue of
variable thickness and variable proportions of fibrous/col­
lagenous and adipose tissue depending on the anatomic site.
Under normal healthy conditions, inflammatory cells are
virtually absent from the subintima apart from a sprinkling
of macrophages. A few mast cells also are present.62 Human
synovial tissue also is a rich source of mesenchymal stem cells,
and although it is unknown which compartment contains
this cell population, some cells have the ability to self-renew,
and differentiate into bone, cartilage, and fat in vitro, a phe­
nomenon that reflects its ability to regenerate in vivo.63-65
the areolar, fibrous, and fatty/adipose types. Under the light
microscope, areolar-type subintima, the most commonly
studied, is generally found in larger joints where there is free
movement (Fig. 2-5A). It is composed of fronds with a cel­
lular intimal lining and loose connective tissue in the sub­
intima, with little in the way of dense collagen fibers, and a
rich vasculature. The fibrous subintima is composed of scant
dense fibrous, poorly vascularized connective tissue and has
an attenuated layer of SLCs (Fig. 2-5B). The adipose type
contains abundant mature fat cells and has a single layer of
SLCs. This is seen more commonly with aging and in intra-
articular fat pads (Fig. 2-5C).
The subintima contains collagen types I, III, V, and
VI; glycosaminoglycans; proteoglycans; and extracellular
matrices including tenascin and laminins. Integrin recep­
tors for collagens, laminin, and vitronectin are absent or
at best weakly expressed by the subintimal cells. In con­
trast, receptors for fibronectin (CD49d and CD49e) are
detected, and CD44, the HA receptor, is strongly expressed
in most subintimal cells. β2 integrins are largely limited to
perivascular areas, particularly in the subintimal zone, as is
CD54.66

A 50 µm B 100 µm

C 100 µm
Figure 2-5  Transmitted light photomicrographs of different morphologic types of synovial tissue. All photomicrographs show an intimal layer of one
to two cells in depth. A, The areolar synovium is composed of villous fronds. Beneath the intimal lining layer, there is cellular loose fibrovascular fatty
subintima. B, The fibrous synovium comprises dense collagenous material in the subintimal layer. C, The subintimal layer of the fatty synovial tissue is
composed of less cellular mature adipose tissue with little collagen deposition.
 28 bresnihan  |  Synovium
Subintimal Vasculature
The vascular supply to the synovium is provided by many
small vessels and is partly shared by the joint capsule, epiph­
yseal bone, and other perisynovial structures. Arteriovenous
anastomoses communicate freely with the vascular supply
to periosteum and to periarticular bone. As large synovial
arteries enter the deep layers of the synovium near the cap­
sule, they branch, and branch again to form microvascular
units in the more superficial subsynovial layers. Precapillary
arterioles probably play a major role in controlling circula­
tion to the lining layer. The surface area of the synovial cap­
illary bed is large, and because it runs only a few cell layers
deep to the surface, it has a role in trans-synovial exchange
of molecules.
Numerous physical factors influence synovial blood flow.
Heat increases blood flow through synovial capillaries. Exer­
cise also increases synovial blood flow to normal joints, but
may reduce the clearance rate of small molecules from the
joint space. Experiments have shown a substantial vascu­
lar reserve capacity in normal articulations. Immobilization
reduces synovial blood flow, and the pressure on synovial
membrane from joint effusions can act to tamponade syno­
vial blood supply.
The vascular endothelial lining cells express CD34 and
CD31 (Fig. 2-6A). They also express receptors for the major
components of basement membrane, including laminin and
collagen IV, and the integrin receptors CD49a (laminin and
collagen receptors), CD49d (fibronectin receptor), CD41,
CD51 (vitronectin receptor), and CD61, the β3 integrin sub­
unit. Endothelial cells also express CD44, the HA receptor,
and CD62, P-selectin, which acts as a receptor that supports
binding of leukocytes to activated platelets and endothe­
lium. They are only weakly positive, however, for expres­
sion of CD54, intercellular adhesion molecule-1, an integral
membrane protein of the immunoglobulin superfamily. The
endothelial cells of capillaries in the superficial zone of the
subintima are strongly positive for HLA-DR expression by
immunohistochemistry, whereas cells in the larger vessels in
the deep aspect of the membrane are negative.32,34
Subintimal Lymphatics
Detailed analysis of the number and distribution of lym­
phatic vessels has been made possible with the use of the
antibody to the lymphatic vessel endothelial HA ­receptor
(LYVE-1) (Fig. 2-6B).67 This antibody is highly specific for
lymphatic endothelial cells in lymphatic vessels and lymph
node sinuses and does not react with endothelial cells of
capillaries and other blood vessels that express CD34 and
factor VIII–related antigen. The expression of LYVE-1 in
lymphatic endothelial cells has been used as a marker to
show that lymphatic vessels are less common in the fibrous
synovium compared with the areolar and adipose variants
of human subsynovial tissue. Detection of this molecule
also reveals that lymphatics are present in the ­ superficial,
intermediate, and deeper layers of synovial membrane from
normal, osteoarthritic, and rheumatoid arthritic joints,
although the number in the superficial subintimal layer is
low in normal synovium. Little difference in the distribu­
tion and number is noted between normal and osteoarthritic
synovium, where there is no villous ­hypertrophy. Lymphatic
channels are plentiful, however, in the subintimal layer
in the presence of villous edema hypertrophy and chronic
inflammation.
Subintimal Nerve Supply
The synovium has a rich network of sympathetic and sensory
nerves. The former, which are myelinated and detected with
the antibody against S100 protein, terminate close to blood
vessels, where they regulate vascular tone (Fig. 2-6C, D, and
E). The sensory nerves respond to proprioception and pain
via large myelinated nerve fibers, and small (<5 μm), either
unmyelinated or myelinated fibers with unmyelinated free
nerve ends (nociceptors). The latter are immunoreactive
in the synovium for neuropeptides, including substance P,
calcitonin gene–related peptide, and vasoactive intestinal
peptides.69,70
FUNCTION
The known synthetic and protective functions of individual
synovial cell populations are multiple and complex. The
composite synovial structure, including the cell populations
and their products, vasculature, nerves, and the intercellular
matrix, possesses several specialized functions that are essen­
tial for normal joint movement, synovial fluid formation,
chondrocyte nutrition, and cartilage protection at multiple
anatomic locations. These functions must be preserved over
a lifetime to maintain maximal mobility and independence.
Absence of essential constituents of synovial fluid, or inad­
equate cartilage protection, results in early articular mal­
function, which may progress to local or generalized joint
failure.
JOINT MOVEMENT
Three characteristics of the synovium are essential for joint
movement—deformability, nonadherence, and cartilage
lubrication. In health, the synovium is a highly deformable
structure that facilitates movement between other adjacent,
nondeformable structures within the joint. This unique facil­
ity of the synovium to enable movement between, rather than
within, tissues has been emphasized,71 and can be attributed
to the presence of a free surface that allows synovial tissue to
remain separated from adjacent tissues. The ensuing space is
maintained by the presence of synovial fluid.
Deformability
The deformability of normal synovium is considerable
because it must accommodate the extreme positional range
available to the joint and its adjacent tendons, ligaments,
and capsule. When flexing a finger, the palmar synovium
of each interphalangeal joint contracts, while the dorsal
synovium expands, and as the finger extends, the reverse
occurs. This normal contraction and expansion of synovium
seems to involve a folding and unfolding component and
an elastic stretching and relaxation of the tissue. It is essen­
tial that during repeated rapid movement, synovial lining
does not become pinched between cartilage surfaces and
can ­successfully retain its integrity and the integrity of the
synovial blood vessels and lymphatics.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE 29

A B 100 µm

C 500 µm D 200 µm

E 50 µm
Figure 2-6  Transmission light photomicrographs of synovium showing lymphovascular and nervous structures by immunohistochemistry. A and 
B, Areolar synovium featuring thin-walled vessels are highlighted with antibody to CD31 (A), and lymphatic vessels in an inflamed synovium are 
highlighted with antibody to LYVE-1 (B). C, Deep in the synovial subintima close to the joint capsule, there are medium-sized neurovascular bundles
with the nerves highlighted by antibody to S100. D, Within the more superficial synovium, small nerves decorated with S100 also are identified. E, The
boxed area in D is shown at higher magnification; upper arrow is nerve; lower arrow is directed at a small vessel.

synovial and cartilage surfaces. The mechanism that pre­

Nonadherence
The second important characteristic of the synovium that
facilitates joint movement is its nonadherence to opposing
surfaces. The intimal cells on the synovial surface adhere to
underlying cells and matrix, but do not adhere to ­opposing
serves this phenomenon of nonadherence is unknown and
may involve the arrangement of cell surface and tissue
matrix molecules, such as collagen, fibronectin, and HA.
Alternatively, nonadherence may result partially from the
regular movements of the normal synovial lining.
 30 bresnihan  |  Synovium
Lubrication
The third characteristic of synovium that is essential for joint
motion is an efficient lubrication mechanism to facilitate
cartilage movement on cartilage. The mechanisms of joint
lubrication are complex and are an integral component of
synovial physiology. In an articulating joint, cartilage is sub­
jected to numerous compressive and frictional forces every
day. Friction and wear can never be eliminated from a func­
tioning joint. Adult chondrocytes do not normally divide
in vivo, and damaged cartilage has limited capacity for self-
repair. For a joint to maintain its function throughout a life­
time of use, there must be protective biologic mechanisms,
such as lubrication, which help minimize the wear and dam­
age that result from normal daily activities.
Boundary lubrication refers to the protective effect of par­
ticular lubricating molecules adsorbing to a surface and repel­
ling its opposing interface.73 Bearing surfaces must generate
a mutual repulsion to be lubricated in the boundary mode.
Boundary lubricants exert their effects by changing the physi­
cochemical characteristics of a surface and reduce articular
friction and wear by providing a smooth and slippery coating.
Friction is reduced by an interposed film of protective fluid
that allows one surface to ride freely over another. The carti­
lage matrix is integral to this phenomenon because it is fluid-
filled and compressible. Loaded cartilage extrudes lubricant
fluid from its surface, and the expressed fluid contributes to the
separation of the two articulating surfaces. Scanning electron
microscopy has shown a continuous film of fluid, only 100 nm
thick, which separates one surface from the other, prevent­
ing direct abrasive contact.74 This ultrathin coating of lubri­
cant also resists distraction of the two articulating surfaces,
enhancing joint stability. Another essential advantage of an
intra-articular lubrication system is the effective prevention of
pinching of adjacent, well-vascularized synovial membrane.
Hyaluronic Acid
HA, a high-molecular-weight polysaccharide, is a major
component of synovial fluid and cartilage.75 It is produced
in large amounts by mechanosensitive, fibroblast-like
sy­noviocytes.76,77 HA, of which there are three mammalian
forms designated HAS1, HAS2, and HAS3,78 is synthesized
by HA synthase at the plasma membrane and extruded
directly into the extracellular compartment. HA synthase
activity and HA secretion are stimulated by proinflamma­
tory cytokines, including interleukin-1β and transforming
growth factor-β.76,79,80 HA also is synthesized by many other
skeletal cells and is an important component of extracellular
matrices. It is simultaneously a solid phase matrix element
of cartilage and other tissues, and a fluid phase element in
the synovial space under normal and abnormal conditions.
HA has many biologic functions, which include effects
on cell growth, migration, and adhesion.72 The regulatory
role of HA is mediated through HA-binding proteins and
receptors, including CD44, which are present on the cell sur­
faces of chondrocytes, lymphocytes, and other mononuclear
cell populations. HA plays a crucial role in morphogenesis
and in wound healing. Additionally, HA is a vital structural
component of the synovial lining, and it has an essential
role in the induction of joint cavitation during embryogene­
sis. HA, produced by synovium, was originally thought to be
primarily a joint lubricant, and it is generally accepted that it
plays a major physiologic role in maintaining synovial fluid
viscosity. It is important in normal joint function, not least
through its capacity to provide effective shock absorption.
It has been suggested that HA is a particularly important
viscohydrodynamic lubricant at low-load interfaces, such as
synovium-on-synovium and synovium-on-cartilage.81 Syno­
vial fluid HA, acting in combination with albumin, also has
a role in the attenuation of fluid loss from the joint cavity,
particularly during periods of increased pressure, which can
occur during sustained joint flexion.82-84
Lubricin. Compelling evidence suggests that lubricin, first
described in the 1970s,85 is the factor primarily responsible
for boundary lubrication of diarthrodial joints.86 Lubricin,
a large secreted, mucin-like proteoglycan with an apparent
molecular weight of 280 kD, is a product of the gene proteo­
glycan 4 (PRG4). It is a major component of synovial fluid
and is present at the cartilage surface. The gene is highly ex­
pressed by human synovial fibroblasts and by superficial zone
chondrocytes.87 Lubricin is closely related to superficial zone
protein, megakaryocyte-stimulating factor, and hemangio­
poietin. Superficial zone protein is expressed by SLCs and
by the superficial zone chondrocytes at the cartilage surface,
but not by intermediate or deep zone chondrocytes.88 It has
been suggested that lubricin may bind to the much longer
hyaluronate polymers, distributing shear stress and stabiliz­
ing essential lubricant molecules.89
In an experimental model, lubricin seemed to have multi­
ple functions in articulating joints and tendons that included
the protection of cartilage surfaces from protein deposition
and cell adhesion and the inhibition of synovial cell over­
growth.90 Prg4−/− mice, consistently normal at birth, showed
progressive loss of superficial zone chondrocytes and increas­
ing synovial cell hyperplasia (Fig. 2-7). The essential role of
lubricin in maintaining joint integrity was shown by the iden­
tification of disease-causing mutations in patients with the
autosomal recessive disorder ­ camptodactyly–­arthropathy–
coxa vara–pericarditis (CACP) syndrome.91 CACP is a
large joint arthropathy associated with the absence of lubri­
cin from synovial fluid and ineffective boundary lubrication
provided by the synovial fluid (Fig. 2-8).89,92 In other studies
of lubricin biology and joint integrity, experimental injury
resulted in reduced synovial fluid lubricin concentrations,
decreased boundary-lubricating ability, and increased carti­
lage matrix degradation, each of which could be attributed
to trauma-induced inflammatory processes.87
Others have argued against the primacy of lubricin in
joint lubrication by proposing that surface-active phos­
pholipid, also secreted by intimal fibroblasts, is the essen­
tial boundary lubricant that reduces cartilage friction to
remarkably low levels.93 It was hypothesized that lubricin
acts as the carrier of surface-active phospholipid to articular
cartilage, but is not the lubricant per se, a function that is
similar to the well-characterized alveolar surfactant binding
proteins in the lung.
FORMATION OF SYNOVIAL FLUID
In health, a constant volume of synovial fluid is important
during joint movement as a cushion for synovial tissue and
as a reservoir of lubricant for cartilage. Many of the soluble
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE 31

p
f
f

t
A –/–
fib

+/+ –/–
E F

B +/+

c
t

ta
h
s

C +/+ D –/– G +/+ H –/–

Figure 2-7  Clinical appearance and radiographic changes in Prg4−/− mice. A and B, Photographs of the hind paws of 6-month-old Prg4−/− (A) and wild-
type (B) mice. Note the curved digits in the mutant mouse and the swelling at the ankle joint. C and D, Radiographs of the ankle joint of 9-month-old
wild-type (C) and Prg4−/− mice (D). The structures corresponding to the tibia (t) and talus (ta) are indicated. Note the calcification of structures adjacent
to the ankle (arrows in D). E, Lateral knee x-ray of a 4-month-old wild-type mouse. The structures corresponding to the patella (p), femoral condyle
(f), tibial plateau (t), and fibula (fib) are indicated. F, Lateral knee x-ray of a 4-month-old Prg4−/− mouse. Note the increased joint space between the
patella and femur (arrow), and osteopenia of the patella, femoral condyles, and tibial plateau. G, Shoulder x-ray of a 4-month-old wild-type mouse. The
structures corresponding to the humeral head (h), glenoid fossa of the scapula (s), and lateral portion of the clavicle (c) are indicated. H, Shoulder x-ray
of a 4-month-old Prg4−/− mouse. Note the increased joint space between the humerus and scapula (arrow), and the osteopenia of the humeral head.
(From Rhee DK, Marcelino J, Baker M, et al: The secreted glycoprotein lubricin protects cartilage surfaces and inhibits synovial cell overgrowth. J Clin Invest 115:
622-631, 2005.)

components and proteins in synovial fluid exit the syno­
vial microcirculation through pores or fenestrations in the
vascular endothelium, then diffuse through the intersti­
tium before entering the joint space. Synovial fluid is par­
tially a filtrate of plasma to which additional components,
including HA and lubricin, are added and removed by the
SLCs (Fig. 2-9).72 The concentrations of electrolytes and
small molecules in synovial fluid are equivalent to those in
plasma. Synovial permeability to most small molecules is
determined by a process of free diffusion through the double
barrier of endothelium and interstitium, limited mainly by
the intercellular space between the SLCs. For most small
molecules, synovial permeability is inversely related to the
dimensions of the molecule.
Experimental evidence suggests that the exchange of small
solutes is determined predominantly by the synovial inter­
stitium, and that permeability to proteins is mainly deter­
mined by the microvascular endothelium. The synovium
should not be regarded as simply an inert membrane, but as
a complex regulatory tissue system. The small physiologic
molecules that traverse the endothelium of synovial blood
vessels and diffuse through the intercellular spaces of the
synovial lining before entering the synovial fluid include
water, glucose, and many other essential nutrients and
waste tissue metabolites. Evidence suggests that the passage
of some solutes across the synovium is facilitated by specific
transport systems providing, possibly, a “pump” mechanism
capable of moving water out of the joint space.
All plasma proteins are capable of crossing the endothe­
lium, traversing the synovial interstitium, and entering the
synovial fluid. The efficiency of this process is determined
by the molecular size of the protein and the diameter of
the endothelial pores. Smaller proteins, such as albumin,
enter easily, whereas larger molecules, such as fibrinogen,
gain access with greater difficulty. In contrast, the clear­
ance or removal of proteins and other synovial fluid con­
stituents is unrestricted, and considerably more efficient,
through lymphatic drainage. The synovial fluid concentra­
tion of any protein reflects the dynamic balance between
ingress and egress at a given time. Because egress is more
efficient than ingress, joint space pressure is normally sub­
atmospheric. The negative intra-articular pressure also is
thought to be important in maintaining joint stability. The
synovial fluid-to-serum ratio of plasma proteins is inversely
related to the molecular size of the protein. When the joint
becomes inflamed, greater endothelial permeability permits
more profuse ingress of all proteins, and the most obvi­
ous changes are in the concentrations of larger molecules.
Increased synovial fluid volume also reduces the stability of
the joint.
In contrast to hydrophilic molecules, fat-soluble mole­
cules can diffuse through and between cell membranes, and
their passage across the synovial surface is less restricted.
The entire surface area of the synovium is available to lipo­
philic molecules that diffuse in and out of the joint space.
Physiologically, the most important fat-soluble molecules
 32 bresnihan  |  Synovium

A B

C
Figure 2-8  Clinical features of camptodactyly–arthropathy–coxa vara–pericarditis (CACP) syndrome. A, The characteristic deformity of the hands is
shown. B, Chest x-ray shows an enlarged cardiac outline caused by pericarditis. C, X-ray of the pelvis highlights coxa vara in a boy with CACP. (B and C
courtesy of Ronald Laxer, MD, Hospital for Sick Children, Toronto.)

Synovium hypoxia and acidosis can have serious implications for the
Synovial fluid Cartilage
Sublining Lining synovial microcirculation and chondrocyte metabolism.
Matrix Hydrophillic molecules:
water, electrolytes, NUTRITION OF CHONDROCYTES
glucose, proteins
Blood Another important function of synovium is to facilitate
vessel Lipophilic molecules: Superficial
O2, CO2 zone the nutrition of chondrocytes, which are resident in artic­
Lymphatic chondrocytes ular cartilage (see Chapter 3). Because articular cartilage
Lubricants is avascular, the delivery of nutrients to chondrocytes and
Hyaluronan
Lubricin
the removal of metabolic breakdown products from the
Egress of SF
components
cartilage are believed to occur through the synovial fluid
unrestricted and the synovial tissue arterioles and venules.33 Morpho­
logic, physiologic, and pathologic studies have confirmed
Figure 2-9  Schematic representation of the formation of synovial fluid.
Many of the soluble components and proteins in synovial fluid exit the
that solutes pass easily from the synovial fluid into carti­
synovial subintimal microcirculation through pores or fenestrations in lage, and that cartilage does not survive without synovial
the vascular endothelium, then diffuse through the interstitium before fluid contact in vivo. Within the cartilage matrix, three
entering the joint space. Synovial permeability to most small molecules potential ­mechanisms for nutrient transfer have been pro­
is determined by a process of free diffusion through the double bar- posed—­diffusion, active transport by chondrocytes, and
rier of endothelium and interstitium, limited mainly by the intercellular
space between the synovial lining cells. Fat-soluble molecules can dif- pumping by intermittent compression of cartilage matrix.
fuse through, and between, cell membranes, and their passage across A large proportion of hyaline cartilage lies within 50 μm
the synovial surface is less restricted. Additional components, including of a synovial surface and its rich supply of blood vessels.
hyaluranon and lubricin, are produced by the synovial lining cells. Chondrocytes are oxygen sensitive and well adapted to liv­
ing in hypoxic conditions. Low oxygen tension promotes
the expression of the chondrocyte phenotype and cartilage-
are the respiratory gases—oxygen and carbon dioxide. When specific matrix formation. Reactive oxygen species also may
the joint is inflamed, synovial fluid may exhibit low partial play a crucial role in the regulation of some normal chon­
pressure of oxygen, high partial pressure of carbon dioxide, drocytic activities, such as cell activation, proliferation, and
decreased pH, and increased lactate production. The resultant matrix remodeling.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
SUMMARY  
The normal human synovial membrane is a highly special­
ized, multifunctional organ that is vital for mobility, inde­
pendence, and survival. The intimal layer is composed of
two distinct cell phenotypes with characteristics of macro­
phage and fibroblast lineages. Synovial macrophages express
CD45, CD163 and CD97, CD68, neuron-specific esterase,
and cathepsins B, L, and D. Cells expressing CD14 are rarely
seen in the healthy intimal layer. FcγRIII (CD16), expressed
by Kupffer cells of the liver and type II alveolar macrophages
of the lung, is expressed on a subpopulation of synovial mac­
rophages. Synovial macrophages also express the MHC class
II molecule, and play a central role in phagocytosis and in
antigen-mediated immune responses.
Synovial intimal fibroblasts possess prominent synthetic
capacity and produce the essential joint lubricants HA and
lubricin. They also synthesize normal matrix components,
including fibronectin, laminin, collagens, proteoglycans,
lubricin, and other identified and unidentified proteins.
They have the capacity to produce large amounts of metal­
loproteinases, metalloproteinase inhibitors, prostaglandins,
and cytokines. The expression of selected adhesion mole­
cules on synovial fibroblasts probably facilitates the traffick­
ing of some cell populations, such as polymorphs, into the
synovial fluid, and the retention of others, such as mono­
nuclear leukocytes, in the synovial tissue.
The subintimal layer is composed of a loose connective
tissue matrix and contains branching blood and lymphatic
vessels; a nerve supply; and a variety of resident cell popu­
lations, including infiltrating macrophages and fibroblasts.
The nerve supply is important in regulating synovial blood
flow. The lymphatic vessels allow egress of metabolic break­
down products from the synovium and synovial fluid. The
morphology of the subintimal layer varies according to the
anatomic location and local functional requirements.
The coordinated functions of the composite synovial
membrane are essential for normal joint movement, forma­
tion of synovial fluid, nutrition of chondrocytes, and protec­
tion of cartilage. These functions must be preserved over
a lifetime at multiple anatomic locations. The absence of
essential constituents of synovial fluid, such as lubricin,
or inadequate cartilage protection results in early articular
malfunction, which may progress to variable degrees of joint
failure. The characteristics of lubricin deficiency have been
elegantly described in animal models and in humans. Fur­
ther studies may define novel clinical categories of degen­
erative polyarthritis that are associated with other specific
disorders of synovial membrane function.
Acknowledgments
The authors thank Suhel Miah, Institute of Orthopaedics and Musculo­
skeletal Science, and Bethany Crane and Steve Crane, Royal National
Orthopaedic Hospital, for preparing many of the images included in this
chapter.
REFERENCES
  1. Nozawa-Inoue K, Takagi R, Kobayashi T, et al: Immunocytochemical
demonstration of the synovial membrane in experimentally induced
arthritis of the rat temporomandibular joint. Arch Histol Cytol
61:451-466, 1998.
33
2. Vandenabeele F, Lambrichts I, Lippens P, et al: In vitro loading of
human synovial membrane with 5-hydroxydopamine: Evidence for
dense core secretory granules in type B cells. Arch Histol Cytol 64:
1-16, 2001.
  3. Castor CW: The microscopic structure of normal human synovial tis­
sue. Arthritis Rheum 3:140-151, 1960.
  4. Barland P, Novikoff A, Novikoff AB, et al: Electron microscopy of the
human synovial membrane. J Cell Biol 14:207-220, 1962.
  5. Krey PR, Cohen AS: Fine structural analysis of rabbit synovial cells in
organ culture. Arthritis Rheum 16:324-340, 1973.
  6. Okada Y, Nakanishi I, Kajikawa K: Ultrastructure of the mouse syno­
vial membrane: Development and organization of the extracellular
matrix. Arthritis Rheum 24:835-843, 1981.
  7. Groth HP: Cellular contacts in the synovial membrane of the cat
and the rabbit: An ultrastructural study. Cell Tissue Res 164:525-541,
1975.
  8. Roy S, Ghadially FN: Ultrastructure of normal rat synovial membrane.
Ann Rheum Dis 26:26-38, 1967.
  9. Wyllie JC, More RH, Haust MD: The fine structure of normal guinea
pig synovium. Lab Invest 13:1254-1263, 1964.
10. Fell HB, Glauet AM, Barratt ME, et al: The pig synovium, I: The
intact synovium in vivo and in organ culture. J Anat 122:663-680,
1976.
11. Watanabe H, Spycher MA, Ruttner JR, et al: Ultrastructural studies of
rabbit synovitis induced by autologous IgG fragments, II: Infiltrating
cells in the sublining layer. Scand J Rheumatol Suppl 15:15-22, 1976.
12. Linck G, Stoerkel ME, Petrovic A, et al: Morphological evidence of a
polypeptide-like secretory function of the B cells in the mouse syno­
vial membrane. Experientia 33:1098-1099, 1977.
13. Johansson HE, Rejno S: Light and electron microscopic investigation
of equine synovial membrane: A comparison between healthy joints
and joints with intraarticular fractures and osteochondrosis dissecans.
Acta Vet Scand 17:153-168, 1976.
14. Ghadially FN: Fine Structure of Joints. London, Butterworths, 1983.
15. Iwanaga T, Shikichi M, Kitamura H, et al: Morphology and functional
roles of synoviocytes in the joint. Arch Histol Cytol 63:17-31, 2000.
16. Graabaek PM: Ultrastructural evidence for two distinct types of
sy­noviocytes in rat synovial membrane. J Ultrastruct Res 78:321-339,
1982.
17. Graabaek PM: Characteristics of the two types of synoviocytes in rat
synovial membrane: An ultrastructural study. Lab Invest 50:690-702,
1984.
18. Edwards JCW: Fibroblast biology: Development of differentiation
of synovial fibroblasts in arthritis. Arthritis Res 2:344-347, 2000.
19. Athanasou NA: Synovial macrophages. Ann Rheum Dis 54:392-394,
1995.
20. Athanasou NA, Quinn J: Immunocytochemical analysis of human
synovial lining cells: Phenotypic relation to other marrow derived
cells. Ann Rheum Dis 50:311-315, 1991.
21. Athanasou NA, Quinn J, Heryet A, et al: The immunohistology
of synovial lining cells in normal and inflamed synovium. J Pathol
155:133-142, 1988.
22. Izumi S, Takeya M, Takagi K, et al: Ontogenetic development of syno­
vial A cells in fetal and neonatal rat knee joints. Cell Tissue Res 262:
1-8, 1990.
23. Edwards JC: The nature and origins of synovium: Experimental
approaches to the study of synoviocyte differentiation. J Anat 184:
493-501, 1994.
24. Lau SK, Chu PG, Weiss LM: CD163: A specific marker of macrophages
in paraffin-embedded tissue samples. Am J Clin Pathol 122:794-801,
2004.
25. Tuijnman WB, van Wichen DF, Schuurman HJ: Tissue distribution of
human IgG Fc receptors CD16. CD32 and CD64: An immunohisto­
chemical study. APMIS 101:319-329, 1993.
26. Edwards JCW, Blades S, Cambridge G: Restricted expression of Fc
gammaRIII (CD16) in synovium and dermis: Implications for tis­
sue targeting in rheumatoid arthritis (RA). Clin Exp Immunol 108:
401-406, 1997.
27. Bhatia A, Blades S, Cambridge G, et al: Differential distribution of Fc
gamma RIIIa in normal human tissues and co-localization with DAF
and fibrillin-1: Implications for immunological microenvironments.
Immunology 94: 56-63, 1998.
28. Walker MG: Z39Ig is co-expressed with activated macrophage genes.
Biochem Biophys Acta 1574:387-390, 2002.
 34 bresnihan  |  Synovium
29. Kim JK, Choi EM, Shin HI, et al: Characterization of monoclonal
antibody specific to the Z39Ig protein, a member of immunoglobulin
superfamily. Immunol Lett 99:153-161, 2005.
30. Lee MY, Kim WJ, Kang YJ, et al: Z39Ig is expressed on macrophages
and may mediate inflammatory reactions in arthritis and atherosclero­
sis. J Leukoc Biol 80:922-928, 2006.
31. el-Gabalawy H, Canvin J, Ma GM, et al: Synovial distribution of alpha
d/CD18, a novel leukointegrin: Comparison with other integrins and
their ligands. Arthritis Rheum 39:1913-1921, 1996.
32. Demaziere A, Athanasou NA: Adhesion receptors of intimal and sub­
intimal cells of the normal synovial membrane. J Pathol 168:209-215,
1992.
33. Edwards JCW: The synovium. In Hochberg MC, Silman AJ, Smolen
JS, et al (eds): Rheumatology, 3rd ed. Edinburgh, Mosby, 2003, pp
159-166.
34. Wilkinson LS, Pitsillides AA, Worrall JG, et al: Light microscopic
characterization of the fibroblast-like synovial intimal cell (synovio­
cyte). Arthritis Rheum 35:1179-1184, 1992.
35. Johnson BA, Haines GK, Haclous LA, et al: Adhesion molecule
expression in human synovial tissue. Arthritis Rheum 36:137-146,
1993.
36. Henderson KJ, Edwards JCW, Worrall JG: Expression of CD44 in nor­
mal and rheumatoid synovium and cultured fibroblasts. Ann Rheum
Dis 53:729-734, 1994.
37. Stevens CR, Mapp PI, Revell PA: A monoclonal antibody (Mab 67)
marks type B synoviocytes. Rheumatol Int 10:103-106, 1990.
38. Pitsillides AA, Wilkinson LS, Mehdizadeh S, et al: Uridine diphospho­
glucose dehydrogenase activity in normal and rheumatoid synovium:
The description of a specialized synovial lining cell. Int J Exp Pathol
74:27-34, 1993.
39. Wilkinson LS, Edwards JD, Paston RN, et al: Expression of vascu­
lar cell adhesion molecule-1 in normal and inflamed synovium. Lab
Invest 68:82-88, 1993.
40. Edwards JC, Wilkinson LS, Speight P, et al: Vascular cell adhesion
molecule 1 and alpha 4 and beta 1 integrins in lymphocyte aggregates
in Sjogren’s syndrome and rheumatoid arthritis. Ann Rheum Dis
52:806-811, 1993.
41. Valencia X, Higgins JM, Kiener HP, et al: Cadherin-11 provides
specific cellular adhesion between fibroblast-like synoviocytes.
J Exp Med 200:1673-1679, 2004.
42. Kiener HP, Brenner MB: Building the synovium: Cadherin-11 medi­
ates fibroblast-like synoviocyte cell-to-cell adhesion. Arthritis Res
Ther 7:49-54, 2005.
43. Kitamura HP, Yanase H, Kitamura H, et al: Unique localization of pro­
tein gene product 9.5 in type B synoviocytes in the joints of the horse.
J Histochem Cytochem 47:343–352, 1999.
44. Hamann J, Wishaupt JO, van Lier RA, et al: Expression of the activa­
tion antigen CD97 and its ligand CD55 in rheumatoid synovial tissue.
Arthritis Rheum 42:650-658, 1999.
45. Hamann J, Vogel B, van Schijadel GM, et al: The seven-span trans­
membrane receptor CD97 has a cellular ligand (CD55, DAF). J Exp
Med 184:1185-1189, 1996.
46. Kiener HP, Lee DM, Agarwal SK, et al: Cadherin-11 induces rheuma­
toid arthritis fibroblast-like synoviocytes to form lining layers in vitro.
Am J Pathol 168:1486-1499, 2006.
47. Lee DM, Kiener HP, Agarwal SK, et al: Cadherin-11 in synovial lining
formation and pathology in arthritis. Science 315:1006-1010, 2007.
48. Mohr W, Beneke G, Mohing W: Proliferation of synovial lining cells
and fibroblasts. Ann Rheum Dis 34:219-224, 1975.
49. Lalor PA, Garcia CH, O’Rourke LM, et al: Proliferative activity of
cells in the synovium as demonstrated by a monoclonal antibody,
Ki67. Rheumatol Int 7:183-186, 1987.
50. Qu Z, Henderson B, Bitensky L, et al: Local proliferation of fibroblast-like
synoviocytes contributes to synovial hyperplasia: Results of proliferating
cell nuclear antigen/cyclin, c-myc, and nucleolar organizer region stain­
ing. Arthritis Rheum 37:212-220, 1994.
51. Coulton LA, Coates PJ, Ansari B, et al: DNA synthesis in human
rheumatoid and nonrheumatoid synovial lining. Ann Rheum Dis
39:241-247, 1980.
52. Hall PA, Edwards JC, Willoughby DA, et al: Regulation of cell num­
ber in the mammalian gastrointestinal tract: The importance of apop­
tosis. J Cell Sci 107:3569-3577, 1994.
53. Edwards JC, Willoughby DA: Demonstration of bone marrow
derived cells in synovial lining by means of giant intracellular gran-
ules as genetic markers. Ann Rheum Dis 41:177-182, 1982.
54. Edwards JC: The nature and origin of synovium: Experimental
approach to the study of synoviocyte differentiation. J Anat 184:
493-501, 1994.
55. Yoshida H, Cecchini MG, Fleisch H, et al: The murine mutation
osteopetrosis is in the coding region of the macrophage colony stimu­
lating factor gene. Nature 345:442-444, 1990.
56. Felix R, Cecchini MG, Fleisch H: Macrophage colony stimulating fac­
tor restores in vivo bone resorption in the op/op osteopetrotic mouse.
Endocrinology 127:2592-2594, 1990.
57. Naito M, Palmer DG, Revell PA, et al: Abnormal differentiation
of tissue macrophage populations in ‘osteopetrosis’ (op) mice defec­
tive in the production of macrophage colony-stimulating factor.
Am J Pathol 139:657-667, 1991.
58. Hogg N, Palmer DG, Revell PA: Mononuclear phagocytes of nor-
mal and rheumatoid synovial membrane identified by monoclonal
antibodies. Immunology 56:673-681, 1985.
59. Li TF, Boesler EW, Jimenez SA, et al: Distribution of tenascin-X in
different synovial samples and synovial membrane-like interface tis­
sue from aseptic loosening of total hip replacement. Rheumatol Int
19:177-183, 2000.
60. Dodge GR, Boesler EW, Jimenez SA: Expression of the basement mem­
brane heparan sulfate proteoglycan (perlecan) in human synovium
and in cultured human synovial cells. Lab Invest 73:649-657, 1995.
61. Konttinen YT, Hoyland JA, Denton J, et al: Expression of laminins
and their integrin receptors in different conditions of synovial mem­
brane and synovial membrane-like interface tissue. Ann Rheum Dis
58:683-690, 1999.
62. Dean G, Kruetner A, Ferguson AB, et al: Mast cells in the synovium
and synovial fluid in osteoarthritis. Br J Rheumatol 32:671-675,
1993.
63. Bentley G, Kreutner A, Ferguson AB: Synovial regeneration and
articular cartilage changes after synovectomy in normal and steroid-
treated rabbits. J Bone Joint Surg (Br) 57:454-462, 1975.
64. De Bari C, Sekiya I, Yagishita K, et al: Multipotent mesenchymal
stem cell from adult human synovial membrane. Arthritis Rheum
44:1928-1942, 2001.
65. Sakaguchi Y, Athanasou NA: Comparison of human stem cells
derived from various mesenchymal tissues: Superiority of synovium as
a cell source. Arthritis Rheum 52:2521-2529, 2005.
66. Demaziere A, Athanasou NA: Adhesion receptors of intimal and sub­
intimal cells of the normal synovial membrane. J Pathol 168:209-215,
1992.
67. Xu H, Edwards J, Banerji S, et al: Distribution of lymphatic vessels
in normal and arthritic human synovial tissues. Ann Rheum Dis
62:1227-1229, 2003.
68. Deleted in press.
69. Bohnsack M: Distribution of substance-P nerves inside the infrapa­
tellar fat pad and the adjacent synovial tissue: A neurohistological
approach to anterior knee pain syndrome. Arch Orthop Trauma Surg
125:592-597, 2005.
70. McDougall JJ: Arthritis and pain: Neurogenic origin of joint pain:
A review. Arthritis Res Ther 10:220-230, 2006.
71. Henderson B, Edwards JCW: Functions of synovial lining. In The
Synovial Lining in Health and Disease. London, Chapman & Hall,
1987, pp 41-74.
72. Deleted in press.
73. Mazzucco D, Spector M: The role of joint fluid in the tribology of total
joint arthroplasty. Clin Orthop 429:17-32, 2004.
74. Clark JM, Norman AG, Kaab MJ, et al: The surface contour of articu­
lar cartilage in an intact, loaded joint. J Anat 195:45-56, 1999.
75. Prehm P: Hyaluronan. In Steinbuchel A (ed): Biopolymers. Wein­
heim, Whiley-VCH-Verlag, 2002, pp 379-400.
76. Momberger TS, Levick JR, Mason RM: Hyaluronan synthesis by rab­
bit synoviocytes is mechanosensitive. Matrix Biol 24:510-519, 2005.
77. Momberger TS, Levick JR, Mason RM: Mechanosensitive synovio­
cytes: A Ca2+-PKCα-MAP kinase pathway contributes to stretch-
induced hyaluronan synthesis in vitro. Matrix Biol 25:306-316, 2006.
78. Weigel PH, Hascall VC, Tammi M: Hyaluronan synthases. J Biol
Chem 272:13997-14000, 1997.
79. Recklies AD, White C, Melching L, et al: Differential regulation and
expression of hyaluronan in human articular cartilage, synovial cells
and osteosarcoma cells. Biochem J 354:17-24, 2001.
80. Tanimoto K, Itoh H, Sagawa N, et al: Cyclic mechanical stretch regu­
lates the gene expression of hyaluronic acid synthetase in cultured rab­
bit synovial cells. Connect Tissue Res 42:187-195, 2001.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
81. Murakami T, Higaki H, Sawae Y, et al: Adaptive multimode lubrica­
tion in natural synovial joints and artificial joints. Proc Inst Mech Eng
212:23-35, 1998.
82. Levick JR: Fluid movement across synovium in healthy joints: Role of
synovial fluid macromolecules. Ann Rheum Dis 54:417-423, 1995.
83. Scott D, Coleman PJ, Mason RM, et al: Molecular reflection by syno­
vial lining is concentration dependent and reduced in dilute effusions
in a rabbit model. Arthritis Rheum 43: 1175-1182, 2000.
84. Sabaratnam S, Mason RM, Levick JR: Hyaluranon molecular reflection
by synovial lining is concentration dependent and reduced in dilute
effusions in a rabbit model. Arthritis Rheum 54:1673-1681, 2006.
85. Swann DA, Sotman S, Dixon M, et al: The isolation and partial char­
acterization of the major glycoprotein from the articular lubricating
fraction from bovine synovial fluid. Biochem J 161:473-485, 1977.
86. Jay GD, Britt DE, Cha C-J: Lubricin is a product of megacaryocyte
stimulating factor gene expression by human synovial fibroblasts.
J Rheumatol 27:594-600, 2000.
87. Elsaid KA, Jay GD, Warman ML, et al: Association of articular car­
tilage degradation and loss of boundary-lubricating ability of synovial
fluid following injury and inflammatory arthritis. Arthritis Rheum
52:1746-1755, 2005.
35
88. Schumacher BL, Hughes CE, Kuettner KE, et al: Immunodetection
and partial cDNA sequence of the proteoglycan, superficial zone
protein, synthesized by cells lining synovial joints. J Bone Joint Surg
17:110-120, 1999.
89. Jay GD, Tantravahi U, Britt DE, et al: Homology of lubricin and
superficial zone protein (SZP): products of megacaryocyte stimulating
factor (MSF) gene expression by human synovial fibroblasts and artic­
ular chondrocytes localized to chromosome 1q25. J Orthop Res 19:
677-687, 2001.
90. Rhee DK, Marcelino J, Baker M, et al: The secreted glycoprotein lubri­
cin protects cartilage surfaces and inhibits synovial cell overgrowth.
J Clin Invest 115:622-631, 2005.
91. Marcelino J, Carpten JD, Suwairi WM, et al: CACP, encoding a
secreted proteoglycan, is mutated in camptodactyly-arthropathy-coxa
vara-pericarditis syndrome. Nat Genet 23:319-322, 1999.
92. Rhee DK, Marcelino J, Sulaiman A-M, et al: Consequences of disease-
causing mutations on lubricin protein synthesis, secretion, and post-
translational processing. J Biol Chem 280:31325-31332, 2005.
93. Hills BA, Crawford RW: Normal and prosthetic synovial joints are
lubricated by surface-active phospholipids: A hypothesis. J Arthro­
plasty 18:499-505, 2003.
 3 Cartilage
KEY POINTS
Articular cartilage matrix is heterogeneous and contains
numerous extracellular matrix (ECM) proteins, of which the
large aggregating proteoglycan aggrecan and collagen types
II, IX, and XI are the major constituents.
The collagen network of cartilage confers tensile strength,
and aggrecan provides resistance to compression.
Adult articular chondrocytes are nonmitotic cells that survive
at low oxygen tension in the absence of a vascular supply.
In response to trauma or inflammation, the metabolic activity
of the chondrocyte is increased in response to catabolic and
anabolic factors that regulate remodeling of the ECM.
Under physiologic conditions, the chondrocyte maintains
low-turnover repair of proteoglycans, but the repair capacity,
responses to anabolic factors, cell survival, and quality of the
matrix decline with age.
Hyaline cartilage, including the articular cartilage of diar-
throdial joints, consists of a single cellular component, the
chondrocyte, which is embedded in a unique and complex
matrix. Adult articular chondrocytes are considered to be
fully differentiated cells that maintain matrix constituents
in a low-turnover state of equilibrium. Chondrocytes serve
diverse functions during development and postnatal life. In
the embryo, the chondrocyte arises from mesenchymal pro-
genitors from diverse sources, including the cranial neural
crest of the neural ectoderm, cephalic mesoderm, sclero-
tome of the paraxial mesoderm, and somatopleure of the
lateral plate mesoderm. The chondrocyte synthesizes the
templates, or cartilage anlagen, for the developing limbs in
a process termed chondrogenesis.
After mesenchymal condensation and chondroprogeni-
tor cell differentiation, the chondrocytes undergo prolifera-
tion, terminal differentiation to chondrocyte hypertrophy,
and apoptosis in a process termed endochondral ossification,
whereby the hypertrophic cartilage is replaced by bone. A
similar sequence of events occurs in the postnatal growth
plate and leads to rapid growth of the skeleton. The pro-
cesses that control the different stages of skeletal develop-
ment are described in Chapter 1.
In adults, the anatomic distribution of cartilage is
restricted primarily to the joints, trachea, and nasal sep-
tum, where the major function is structural support. In
joints, cartilage has the additional function of providing
low-friction articulation. Adult articular cartilage comprises
a specialized matrix of collagens, proteoglycans, and other
cartilage-­specific and nonspecific proteins. Adult articular
chondrocytes, remnants of the resting, or reserve, chon-
drocytes that laid down the original cartilage matrix during
and Chondrocytes
Mary B. Goldring
chondrogenesis, are inactive metabolically, owing partially
to the absence of a vascular supply and innervation in the
tissue.1 The clinical importance of the adult chondrocyte
resides in its capacity to respond to mechanical stimuli,
growth factors, and cytokines that may influence normal
homeostasis in a positive or negative manner. In rheumatoid
arthritis (RA), cartilage destruction occurs primarily in areas
contiguous with the proliferating synovial pannus, although
there is evidence that the chondrocyte can respond to the
inflammatory milieu and participate in degrading its own
matrix.2 In osteoarthritis, the chondrocyte plays a key role
by reacting to structural changes in the surrounding carti-
lage matrix through the production of catabolic cytokines
and anabolic factors, which act in an autocrine-paracrine
manner.3,4 Nevertheless, the chondrocyte has limited capac-
ity, which declines with age, to regenerate the normal car-
tilage architecture with the zonal variations in the matrix
network that was formed originally. This chapter focuses on
the structure and function of normal articular cartilage and
the role of the chondrocyte in maintaining cartilage homeo-
stasis and responding to adverse environmental insults that
may modify cartilage integrity.
CARTILAGE STRUCTURE
Normal articular cartilage is a specialized tissue character-
ized macroscopically by its milky, shelled-almond (hyaline)
appearance. It is an avascular tissue nourished by diffusion
from the vasculature of the subchondral bone and from the
synovial fluid. Articular cartilage is more than 70% water,
and it is hypocellular compared with other tissues; chondro-
cytes constitute only 1% to 2% of its total volume.5-7 Most of
the dry weight of cartilage consists of two components: type
II collagen and the large aggregating proteoglycan, aggrecan.
Several “minor” collagens and small proteoglycans also seem
to play a role in cartilage-matrix organization, however.8,9
Organic constituents represent only about 20% of the wet
weight. Collagen, primarily type II, accounts for approxi-
mately 15% to 25% of the wet weight and about half of the
dry weight except in the superficial zone, where it represents
most of the dry weight. Proteoglycans, primarily aggrecan,
account for 10% of the wet weight and about 25% of the dry
weight. The highly cross-linked type II collagen–containing
fibrils form a systematically oriented network that traps the
highly negatively charged proteoglycan aggregates.10 Histo-
chemical analysis of cartilage shows that proteoglycans can
be stained reliably with safranin O, toluidine blue, or alcian
blue, although at low substrate concentrations, these meth-
ods are not stoichiometric.11 Collagen also can be stained
efficiently, but differentiation of collagen types requires imm­
unostaining with specific antibodies.
37
 38 Goldring  |  Cartilage and Chondrocytes

Articular Superficial cell

surface protein (also
known as lubricin)
Superficial zone Decorin and biglycan

Pericellular region
(decorin, type VI collagen)

Middle zone

Territorial region
(more intact aggrecan)

Deep zone Interterritorial region

Aggrecan most (degraded aggrecan)
concentrated and
collagen content at
its lowest here

Tidemark
Type X collagen
Calcified zone

Hypertrophic chondrocyte

Subchondral bone

Subchondral bone marrow

Figure 3-1  The structure of human adult articular cartilage, showing the zones of cellular distribution and the pericellular, territorial, and
interterritorial regions of matrix organization. Insets show the relative diameters and orientations of collagen fibrils in the different zones. The
positions of the tidemark and subchondral bone and other special features of matrix composition also are noted. (From Poole AR, Kojima T, Yasuda
T, et al: Composition and structure of articular cartilage: A template for tissue repair. Clin Orthop 391S:S26-S33, 2001. Copyright Lippincott Williams
& Wilkins.)

Despite its thinness (≤7 mm) and apparent homogene-
ity, mature articular cartilage is a heterogeneous tissue with
four distinct regions: (1) the superficial tangential (or glid-
ing) zone, (2) the middle (or transitional) zone, (3) the deep
(or radial) zone, and (4) the calcified cartilage zone, which
is located immediately below the tidemark and above the
subchondral bone (Fig. 3-1).7,10,12,13 In the superficial zone,
the chondrocytes are flattened, and the matrix comprises
thin collagen fibrils in tangential array, associated with a
high concentration of the small proteoglycan decorin and
a low concentration of aggrecan. The middle zone, compos-
ing 40% to 60% of the cartilage weight, consists of rounded
chondrocytes surrounded by radial bundles of thick colla-
gen fibrils. In the deep zone, the chondrocytes frequently
are grouped in columns or clusters (Fig. 3-2). In this region,
the collagen bundles are the thickest and are arranged in a
radial fashion.
The cell density progressively decreases from the surface
to the deep zone, where it is one half to one third of the den-
sity in the superficial zone14; the chondrocytes in the deep
and middle zones have a cell volume that is twice that of the
superficial chondrocytes.15 Water is 75% to 80% of the wet
weight in the superficial zone and progressively decreases
to 65% to 70% with increasing depth. Greater amounts of
collagen relative to proteoglycans are present in the super-
ficial zone, compared with the middle and deep zones, and
type I collagen may be synthesized in addition to type II
collagen.16,17 With increasing depth, the proportion of pro-
teoglycan increases to 50% of the dry weight in the deep
zone.15,18-20 The calcified zone is formed as a result of endo-
chondral ossification and persists after growth plate closure
as the tidemark.21 The calcified zone serves as an important
mechanical buffer between the uncalcified articular carti-
lage and the subchondral bone.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE 39

S in cartilage abnormalities.27-32 Deficiencies or disruptions in

genes that encode the cartilage-specific collagens result, in
some cases, in premature osteoarthritis.32 Knowledge of
T the composition of the cartilage matrix has permitted the
development of methods for identifying molecular mark-
ers in serum and synovial fluid that can be used to monitor
changes in cartilage metabolism and to assess cartilage dam-
age in osteoarthritis or RA.33-36 Changes in the structural
U composition of cartilage can markedly affect its biomechan-
ical properties (see Chapter 6).

L ular cartilage is the triple-helical type II collagen molecule,
M residues that can form cross-links and the presence of other
B IIB collagen in articular cartilage is a product of alternative
1 splicing and lacks a 69-amino acid, cysteine-rich domain of
Figure 3-2  Light micrograph of vertically sectioned adult human car-
tilage (femoral condyle), illustrating its subdivision into superficial (S),
transitional (T), upper radial (U), lower radial (L), and calcified cartilage
(M) zones; the last-mentioned abuts on the subchondral bone plate
(B). Saw-cut, 100-μm thick, surface-stained with basic fuchsin, McNeil’s
tetrachrome, and toluidine blue O. (From Hunziker EB: Articular cartilage
structure in humans and experimental animals. In Kuettner KE, Schleyerbach
R, Peyron JG, et al [eds]: Articular Cartilage and Osteoarthritis. New York,
Raven, 1992, pp 183-199.)
The physical properties of articular cartilage are deter-
mined by the unique fibrillar collagen network, which
provides tensile strength, interspersed with proteoglycan
aggregates that bestow compressive resilience.22-24 The pro-
teoglycans are ass­ociated with large quantities of water bound
to the hydrophilic glycosaminoglycan chains. This carti-
laginous extracellular matrix (ECM), with its tightly bound
water, provides a high degree of resistance to deformation by
compressive forces. The capacity to resist compressive forces
is associated with the ability to extrude water as the cartilage
compresses. When the compression is released, the proteo-
glycans (now depleted of balancing counter ions that were
removed with the water) contain sufficient fixed charge to
reabsorb osmotically the water and small solutes into the
matrix, which then rebounds to its original dimensions.25,26
STRUCTURE-FUNCTION RELATIONSHIPS
OF CARTILAGE MATRIX COMPONENTS
The ECM components synthesized by chondrocytes include
highly cross-linked fibrils of triple-helical type II collagen
molecules that interact with other collagens, aggrecan,
small proteoglycans, and other cartilage-specific and non-
specific matrix proteins (Table 3-1).6,8,9,12 The importance
of these structural proteins may be observed in heritable dis-
orders, such as chondrodysplasias, or in transgenic animals
in which mutations or deficiencies in cartilage genes result
CARTILAGE COLLAGENS
The major component of the collagen network in adult artic-
which is composed of three identical α chains, [α1(II)]3.
These molecules are assembled in fibrils in a quarter-­stagger
array that can be observed by electron microscopy.8,37,38
These fibrils are thinner than the type I collagen–containing
fibrils in skin because of the higher numbers of hydroxylysine
collagen and noncollagen components in the fibril. The type
the amino-terminal propeptide, which is encoded by exon
2 in the human type II collagen gene (COL2A1).39 This
domain is found in type IIA procollagen, which is expressed
by chondroprogenitor cells during development, and in the
amino propeptides of other interstitial collagen types, and
may play a feedback-inhibitory role in collagen biosynthe-
sis. The reappearance of type IIA collagen in the midzone
pericellular matrix and type X collagen, the hypertrophic
chondrocyte marker, in the deep zone of osteoarthritis carti-
lage suggests reversion to a developmental phenotype as an
attempt to repair the damaged matrix.40,41
Although collagen types VI, IX, XI, XII, and XIV are
quantitatively minor components, they may have impor-
tant structural and functional properties. Types IX and XI
are specific to cartilage, whereas types VI, XII, and XIV are
widely distributed in other connective tissues.8 Type VI col-
lagen, which is present in cartilage as microfibrils in very
small quantities in the pericellular matrix, may play a role
in cell attachment and interacts with other matrix proteins,
such as hyaluronan, decorin, and biglycan. There are small
amounts of type III collagen in cartilage, and type III and
type VI collagens may increase in osteoarthritis cartilage.41
Type IX collagen is a proteoglycan and a collagen because
it contains a chondroitin sulfate chain attachment site in
one of the noncollagen domains. The helical domains of the
type IX collagen molecule form covalent cross-links with
type II collagen telopeptides and are attached to the fibril-
lar surface, as observed by the electron microscope. Type IX
collagen may function as a structural intermediate between
type II collagen fibers and the proteoglycan aggregates, serv-
ing to enhance the mechanical stability of the fibril network
and resist the swelling pressure of the trapped proteoglycans.
Destruction of type IX collagen accelerates cartilage degra-
dation and loss of function.8,41
The α3 chain of type XI collagen has the same primary
sequence as the α1(II) chain, and the heterotrimeric type XI
collagen molecule is buried in the same fibril as type II col-
lagen. Type XI collagen may have a role in regulating fibril
 40 Goldring  |  Cartilage and Chondrocytes

Table 3-1  Extracellular Matrix Components of Cartilage

Molecule Structure and Size Function and Location
Collagens
Type II [α1(II)]3; fibril-forming Tensile strength; major component of collagen fibrils
Type IX [α1(IX)α2(IX)α3(IX)]; single CS or DS chain;   Tensile properties, interfibrillar connections; cross-links to
α1(II) gene encodes α3(IX); FACIT surface of collagen fibril, NC4 domain projects into matrix
Type XI [α1(XI)α2(XI)α3(XI)]; fibril-forming Nucleation/control of fibril formation; within collagen fibril
Type VI [α1(VI)α2(VI)α3(VI)]; microfibrils Forms microfibrillar network, binds hyaluronan, biglycan,
­decorin; pericellular
Type X [α1(X)]3; hexagonal network Support for endochondral ossification; hypertrophic zone and
­calcified cartilage
Type XII [α1(XII)]3; FACIT large cruciform NC3 domain Associated with collagen I fibrils in perichondrium and articular
surface
Type XIV [α1(XIV)]3; FACIT Associated with type I collagen; superficial zone
Type XVI [α1(XVI)]3; FACIT Integrates with collagen II/XI fibrils
Type XXVII Col27a1 gene: 156 kb, 61 exons Fibril-forming; developing cartilage
Proteoglycans
Aggrecan 255-kD core protein; CS/KS side chains;   Compressive stiffness through hydration of fixed charge density;
C-terminal EGF and lectin-like domains ­binding through G1 domain to HA stabilized by link protein
Versican 265-370 kD core protein; CS/DS side chains;   Low levels in articular cartilage throughout life; calcium-binding
C-terminal EGF, C-type lectin, and CRP-like and selectin-like properties
­domains
Perlecan 400-467 kD core protein; HS/CS side chains; no   Cell-matrix adhesion; pericellular
HA-binding
Biglycan 38 kD; LRR core protein with two DS chains (76 kD) Binds collagen VI and TGF-β; pericellular
Decorin 36.5 kD; LRR core protein with one CS or   Controls size/shape of collagen fibrils, binds collagen II and  
DS side chain (100 kD) TGF-β; interterritorial
Asporin 40 kD; LRR core protein; N-terminal extension Binds collagen, modulates TGF-β function
of 15 aspartate residues
Fibromodulin 42 kD; containing KS chains in central LRR region Same as decorin
and N-terminal tyrosine sulfate domains
Lumican 38 kD; structure similar to fibromodulin Same as decorin
PRELP 44 kD; LRR core protein; proline-rich and   Mediates cell binding through HS sulfate in syndecan
arginine-rich N-terminal binding domain  
for heparin and HS
Chondroadherin 45 kD; LRR core protein without N-terminal   Binding to cells via α2β1 integrin
extension
Other Molecules
Hyaluronic acid   1000-3000 kD Retention of aggrecan within matrix
(HA; hyaluronan)
Link protein 38.6 kD Stabilizes attachment of aggrecan G1 domain to HA
Cartilage oligomeric 550 kD; five 110-kD subunits;   Interterritorial in articular cartilage; stabilizes collagen network
­matrix protein (COMP) thrombospondin-like or promotes collagen fibril assembly; calcium binding
Cartilage matrix ­protein Three 50-kD subunits with vWF and EGF domains Tightly bound to aggrecan in immature cartilage
(CMP, or matrilin-1);
­matrilin-3
Cartilage ­intermediate 92 kD; homology with nucleotide Restricted to middle/deep zones of cartilage; increase in early
layer protein (CILP) ­pyrophosphohydrolase without active site and late osteoarthritis
Glycoprotein (gp)-39,   39 kD; chitinase homology Marker of cartilage turnover; chondrocyte proliferation;
YKL-40, or chitinase   ­superficial zone of cartilage
3-like protein 1 (CH3L1)
Fibronectin Dimer of 220-kD subunits Cell attachment and binding to collagen and proteoglycans;
increased in osteoarthritis cartilage
Tenascin-C Six 200-kD subunits forming hexabrachion   Binds syndecan-3 during chondrogenesis; angiogenesis
structure
Superficial zone ­protein 225 kD, 200 nm length Joint lubrication; superficial zone only
(SZP), ­lubricin, or
­proteoglycan (PRG) 4
Membrane Proteins
CD44 Integral membrane protein with extracellular   Cell-matrix interactions; binds HA
HS/CS side chains
Syndecan-3 N-terminal HS attachment site; cytoplasmic Receptor for tenascin-C during cartilage development; cell-matrix
tyrosine residues interactions
Anchorin CII   34 kD; homology to calcium-binding proteins   Cell surface attachment to type II collagen; calcium binding
(or annexin V) calpactin and lipocortin
Integrins (α1, α2, α3, α5, Two noncovalently linked transmembrane   Cell-matrix binding: α1β1/collagen 1 or VI, α2β1 or α3β1/collagen II,
α6, α10; β1, β3, β5) glycoproteins (α and β subunits) α5β1/fibronectin; intracellular signaling
Discoidin domain   Receptor tyrosine kinase Binds native type II collagen fibrils; Ras/ERK signaling
receptor 2
CRP, complement regulatory protein; CS, chondroitin sulfate; DS, dermatan sulfate; EGF, epidermal growth factor; FACIT, fibril-associated collagens with interrupted
triple helices; HA, hyaluronic acid; HS, heparan sulfate; KS, keratan sulfate; LRR, leucine-rich repeat; NC, noncollagen; PRELP, proline-rich and arginine-rich end
leucine-rich repeat protein; TGF, transforming growth factor; vWF, von Willebrand factor.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
diameter. The more recently discovered nonfibrillar fibril-
associated collagens with interrupted triple helices, XII and
XIV, which are structurally related to type IX collagen, do
not form fibrils by themselves, but coaggregate with fibril-
forming collagens and modulate the packing of collagen
fibers by domains projecting from their surfaces.8,37,38
CARTILAGE PROTEOGLYCANS
The major proteoglycan in articular cartilage is the large
aggregating proteoglycan, or aggrecan, which consists of a
core protein of 225 to 250 kD with covalently attached side
chains of glycosaminoglycans, including approximately 100
chondroitin sulfate chains, 30 keratan sulfate chains, and
shorter N-linked and O-linked oligosaccharides.6,9,42,43 Link
protein, a small glycoprotein, stabilizes the noncovalent
linkage between aggrecan and hyaluronic acid (also called
hyaluronan) to form the proteoglycan aggregate that may
contain 100 aggrecan monomers. The G1 and G2 N-terminal
globular domains of aggrecan and its C-terminal G3 domain
have distinct structural properties that function as integral
parts of the aggrecan core protein and as cleavage prod-
ucts that accumulate with age or in osteoarthritis. The
G2 domain is separated from G1 by a linear interglobular
domain and has two proteoglycan tandem repeats, but it
does not bind to hyaluronic acid. The G3 domain contains
sequence homologies to epidermal growth factor, lectin, and
complement regulatory protein, and it participates in growth
regulation; cell recognition; intracellular trafficking; and the
recognition, assembly, and stabilization of the ECM. About
half of the aggrecan molecules in adult cartilage lacks the
G3 domain, probably as a result of proteolytic cleavage dur-
ing matrix turnover. Small amounts of other large proteo-
glycans are found in cartilage, including versican, which
forms aggregates with hyaluronic acid, and perlecan, which
is nonaggregating; however, these proteoglycans function
primarily during skeletal development, where versican is
expressed in prechondrogenic condensations, and perlecan is
expressed in the cartilage anlagen after expression of type II
collagen and aggrecan.43
The nonaggregating small proteoglycans are not spe-
cific to cartilage, but in cartilage they may serve impor-
tant roles in matrix structure and function, primarily by
modulating collagen-fibril formation.44-46 Of the more
than 10 leucine-rich repeat (LRR) proteoglycans discov-
ered so far, only osteoadherin is not present in cartilage.
The 24-amino acid central LRR domain is conserved, but
the N-terminal and C-terminal domains have patterns of
cysteine residues involved in intrachain disulfide bonds
that distinguish the four subfamilies: (1) biglycan, deco-
rin, fibromodulin, and lumican; (2) keratocan and pro-
line and arginine-rich end leucine-rich repeat protein
(PRELP); (3) chondroadherin; and (4) epiphycan/PG-Lb
and mimecan/osteoglycin. Biglycan has two glycosami-
noglycan chains, either chondroitin sulfate or dermatan
sulfate, or both, attached near the N-terminus through
two closely spaced serine-glycine dipeptides. Decorin
contains only one chondroitin sulfate or dermatan sul-
fate chain. Fibromodulin and lumican contain keratan
sulfate chains linked to the central domain of the core
protein and several sulfated tyrosine residues in the
N-terminus. The negatively charged glycosaminoglycan
41
side chains contribute to fixed charge density of the matrix
and, together with the highly anionic tyrosine-sulfation
sites, permit multiple-site linkage between adjacent col-
lagen fibrils, stabilizing the network. Decorin, the most
extensively studied LRR proteoglycan, binds to several
collagens present in cartilage, including types II, VI, XII,
and XIV, and to fibronectin and thrombospondin. Bigly-
can, decorin, and fibromodulin bind transforming growth
factor (TGF)-β and the epidermal growth factor receptor
and may modulate growth, remodeling, and repair. PRELP
and chondroadherin may regulate cell-matrix interactions
through binding to syndecan and α2β1 integrin.
OTHER EXTRACELLULAR MATRIX AND CELL
SURFACE PROTEINS
Several other noncollagenous matrix proteins may play impor-
tant roles in determining cartilage matrix integrity. Cartilage
oligomeric protein (COMP), a member of the thrombospon-
din family, is a disulfide-bonded, pentameric, 550-kD, cal-
cium-binding protein that constitutes approximately 10%
of the noncollagenous, nonproteoglycan protein in nor-
mal adult cartilage. COMP is located in the interterrito-
rial matrix of adult articular cartilage where it interacts
with the col3 and NC4 domains of type IX collagen that
protrude from the fibril, stabilizing the collagen network.
COMP is pericellular in the proliferating region of the
growth plate, where it may have a role in cell-matrix inter-
actions.47 The cartilage matrix protein (or matrilin-1) and
matrilin-3 are expressed in cartilage at certain stages of
development and are present in tracheal cartilage, but not
in mechanically loaded adult articular or intervertebral
disk cartilage.48,49
Tenascin-C, a glycoprotein that is regulated in develop-
ment, is characteristic of nonossifying cartilage.50 Similar
to fibronectin, alternative splicing of tenascin-C mRNA
gives rise to different protein products at different stages
of chondrocyte differentiation. Both proteins are increased
in osteoarthritis cartilage and may serve specific functions
in remodeling and repair. A splice variant of tenascin-C
mRNA is found in chondrosarcomas.51 The cartilage inter-
mediate-layer protein is expressed by chondrocytes in the
middle to deep zones of articular cartilage as a precursor pro-
tein. When cleaved during secretion, cartilage intermediate-
layer protein has structural similarities with nucleotide
pyrophosphohydrolase, although it lacks the catalytic site,
and it may play a role in pyrophosphate metabolism and
calcification.52,53 Asporin is related to decorin and ­biglycan
and, similar to those other LRR proteins, may interact with
and sequester growth factors such as TGF-β.54-56 YKL-40/
HC-gp39, also known as chitinase 3-like protein 1, is found
only in the superficial zone of normal cartilage and stimu-
lates proliferation of chondrocytes and synovial cells.57
Chitinase 3-like protein 1 is induced by inflammatory cyto-
kines and may function as a feedback regulator because it
inhibits cytokine-induced cellular responses.58,59 Synthesis
or release of these proteins or fragments is often increased
in cartilage that is undergoing repair or remodeling, and
they have been investigated as markers of cartilage damage
in arthritis.33,34 A related member of the chitinase family,
YKL-39, may be a more specific serum marker as a cartilage-
derived ­autoantigen.60,61
 42 Goldring  |  Cartilage and Chondrocytes
MORPHOLOGY, CLASSIFICATION, AND
NORMAL FUNCTION OF CHONDROCYTES
MORPHOLOGY
The characteristic feature of the chondrocyte embedded
in cartilage matrix is its rounded or polygonal morphol-
ogy. The exception occurs at tissue boundaries, such as the
articular surface of joints, where chondrocytes may be flat-
tened or discoid. Intracellular features, including a rough
endoplasmic reticulum, a juxtanuclear Golgi apparatus, and
deposition of glycogen, are characteristic of a synthetically
active cell. Stockwell and Meachim62 calculated that the
cell density of full-thickness, human, adult, femoral condyle
cartilage is maintained at 14.5 (± 3.0) × 103 cells/mm2 from
age 20 to 30 years. Because senescence of chondrocytes is
known to occur with aging, it is logical to suppose that dead
chondrocytes are replaced by mitosis. Mitotic figures are not
observed, however, in normal adult articular cartilage.
The morphology, density, and synthetic activity of an
adult chondrocyte vary according to its position within
the different zones of articular cartilage.63-65 In the region
of highest cell density, the superficial zone, the cells are
flattened and oriented parallel to the surface along with
the collagen fibers. Chondrocytes within the middle zone
appear larger and more rounded and display a random distri-
bution within the matrix, where the collagen fibers also are
more randomly arranged. The chondrocytes in the deeper
zones form columns that, along with the collagen fibers, are
oriented perpendicular to the cartilage surface. The chon-
drocytes may exhibit different behaviors depending on their
position within the different layers, and these zonal differ-
ences in synthetic properties may persist in primary chon-
drocyte cultures.63,64 Chondrocyte volume in situ increases
from the superficial through the deep zones and with the
degree of cartilage degeneration.7 A study using confo-
cal scanning laser microscopy of live, unfixed cartilage has
revealed fine cytoplasmic processes extending from the cell
bodies of 40% of chondrocytes.66 These processes are pro-
posed to permit interactions among the chondrocytes and
the cartilage matrix at near and remote sites. They are dis-
tinct from the cilia, which are observed by electron micros-
copy,67 but not by confocal scanning laser microscopy.66
CLASSIFICATION: CELL ORIGIN
AND DIFFERENTIATION
The chondrocyte arises in the embryo from mesenchymal ori-
gin during chondrogenesis, which is the earliest phase of skel-
etal development involving mesenchymal cell recruitment,
migration, and condensation and differentiation of the mes-
enchymal chondroprogenitor cells.68 As described in detail
in Chapter 1, chondrogenesis results in the ­formation of car-
tilage anlagen, or templates, at sites where skeletal elements
form. This process is controlled by cell-cell and cell-matrix
interactions and by growth and differentiation factors that
initiate or suppress cellular signaling ­pathways and transcrip-
tion of specific genes in a temporospatial ­manner.
Vertebrate limb development is controlled by interact-
ing patterning systems involving fibroblast growth factor
(FGF), hedgehog, bone morphogenetic protein (BMP), and
Wnt pathways. Wnt signaling, via the canonical β-catenin
pathway and activation of TCF/Lef transcription factors,
functions in a cell-autonomous manner to induce osteoblast
differentiation and suppress chondrocyte differentiation in
early chondroprogenitors.69 During chondrogenesis, Wnt/
β-catenin acts at two stages, at low levels to promote chondro-
progenitor differentiation and later at high levels to promote
chondrocyte hypertrophic differentiation and subsequent
endochondral ossification.70,71 The transcription factor, Sry-
type high mobility group box 9 (Sox9) is an early marker of
the differentiating chondrocyte that is required for the onset
of expression of type II collagen, aggrecan, and other carti-
lage-specific matrix proteins, such as type IX ­collagen.72 Two
other members of the SOX family, L-Sox5 and Sox6, are not
present in early mesenchymal condensations, but are required
during overt chondrocyte differentiation, forming heterodi-
mers that induced transcription more efficiently than Sox9
by itself.72 The expression of SOX proteins depends on BMP
signaling via SMADs, which are functionally redundant and
active in differentiating chondrocytes.73 A long form of c-Maf
interacts with Sox9 at early stages to upregulate COL2A1
gene expression,74 whereas C/EBPβ and C/EBPγ and AP-2α
may inhibit chondrocyte differentiation by blocking tran-
scription of COL2A1, aggrecan, and other cartilage-specific
genes by direct or indirect mechanisms.75,76
In the embryonic or postnatal epiphyseal growth plates,
the upregulation of molecules that promote matrix remodel-
ing and angiogenesis facilitates endochondral ossification,
whereby bone replaces the calcified cartilaginous matrix in
the hypertrophic zone (see Chapter 1). The differentiated
chondrocytes that remain in the reserve, or resting, zone
become the cartilage elements in articular joints, or they
can proliferate and undergo the complex process of terminal
differentiation to hypertrophy marked by type X collagen.
Indian hedgehog and parathyroid hormone–related protein
transiently induce proliferation and repress differentiation,
determining the number of cells that enter the hypertrophic
maturation pathway.77 The runt-domain transcription factor,
Runx2 (also known as core binding factor or Cbfa1), serves
as a positive regulatory factor in chondrocyte maturation to
the hypertrophic phenotype and subsequent osteogenesis.78
Runx2 is expressed in the adjacent perichondrium and in
prehypertrophic chondrocytes, but less in late hypertrophic
chondrocytes, and is required for the expression of type X
collagen and other markers of terminal differentiation.79
Numerous other transcription factors positively or nega-
tively regulate chondrocyte terminal differentiation by reg-
ulating the expression or activity of Runx2.80 BMP-induced
Smad 1 and interactions between Smad 1 and Runx2 are
required for the induction of chondrocyte hypertrophy.
Because there is no SMAD site on the Runx2 promoter, it
has been proposed that homeodomain proteins such as Dlx3
may activate Runx2 signaling in response to BMP-2 during
endochondral ossification, whereas Dlx5, Dlx6, and Msx2
are known to inhibit Runx2-mediated activation of genes
such as osteocalcin at later stages.81 The homeodomain pro-
tein Nkx3.2, which is an early BMP-induced signal required
at the onset of chondrogenesis, is a direct transcriptional
repressor of Runx2 promoter activity.82 The bHLH factor
Twist transiently inhibits Runx2 function and prevents pre-
mature osteoblast differentiation,83 whereas cooperation of
the Groucho homologue Grg5 or the leucine zipper protein
ATF4 with Runx2 promotes chondrocyte maturation.84
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
­ is­tone deacetylase 4 (HDAC4), which is expressed later
H tissue growth factor.98,99 In the growth plate, hypoxia and
in prehypertrophic chondrocytes, prevents premature chon-
drocyte hypertrophy by interacting with Runx2 and inhib-
iting its activity.85 The hypoxia-inducible factor (HIF)-1α
is required for chondrocyte survival during hypertrophic
differentiation, owing partially to its regulation of vascular
endothelial cell growth factor (VEGF) expression.86
The leucine zipper proteins Fra2, ATF2, and c-Maf are
required for gene expression during late-stage hypertro-
phy.87 One major function of the chondrocyte is growth of
the skeleton through increases in cell proliferation, produc-
tion of ECM, and cell volume through hypertrophy. After
cessation of growth, the resting chondrocyte remains as part
of the supporting structures in articular, tracheal, and nasal
cartilages, indicating that the fate of a chondrocyte depends
on origin and location (Fig. 3-3).
NORMAL FUNCTION OF THE ADULT ARTICULAR
CHONDROCYTE
The mature articular chondrocyte embedded in its ECM is
a resting cell with no detectable mitotic activity and a low
rate of synthetic activity. Because articular cartilage is not
vascularized, the chondrocyte must rely on diffusion from
the articular surface or subchondral bone for the exchange
of nutrients and metabolites. Chondrocytes maintain active
membrane transport systems for exchange of cations, includ-
ing Na+, K+, Ca2+, and H+, whose intracellular concentra-
tions fluctuate with load and changes in the composition
of the cartilage matrix.88 The chondrocyte cytoskeleton is
composed of actin, tubulin, and vimentin filaments, and the
composition of these filament systems varies in the different
cartilage zones.89,90
Chondrocyte metabolism operates at low oxygen tension
within the cartilage matrix, ranging from 10% at the sur-
face to less than 1% in the deep zone. The consumption of
oxygen by cartilage on a per-cell basis is only 2% to 5% of
that in liver or kidney, although the amounts of lactate pro-
duced are comparable. Chondrocytes do not normally con-
tain abundant mitochondria, energy metabolism depends
strongly on the glucose supply, and the energy requirements
may be modulated by mechanical stress.91 Glucose serves
as the major energy source for the chondrocytes and as an
essential precursor for glycosaminoglycan synthesis.92 Facili-
tated glucose transport in chondrocytes is mediated by sev-
eral distinct glucose transporter proteins (GLUTs) that are
either constitutively expressed (GLUT3 and GLUT8) or
cytokine-inducible (GLUT1 and GLUT6).93,94 A proteomic
study of chondrocytes identified 93 different intracellular
proteins known to be involved in cell organization (26%),
energy (16%), protein fate (14%), metabolism (12%), and
cell stress (12%).95 The relative expression of these pro-
teins may determine the capacity of chondrocytes to survive
in cartilage matrix and to modulate metabolic activity in
response to the environmental changes.
When cultured in a range of oxygen tensions between severe
hypoxia (0.1% oxygen) and normoxia (21% oxygen), chon-
drocytes adapt to low oxygen tensions by upregulating HIF-
1α. Hypoxia via HIF-1α can stimulate chondrocytes to express
GLUTs94 and angiogenic factors such as VEGF96,97 and numer-
ous genes associated with cartilage anabolism and chondro-
cyte differentiation, including Sox9, TGF-β, and connective
43
HIF-1α are associated with type II collagen production.100
HIF-1α is expressed in normal and osteoarthritis articular
cartilage, where it maintains tonic activity during physiologic
hypoxia in the deeper layers associated with increased pro-
teoglycan synthesis.101 It is not completely degraded, how-
ever, as it is in other tissues, when normoxic conditions are
applied.102 Long-term systemic hypoxia (13%) may downreg-
ulate collagen and aggrecan gene expression in articular car-
tilage,103 whereas hyperoxia (55% oxygen) may increase the
breakdown of cartilage collagens in articular cartilage in the
presence of vascularized rheumatoid synovium.104 By modu-
lating the intracellular expression of survival factors such as
HIF-1α, chondrocytes have a high capacity to survive in the
avascular cartilage matrix and to respond to environmental
changes. Findings that catabolic stress and inflammatory
cytokines upregulate HIF-1α also suggest that it may serve as
a survival factor in osteoarthritis cartilage.101,105,106
CHONDROCYTE SYNTHETIC FUNCTION
The chondrocyte maintains a steady-state metabolism
secondary to equilibrium between anabolic processes and
catabolic processes that results in the normal turnover of
matrix molecules. In normal adult articular cartilage, the
turnover of matrix components is low. The turnover of col-
lagen has been estimated to occur with a half-life of more
than 100 years.107,108 In contrast, the glycosaminoglycan
constituents on the aggrecan core protein are more readily
replaced; the half-life of aggrecan subfractions has been esti-
mated to range from 3 to 24 years.109 Other cartilage ECM
components, including biglycan, decorin, COMP, tenascins,
and matrilins, incorporated previously into the matrix dur-
ing ­development, also may be synthesized by chondrocytes
under low-turnover conditions. There are regional differ-
ences in the remodeling activities of chondrocytes, however,
and matrix turnover may be more rapid in the immediate
pericellular zones.110 The metabolic potential of these cells
also is indicated by their capacity to proliferate in culture
and synthesize matrix proteins after enzymatic release from
the cartilage of even elderly individuals.
The complex composition of the articular cartilage matrix
is more difficult for the chondrocyte to replicate if severe dam-
age to the collagen network occurs. During the initial stages
of osteoarthritis, chondrocytes in vivo respond to structural
changes in the surrounding cartilage matrix by increasing cell
proliferation and synthesis of matrix proteins, proteinases,
and anabolic and catabolic factors. The aberrant behavior
of osteoarthritis chondrocytes is reflected in the appearance
of fibrillations; matrix depletion; cell clusters; and changes
in quantity, distribution, or composition of matrix pro-
teins.111,112 Evidence of phenotypic modulation is reflected
in increased type I and III collagens113 and the appearance of
the hypertrophic chondrocyte marker, type X collagen, and
other chondrocyte differentiation genes, suggesting a recapit-
ulation of a developmental program.40,114 There is evidence,
however, of compensatory increases in type II collagen syn-
thesis in deeper regions of the articular cartilage.112
Genomic and proteomic analyses of global gene expres-
sion in cartilage have confirmed the increased levels of type
II collagen (COL2A1) mRNA levels in early ­osteoarthritis
cartilage.115-117 The increased levels of anabolic factors,
 44 Goldring  |  Cartilage and Chondrocytes

Mesenchymal Type I collagen

precursor cell Hyaluronan
TGF-β
FGF-2,4,8,10
Wnt-3A,7A
Condensation
Sonic Hh
BMP-2,4,7
Collagen IIA
Chondroprogenitor Tenascin
Chondrogenesis Vimentin
BMP-2,4,7,14
IGF-1
Chondrocyte FGF-2/FGFR2
proliferation
and differentiation
Collagen II, IX, XI
Proliferating Aggrecan
chondrocyte S-100

FGF-18/FGFR3
BMP-2,7 Resting BMP-2,7,13
TGF-β IGF-1
chondrocyte
PTHrP
Indian Hh
VEGF
Differentiated
Hypertrophy chondrocyte

Hypertrophic
chondrocyte Retinoic acid Collagen II, IX, XI
In vitro
Serum/FGF-2 Aggrecan
IL-1 S-100
Collagen X
Osteocalcin Collagen I, III
Alkaline
phosphate
MMP-13 Dedifferentiated
chondrocyte
Figure 3-3  Schematic representation of cellular phenotypes associated with developmental fates during condensation, chondrogenesis, chondro-
cyte proliferation, differentiation, and hypertrophy. Some of the regulatory factors active at different stages are listed to the left of the arrows. The
major extracellular matrix genes are listed to the right of each cell type in which they are differentially expressed. BMP, bone morphogenetic protein;
FGF, fibroblast growth factor; Hh, hedgehog; IGF, insulin-like growth factor; IL-1, interleukin-1; MMP, matrix metalloproteinase; PTHrP, parathyroid 
hormone–related protein; TGF-β, transforming growth factor-β; VEGF, vascular endothelial growth factor; Wnt, wingless type.

BMP-2 and inhibin βA/activin, members of the TGF-β
superfamily,116,118,119 and prostaglandins120 suggest a pos-
sible mechanism. Nevertheless, Aigner and coworkers117
have shown that expression of the type II collagen gene
(COL2A1) is suppressed in upper zones of osteoarthri-
tis cartilage with progressing matrix destruction, whereas
global COL2A1 gene expression is increased in late-stage
osteoarthritis cartilage compared with normal and early
degenerative cartilage. The capacity of the adult articular
chondrocyte to regenerate the normal cartilage matrix
architecture is limited, and the damage becomes irrevers-
ible, unless the destructive process is interrupted.
CULTURE MODELS FOR STUDYING
CHONDROCYTE METABOLISM
Primary cultures of articular chondrocytes isolated from var-
ious animal and human sources have served as useful mod-
els for studying the mechanisms controlling responses to
growth factors and cytokines.121-124 A challenge to research-
ers in cartilage biology is the maintenance of chondrocyte
morphology and cartilage-specific gene expression during in
vitro studies. In primary monolayer cultures, chondrocytes
maintain a rounded, polygonal morphology (Fig. 3-4), but
there is a progressive loss of cartilage phenotype with passage
of time and after subculture. High-density cultures maintain
the gene expression and synthesis of cartilage-specific matrix
proteins until they are subcultured, although gene expres-
sion of type II collagen is generally more labile than that of
aggrecan. During this loss of phenotype or dedifferentiation,
chondrocytes lose the rounded, polygonal morphology and
express some, but not all, characteristics of the fibroblast
phenotype, such as type I collagen. It is possible to expand
the cultures through a limited number of subcultures and
“redifferentiate” the cells in fluid or gel suspension cultures,
in which the chondrocytes regain morphology, and the ces-
sation of proliferation is associated with increased expres-
sion of cartilage-specific matrix proteins. ­ Alternatively,
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
A and readily respond to serum growth factors that stimulate
B these cells to acquire a fibroblast-like morphology associ-
Figure 3-4  Morphology of human articular chondrocytes grown in
monolayer culture on plastic. Chondrocytes were isolated from articu-
lar cartilage and cultured in growth medium containing 10% fetal calf
serum until confluent. The cultures were changed to serum-free defined
­medium, interleukin-1β (IL-1β) was added the next day, and incubation
was continued for 24 hours. A, Untreated chondrocytes display the char-
acteristic cobblestone morphology. B, IL-1β-treated cultures respond
with a dramatic morphologic change.
explant cultures of articular cartilage in which the chon-
drocytes remain encased within their own ECM have been
used as in vitro models to study cartilage biochemistry and
metabolism, as described in the following section.
ARTICULAR CHONDROCYTES
Cartilage Explant (Organ) Cultures
Based on the pioneering work of Fell,125 who showed that
it was possible to maintain pieces of cartilage in culture,
the explant culture system was developed to characterize
chondrocyte function in cartilage from various species,
including humans, at different ages. The early work in
bovine cartilage established the mechanisms of biosynthe-
sis of cartilage proteoglycans under the influence of dif-
ferent serum concentrations and determined the turnover
rate whereby the chondrocyte could maintain the bal-
ance between anabolic and catabolic pathways.126 Meth-
ods developed for measuring the proteoglycan content in
cartilage and the incorporation of 35S-sulfate into newly
synthesized proteoglycans are used widely as the standard
assays for assessing cartilage metabolism.127 Cartilage
organ cultures also maintain ­constant ­levels of type II col-
lagen during several weeks of ­ culture and the character-
istic morphology and banding pattern of collagen fibers.
These cultures have been useful for studying the regulation
45
of cartilage matrix synthesis and degradation by protein-
ases, inflammatory cytokines, retinoic acid, and anabolic
growth factors.
Monolayer Cultures
Primary monolayer cultures of chondrocytes isolated from
young animals that maintain the cartilage-specific phenotype
at least throughout primary culture are easily obtained and
have been used widely to assess differentiated chondrocyte
functions. When chondrocytes are isolated from their matrix
and cultured in monolayer, they adhere to the culture dish
proliferation of the normally quiescent cells. Freshly isolated
human articular or costal chondrocytes express cartilage-
specific type II collagen and continue to do so for several days
to weeks in primary monolayer culture.128,129 In addition to
cartilage-specific collagens and aggrecan, chondromodulin
and protein S-100 are useful markers expressed in primary
chondrocyte cultures.130-132 The more recent identification
of cell surface markers that determine chondrogenic capac-
ity is expected to enable the enrichment of subpopulations
for further characterization.133
Early attempts to culture chondrocytes from various ani-
mal and human sources were frustrated by the tendency of
ated with the appearance of type I collagen synthesis.4,123
When plated at high density, the cells maintain a polygonal,
although flattened, morphology. At low plating densities,
with prolonged culture, and on expansion in serial subcul-
ture, the cells gradually assume a more elongated, “fibroblast-
like” morphology. Early work suggested that this change in
morphology is associated with a loss of phenotype, whereby
the synthesis of cartilage-specific matrix molecules, such as
type II collagen and aggrecan, decreases or disappears. This
“dedifferentiated phenotype,” which has been described so
far only in vitro, is marked by the appearance of synthesis
of type I and type III collagens, and it can be accelerated by
plating the cells at low densities or by treatment with cyto-
kines such as interleukin (IL)-1 or retinoic acid. A lack of
correlation between cell shape and chondrocyte phenotype
has been reported. The dedifferentiation of chondrocytes in
monolayer culture seems to be associated, however, with the
increased expression of genes involved in cell proliferation,
such as cyclin D1.130 The substrate on which the chondro-
cytes are plated can influence the differentiation capacity of
articular chondrocytes.134
The use of chondrocytes of adult human origin in stud-
ies related to the pathogenesis of joint diseases has been
problematic because the source of the cartilage cannot
be controlled, sufficient numbers of cells are not readily
obtained from random operative procedures, and the phe-
notypic stability of adult human chondrocytes is lost more
quickly on expansion in serial monolayer cultures than in
cells of juvenile human or embryonic or postnatal animal
origin. High-density micromass cultures are useful if suf-
ficient numbers of chondrocytes can be obtained, particu-
larly for studying proteoglycan biosynthesis.135 Serum-free
defined media of varying compositions, but usually includ-
ing insulin, also have been used, frequently in combination
with monolayer and other culture systems mentioned in
the following section.136,137
 46 Goldring  |  Cartilage and Chondrocytes
Three-Dimensional Culture Systems
Early studies showed that phenotype could be maintained if
the isolated chondrocytes are placed in suspension cultures
in spinner flasks or in dishes coated with nonadherent sub-
strates.4,123 Freshly isolated or subcultured chondrocytes also
can be embedded in three-dimensional matrices, such as col-
lagen gels or sponges,138,139 agarose,140,141 or alginate.142,143
In these three-dimensional matrices, chondrocytes have the
normal spherical shape, synthesize and secrete abundant
cell-associated ECM components, and may maintain phe-
notypic stability for several months. Because articular chon-
drocytes are unable to proliferate in fluid or gel suspension
culture, expansion in monolayer culture followed by trans-
fer to alginate or other suspension culture has been used
as a strategy to obtain sufficient numbers of differentiated
chondrocytes for study. After prolonged culture in mono-
layer, however, dedifferentiated chondrocytes may lose irre-
versibly their chondrogenic potential.144 The high-density
pellet culture system, originally developed to study growth
plate hypertrophy, also has been used as a three-dimensional
model because it permits articular chondrocytes to deposit a
well-organized ECM containing type II collagen and aggre-
can.133,145,146 Isolated chondrons containing one or more
chondrocytes within a capsule of pericellular matrix also
have been used for in vitro studies of chondrocyte metabo-
lism within a three-dimensional environment.
PREHYPERTROPHIC AND HYPERTROPHIC
CHONDROCYTES: MODELS OF THE GROWTH
PLATE AND TERMINAL DIFFERENTIATION
Tissues or cells from embryonic or young animals at specific
developmental stages or with different developmental fates
have been used widely to recapitulate in vitro the transi-
tional stages of chondrogenesis, chondrocyte hypertrophy,
and endochondral ossification.4,123 A common feature of
these models is the requirement for deposition of a col-
lagenous matrix by sufficient numbers of cells after cessa-
tion of proliferation of chondrogenic cells in high-density
micromass, pellet, or three-dimensional matrix cultures. Epi­
physeal chondrocytes isolated from the long bones of post-
natal immature rats and rabbits and cultured at high density
­progress through a differentiation pathway that mimics the
transition from a type II collagen–producing, proliferating
chondrocyte phenotype to the terminally differentiated type
X collagen–producing hypertrophic phenotype associated
with growth plate formation and endochondral ossification.
Alkaline phosphatase, osteocalcin, and osteopontin also
have been used as markers of terminal differentiation. The
pellet culture system has been used widely to study terminal
differentiation and hypertrophy because it mimics the dis-
tribution of cells within the growth plate and is sufficiently
organized to permit calcification in situ. Arrest of cell prolif-
eration and activation of type X collagen expression occur
when the serum concentration is reduced from 10% to 2% or
lower. Insulin-like growth factor (IGF)-I or insulin added in
serum-free medium or as a constituent of serum seems to be
a universal basal requirement in these culture systems. Ascor-
bic acid and treatments such as thyroxine 1,25-­dihydroxyvitamin
D3, retinoic acid, or dexamethasone promote terminal differ-
entiation in vitro. Ectopic matrix mineralization also may
require a phosphate donor such as β-glycerophosphate. In
contrast, certain BMPs alone or in the presence of ascor-
bic acid may induce hypertrophy and mineralization in the
absence of other additives, if the appropriate progenitor cell
population is used.
CHONDROCYTE CELL LINES
Because primary human chondrocytes in monolayer cultures
maintain phenotype only until they are passaged, researchers
have attempted to develop chondrocyte cell lines with vari-
able success. Immortalization of chondrocytes from mice,
rats, and other species with viral oncogenes has generated
cell lines with high proliferative capacities and at least some
differentiated chondrocyte properties.147,148 Attempts to
generate cell lines that can undergo chondrogenesis and ter-
minal differentiation to the hypertrophic phenotype have
been more successful because of the availability and plas-
ticity of progenitor cell populations. Cells derived from the
ribs of transgenic mice harboring the temperature-sensitive
mutant of simian virus 40 (SV40) large T antigen (TAg)
are able to undergo hypertrophic differentiation,149,150 and
bone marrow–derived mesenchymal stem cells derived from
these mice contain osteogenic, adipogenic, myogenic, and
chondrogenic progenitor cells. Several chondrogenic cell
lines from mice or rats, including ATDC5, C3H10T1/2,
RCJ3.1, CK2, and C1, are now used widely in the field, as
reviewed by Johnstone and colleagues.151
Human chondrosarcoma cell lines express some aspects
of the chondrocyte phenotype, but are tumorigenic.152,153
Stable expression of SV40-TAg using plasmid or retrovi-
ral vectors has yielded immortalized human chondrocyte
cell lines that are useful for studying the regulation of
gene expression under defined conditions, but do not pro-
duce sufficient amounts of matrix proteins to form carti-
lage matrix owing to high rates of proliferation.154 Human
articular chondrocyte cell lines also have been established
using temperature-sensitive SV40-TAg,155 the human pap-
illomavirus type 16 early function genes E6 and E7,156 and
telomerase.157 A general observation is that phenotypic
stability of immortalized chondrocytes is lost during serial
subculture in monolayer, but can be restored by transfer to
three-­dimensional culture in alginate155 or hyaluronan156
or to suspension culture in poly-2-hydroxyethyl methacry-
late–coated dishes.157 Immortalized chondrocytes may con-
tinue to proliferate in three-dimensional culture, however,
and if the scaffold cannot be remodeled, necrotic clusters
form. The studies to date indicate that mature chondrocytes
removed from the ideal cartilage environment in vivo are
incapable of replicating normal phenotype in vitro, and
the perfect chondrocyte culture model that reproduces the
human articular chondrocyte has yet to be fabricated, as
confirmed by gene profiling studies.158,159
INTERACTIONS OF CHONDROCYTES
WITH THE EXTRACELLULAR MATRIX
Chondrocytes in vivo respond to structural changes in the
cartilage ECM. The ECM not only provides a framework for
the chondrocytes suspended within it, but also its constitu-
ents interact with cell surface receptors and provide signals
that regulate many chondrocyte functions.90,160,161
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
INTEGRINS
The most prominent of the ECM receptors are the integ-
rins, which bind specifically with different cartilage matrix
components and induce the formation of intracellular sig-
naling complexes that regulate cell proliferation, differen-
tiation, survival, and matrix remodeling. Integrins also may
serve as mechanoreceptors and mediate responses to nor-
mal and abnormal loading of cartilage.162-164 Chondrocytes
express many different integrins that interact with cartilage
ECM ligands, although most are not specific to this cell
type.37,90,160,165 They include the integrins that are receptors
for collagen (α1β1, α2β1, α3β1, α10β1), fibronectin (α5β1,
αvβ3, αvβ5), and laminin (α6β1). The integrin α1β1 has
broader ligand specificity than the other collagen-binding
integrins and mediates chondrocyte adhesion to the pericel-
lular type VI collagen and to the cartilage matrix protein,
matrilin-1. The α2β1 integrin also binds to chondroadherin.
The αv-containing integrins bind to vitronectin and osteo-
pontin in addition to serving as alternative fibronectin
receptors. The α5β1 and αvβ3 integrins also serve as recep-
tors for different conformations of COMP.166
Because α1β1, α2β1, and α10β1 all are receptors for
cartilage-specific type II collagen, there is great interest in
determining whether they mediate differential responses of
chondrocytes to changes in the ECM resulting from normal
loading or pathologic changes.162-164 The α5β1 integrin is
the prominent integrin in human adult articular cartilage.
Depending on the method of analysis, adult chondrocytes
also express α1β1 and αvβ5 integrins accompanied by weaker
expression of α3β1 and αvβ3. Normal adult articular chon-
drocytes express little or no α2β1, whereas the expression of
α2β1 and α3β1 integrins is associated with a proliferative
phenotype, as in fetal chondrocytes and in chondrosarcoma
and chondrocyte cell lines.167,168 In growth plate chon-
drocytes, α5β1, αvβ5, and α10β1 are important for joint
formation, chondrocyte proliferation, hypertrophy, and sur-
vival.169-173 Knockout of β1 integrin results in severe growth
plate abnormalities and chondrodysplasia,174 whereas α1
integrin knockout mice develop osteoarthritis without
growth plate abnormalities.175
Cellular binding to immobilized ECM proteins or integ-
rin receptor aggregation with activating antibodies can pro-
mote numerous intracellular signaling events.90 As in other
cell types, integrin signaling is mediated by interaction with
intracellular protein tyrosine kinases, such as pp125 focal
adhesion kinase and pyk2, which interact with the integrin
cytoplasmic tail and induce a conformational change in the
receptor subunits. Changes in organization of the cytoskel-
eton are associated with the formation of integrin signaling
complexes, which contain scaffolding proteins such as talin,
paxillin, and α-actinin in addition to focal adhesion kinase
and the integrin-linked kinase. Mice lacking integrin-linked
kinase in cartilage display chondrodysplasia, a phenotype
similar to that of the cartilage-specific β1 integrin knockout
mice.174 Other signaling kinases, such as Src, Ras/Raf, Sos,
and Mek family members, may be associated with integrin
signaling complexes and mediate downstream signaling cas-
cades in a cell type–specific and ligand-specific manner.
Cooperative signaling among integrins and growth fac-
tors is a fundamental mechanism in the regulation of cellu-
lar functions. Integrin aggregation and receptor occupancy
47
enhance phosphorylation of growth factor receptors and
activation of mitogen-activated protein kinases (MAPKs)
in many cell types. The anchorage-dependent mitogenic
response to growth factors is thought to be due to synergy
between integrin and growth factor signaling. The induc-
tion of chondrocyte proliferation by FGF requires fibronec-
tin binding to α5β1 integrin.169 The β1 integrin subunit also
interacts with the IGF-I receptor on treatment of chondro-
cytes with IGF-I.176 Fibronectin increases FGF-stimulated
and IGF-I-stimulated proteoglycan synthesis in chondro-
cytes, and nitric oxide, which disrupts focal adhesion sig-
naling complexes, inhibits this process.177 Type II collagen
increases TGF-β-induced type II collagen and aggrecan gene
expression by a feedback mechanism that is mediated by β1
integrin.178
The primary fibronectin receptor, α5β1, may play a role
in cartilage degradation through binding to fibronectin frag-
ments that upregulate matrix metalloproteinases (MMPs),
such as MMP-3 and MMP-13.179-181 Extensive studies in
cultured chondrocytes have shown that α5β1 is crucial in
the hyperpolarization response to mechanical load.164 Nor-
mal chondrocytes use α5β1 as a mechanoreceptor, and sub-
sequent to activation of the integrin-signaling cascade by
mechanical stimulation, there is secretion of IL-4, which acts
in an autocrine manner via the janus activating kinase (JAK)/
signal transducer and activator of transcription (STAT)
pathway to increase aggrecan mRNA and decrease MMP-3
mRNA levels.182
Chondrocyte adhesion to fibronectin or binding to fibro­
nectin fragments also increases the production of cytokines,
such as IL-1, tumor necrosis factor (TNF), IL-6, and gran-
ulocyte-macrophage colony-stimulating factor. Synergies
between fibronectin/α5β1 and IL-1 have been shown in
chondrocytes.183,184 Osteoarthritis chondrocytes seem to
respond to α5β1 ligation by production of IL-1β and other
proinflammatory mediators, whereas αvβ3 integrin ligation
attenuates these responses.183 Nevertheless, there is good
evidence that fibronectin fragments or blocking antibodies
to α2β1 and α5β1 integrins can directly stimulate signaling
via extracellular signal-regulated kinase (Erk)-1 or Erk-2,
c-Jun N-terminal kinase (JNK), and p38 MAPK in chon-
drocytes and increase MMP-13 production independent of
autocrine production of IL-1β.180 This response requires
reactive oxygen species.185 Collagen binding to α1β1 and
α2β1 integrins also results in activation of distinct signal-
ing pathways and may lead to opposite cellular responses.186
The downregulation of IL-1-induced responses by dynamic
compression also is mediated by integrins.187 The specific-
ity of the response may depend on the relative expression
of α-integrin subunits on the chondrocyte cell surface.
OTHER CELL SURFACE RECEPTORS
IN CHONDROCYTES
Other integral membrane proteins found in chondrocytes
include cell determinant 44 (CD44), anchorin CII, and
syndecan-3.188 CD44 is a receptor for hyaluronan and
binds collagen and fibronectin. Through specific interac­
tions with hyaluronan, CD44 has a role in assembly,
organization, and maintenance of the chondrocyte pericel-
lular matrix.189 In chondrocyte cultures, the assembly of a
newly synthesized pericellular matrix can be prevented or
 48 Goldring  |  Cartilage and Chondrocytes
reversed by ­ incubation with hyaluronan hexasaccharides
or with a CD44 monoclonal antibody. CD44 expression is
upregulated in chondrocytes in articular cartilage from RA
patients and in experimental osteoarthritis.190 Hyaluronan
binding to CD44 increases MMP-13 and nitric oxide pro-
duction by chondrocytes.191 Although blocking CD44 has
no effect on attachment of chondrocytes to a cut cartilage
surface, more recent evidence indicates that CD44 mediates
the expression of MMP-1, MMP-2, MMP-9, and MMP-13,
the MMP-specific cleavage of type II collagen and nitric
oxide production induced by the heparin-binding fibronec-
tin fragment in articular cartilage.192,193 Because fibronectin
fragments and IL-l enhance CD44 expression in chondro-
cytes, cell-matrix interactions mediated by such cell surface
receptors represent alternative mechanisms for cartilage
damage in joint disease. During joint disease, CD44-medi-
ated co-internalization with hyaluronic acid may be one
important mechanism for the elimination of residual aggre-
can fragments after extracellular degradation.194 Aggreca-
nase-mediated depletion of proteoglycan does not require
CD44, however.195
Anchorin CII, also known as annexin V, is a 34-kD inte-
gral membrane protein that binds type II collagen and shares
extensive homology with the calcium-binding proteins cal-
pactin and lipocortin.196,197 Three types, annexins II, V, and
VI, have been detected in chondrocytes, where they likely
play roles in physiologic mineralization of skeletal tissues
and in pathologic mineralization of articular cartilage.198
Annexin V, or anchorin CII, was first detected in chick car-
tilage and described as a type II collagen–binding protein
that anchors the chondrocytes to the ECM. In growth plate
chondrocytes, annexins are required for calcium ion uptake
and subsequent mineralization. Annexin V antibodies block
chondrocyte attachment to immobilized type II collagen
more effectively than integrin antibodies, but not to a cut
cartilage surface, where the N-terminal collagen binding
site may not be exposed.188 In contrast to integrins, annexin
V binds to the N-telopeptide of type II collagen, but not to
triple-helical fragments.199
Syndecan-3 links to the cell surface via glycosyl phos-
phatidylinositol and binds tenascin-C and growth factors,
proteases and inhibitors, and other matrix molecules, through
heparan sulfate side chains on the extracellular domain. Syn-
decans have important roles during cartilage development.
Syndecan-1 and syndecan-3 are upregulated in human and
mouse osteoarthritis.200,201
In contrast to mammalian collagen receptors that bind
collagen fragments, discoidin domain receptor 2 binds spe-
cifically to type II and X collagen fibrils, leading to activa-
tion of its integral receptor tyrosine kinase.202,203 Discoidin
domain receptor 2 is upregulated in osteoarthritis cartilage
and induces specifically the expression of MMP-13 associ-
ated with cleavage of type II collagen.204
ANGIOGENIC AND ANTIANGIOGENIC
FACTORS
Adult articular cartilage is among the few avascular tissues
in mammalian organisms, and this property and the pres-
ence of angiogenesis inhibitors make it resistant to vascular
angiogenesis and invasion by inflammatory and neoplastic
cells. In conditions in which there is extensive remodeling
of ECM, as in arthritis, the cartilage becomes susceptible
to invasion by vascular endothelial and mesenchymal cells
from the synovium and subchondral bone.205 In osteoarthri-
tis, the upregulation of angiogenic factors may contribute to
ingrowth of blood vessels, tidemark advancement, and car-
tilage calcification in the deep zone. In RA, the ingrowth of
blood vessels and synovial pannus into cartilage contributes
to the degradation of the cartilage matrix. Troponin I, MMP
inhibitors, chondromodulin-I, and endostatin, a 20-kD
proteolytic fragment of type XVIII collagen, all function as
endogenous angiogenic inhibitors.206-209 VEGF, which is an
essential mediator of angiogenesis during endochondral ossi-
fication (see Chapter 1), is induced by hypoxia and mechan-
ical overload.210,211 In osteoarthritis, in which abnormal
biomechanics and joint effusions cause severe hypoxia,
chondrocytes may produce VEGF, inducing angiogenesis at
the chondro-osseous junction and contributing to cartilage
destruction.212,213
Intercellular adhesion molecules also contribute
to angiogenesis. These include vascular cell adhesion
molecule-1 and intercellular adhesion molecule-1, which
are expressed by human articular chondrocytes and syno-
vial and endothelial cells. Their function on chondro-
cytes may not be significant, however, unless damage
to the matrix permits cell-cell interactions.214 Vascular
cell adhesion molecule-1, VEGF, FGF, and TNF-α con-
tribute to angiogenesis during synovitis and to activa-
tion of chondrocytes during cartilage degradation.215,216
In RA, VEGF expression may be upregulated by inflam-
matory cytokines in chondrocytes and synovial cells and
by hypoxia.217,218 The importance of this mechanism is
supported by findings in Vegfb knockout mice, which are
protected against synovial angiogenesis in experimental
inflammatory models.219
ROLES OF GROWTH AND
DIFFERENTIATION, OR
ANABOLIC, FACTORS IN
NORMAL CARTILAGE METABOLISM
Growth and differentiation factors generally are considered
positive regulators of homeostasis of mature articular carti-
lage because of their capacity to stimulate chondrocyte ana-
bolic activity and, in some cases, inhibit catabolic activity
(see Chapter 1).220 The most well-characterized anabolic
factors in the context of their production and action in
articular cartilage include IGF-I, FGFs, and members of the
TGF-β/BMP family.221-225 Many of these factors also regu-
late chondrogenesis and endochondral ossification during
skeletal development.68 In adult cartilage, their expression
declines with age, a risk factor for osteoarthritis, and their
activities are downregulated.220
INSULIN-LIKE GROWTH FACTOR
IGF-I, also known as somatomedin C, was first discovered as
a serum factor controlling sulfate incorporation by articular
cartilage in vitro and was later found to have the capacity
specifically to stimulate or maintain chondrocyte phenotype
in vitro by promoting the synthesis of type II collagen and
aggrecan. IGF-I is categorized more appropriately as a dif-
ferentiation factor because its limited mitogenic activity
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
seems to depend on the presence of other growth fac-
tors, such as FGF-2 or BMP-7.142,226 IGF-I is considered
an essential mediator of cartilage homeostasis through its
capacity to stimulate proteoglycan synthesis, promote chon-
drocyte survival, and oppose the activities of catabolic cyto-
kines.143,227,228 IGF-I and insulin can activate either the cell
surface IGF-I tyrosine kinase receptor or the type I insulin
receptor at concentrations proportional to their binding
affinities.
Specific IGF-binding proteins (IGFBPs) that do not rec-
ognize insulin also regulate IGF-I activity. Chondrocytes
at different stages of differentiation express IGF-I and IGF
receptors and different arrays of IGFBPs, providing a unique
system by which IGF-I can exert different regulatory effects
on these cells. IGFBP-2 seems to be a positive regulator in
chondrocytes because its induction by TGF-β or estrogen is
associated with increased proteoglycan synthesis.229 Binding
of IGFBP-3 to IGF-I is thought to regulate negatively the
anabolic functions of IGF-I, although IGFBP-3 may directly
inhibit chondrocyte proliferation in an IGF-­independent
manner.
In osteoarthritis cartilage, the normal anabolic func-
tion of IGF-I may be disrupted because chondrocytes from
animals with experimental arthritis and from patients
with osteoarthritis are hyporesponsive to IGF-I, despite
normal or increased IGF-I receptor levels. This hypore-
sponsiveness has been attributed to increased levels of
IGFBPs that may interfere with IGF-I actions.230,231 Dis-
turbances in the balance of IGF-I to IGFBPs that have
been reported in osteoarthritis and RA joints may con-
tribute to defective chondrocyte responses to IGF-I.230-233
Small-molecule inhibitors of IGF-I/IGFBP interactions
that could restore IGF-I-dependent proteoglycan synthesis
in cartilage have been proposed for treatment of osteoar-
thritis.228 Although IGF-I can oppose the effects of inflam-
matory cytokines that promote cartilage degradation and
inhibit proteoglycan synthesis,234 these cytokines also
increase the production of IGFBP-3 by chondrocytes.235
Overproduction of nitric oxide also may contribute to
IGF-I resistance by chondrocytes through disruption of
integrin signaling, reducing phosphorylation of the IGF-I
receptor, stimulation of cyclic guanosine monophosphate
production, or suppression of mitochondrial oxidative
metabolism.227,236-239 More recent evidence indicates that
suppressor of cytokine signaling 3 acts as a negative feed-
back regulator during IGF-I desensitization in the absence
of nitric oxide by inhibiting insulin receptor substrate-1
phosphorylation.240
FIBROBLAST GROWTH FACTOR
Members of the FGF family, including FGF-2, FGF-4,
FGF-8, FGF-9, FGF-10, and FGF-18, together with the
FGF receptors, FGFR1, FGFR2, and FGFR3, coordinate
patterning and cell proliferation during chondrogenesis
and endochondral ossification in embryonic and postnatal
growth plates.241 The most extensively studied is FGF-2,
or basic FGF, which is a potent mitogen for adult articu-
lar chondrocytes,224 but findings on its effects on the
synthesis of cartilage matrix are contradictory, showing
stimulation, inhibition, or no effect on proteoglycan syn-
thesis.242
49
Early studies suggested that low concentrations of FGF-2
could stimulate chondrocyte mitogenesis and proteoglycan
synthesis, whereas high concentrations might have opposite
effects.243 More recent studies showing that FGF-2 stored in
the adult cartilage matrix is released with mechanical injury
or with loading suggest a mechanism for modulating chon-
drocyte proliferation and anabolic activity.244,245 Although
FGF-2 and FGF-9 stimulate the expression of Sox9 and
increase the activity of the Sox9-dependent, chondrocyte-
specific enhancer in the type II collagen gene,246,247 FGF-2
can inhibit the anabolic activities of IGF-I and osteogenic
protein-1 (OP-1) in vitro.142 FGF-9 and FGF-18 increase
matrix synthesis by mature chondrocytes.248-251 A study
showed that FGF-18 promotes cartilage repair in a rat
meniscal tear model of osteoarthritis.252 FGF-2 stimulates,
whereas IGF-I inhibits, expression of matrix Gla protein,
which is a marker for the chondrocyte survival during endo-
chondral ­ ossification.253,254 FGFs and FGF receptors have
important roles in cartilage homeostasis during prenatal and
postnatal life.
TRANSFORMING GROWTH FACTOR-β/BONE
MORPHOGENETIC PROTEIN SUPERFAMILY
Activities of the TGF-β/BMP superfamily in the skeleton
were first discovered as constituents of demineralized bone
that induced new bone formation when implanted into
extraskeletal sites in rodents.255 These bioactive morphogens
subsequently were extracted, purified, and cloned and found
to regulate the early commitment of mesenchymal cells to the
chondrogenic and osteogenic lineages during cartilage devel-
opment and endochondral bone formation (Table 3-2).68,256
The TGF-β/BMP superfamily includes activins, inhibins,
müllerian duct inhibitory substance, nodal, glial-derived
neurotrophic factor, OP-1 (or BMP-7), and growth differen-
tiation factors (GDFs), also called cartilage-derived morpho-
genetic proteins (CDMPs). In addition to regulating cartilage
condensation and chondrocyte differentiation, members of
this superfamily play key roles in site specification and cavi-
tation of synovial joints (see Chapter 1) and participate in
the development of other organ systems. Many of these fac-
tors, including BMP-2, BMP-6, BMP-7, BMP-9, TGF-β, and
CDMP-1, are able to induce chondrogenic differentiation of
mesenchymal progenitor cells in vitro. They also may have
direct effects on mature articular chondrocytes in vivo and
in vitro.
Transforming Growth Factor-β
TGF-β was named based on its discovery as a factor that
could transform cells to grow in soft agar. TGF-β is not
a potent inducer of chondrocyte proliferation, however;
rather, it controls early mesenchymal cell condensa-
tion and differentiation to chondrocytes at early and
late stages of endochondral ossification (see Chapter 1).
Inhibition and stimulation of the synthesis of aggrecan
and type II collagen by TGF-β have been observed in
vitro. TGF-β, by itself, cannot rescue the type II colla-
gen phenotype, however, when the cells have undergone
dedifferentiation during serial passaging. The levels of
TGF-β measured in synovial fluids of osteoarthritis and
RA patients may reflect anabolic processes in cartilage
 50 Goldring  |  Cartilage and Chondrocytes

Table 3-2  Bone Morphogenetic Protein  

Bone Morphogenetic Proteins
Superfamily
Bone The BMPs constitute a large subclass of the TGF-β super-
Morphogenetic family that are essential for normal appendicular skeletal and
Protein Other Names Potential Function joint development.68,221,256,266 The isolation and cloning of
BMP-2 BMP-2A Cartilage and bone the first BMP family members from bone prompted a search
morphogenesis for the cartilage-derived BMPs, CDMPs, CDMP-1, CDMP-2,
BMP-3 Osteogenin, GDF-10 Bone formation and CDMP-3, which are classified as GDF-5, GDF-6,
BMP-4 BMP-2B Cartilage and bone
and GDF-7. The BMPs may be divided into four distinct
morphogenesis subfamilies based on the similarity of primary amino acid
BMP-5 Bone morphogenesis
sequences: (1) BMP-2 and BMP-2B (BMP-4), which are
92% identical in the 7-cysteine region; (2) BMP-3 (osteo-
BMP-6 Vegetal-related-1   Cartilage hypertrophy
(Vgr-1)
genin) and BMP-3B (GDF-10); (3) BMP-5, BMP-6, BMP-7
(OP-1), BMP-8 (OP-2), BMP-9 (GDF-2), BMP-10, and
BMP-7 Osteogenic   Cartilage and bone
protein-1 (OP-1) morphogenesis
BMP-11 (GDF-11); and (4) BMP-12 (GDF-7 or CDMP-3),
BMP-13 (GDF-6 or CDMP-2), BMP-14 (GDF-5 or CDMP-
BMP-8 Osteogenic   Bone morphogenesis
protein-2 (OP-2)
1), and BMP-15. BMP-1 is not a member of this family, but
is an astacin-related MMP that cleaves the BMP inhibitor
BMP-9 GDF-2 Cartilage
­morphogenesis
chordin and acts as a procollagen C-proteinase.267
Several BMPs, including BMP-2, BMP-7 (OP-1), and
BMP-10 Unknown
GDF-5/CDMP-1, can stimulate differentiation of mes-
BMP-11 GDF-11 Unknown enchymal precursors into chondrocytes and promote the
BMP-12 GDF-7, CDMP-3 Cartilage ­differentiation of hypertrophic chondrocytes.268,269 BMP-2,
­morphogenesis BMP-4, BMP-6, BMP-7, BMP-9, and BMP-13 can enhance
BMP-13 GDF-6, CDMP-2 Cartilage the synthesis of type II collagen and aggrecan by articular
­morphogenesis chondrocytes in vitro.223,270,271 In addition, BMP-7 reverses
BMP-14 GDF-5, CDMP-1 Cartilage many of the catabolic responses induced by IL-1β, including
­morphogenesis induction of MMP-1 and MMP-13, downregulation of TIMP
CDMP, cartilage-derived morphogenetic protein; GDF, growth and differen- expression, and downregulation of proteoglycan synthesis in
tiation factor. primary human articular chondrocytes.223 CDMP-2 is found
in articular cartilage, skeletal muscle, placenta, and hypertro-
and other joint tissues. TGF-β also may promote anab- phic chondrocytes of the epiphyseal growth plate. CDMP-1
olism by inducing the expression of tissue inhibitors of and CDMP-2 maintain the synthesis of type II collagen and
MMP (TIMP). aggrecan in mature articular chondrocytes, although they are
Nevertheless, TGF-β1, TGF-β2, and TGF-β3 generally less effective initiators of chondrogenesis than other BMPs
are considered as potent stimulators of proteoglycan and in early progenitor cell populations in vitro.272
type II collagen synthesis in primary chondrocytes and car- BMP-7 is expressed in mature articular cartilage and is pos-
tilage explants in vitro.257 Microarray analysis of chondro- sibly the strongest anabolic stimulus for adult chondrocytes in
cytes in vitro indicates that TGF-β is able to counteract the vitro because it increases aggrecan and type II collagen syn-
expression of numerous IL-1-induced genes involved in car- thesis more strongly than IGF-I.223 BMP-2 also is expressed
tilage injury.258 Although intra-articular injection of TGF-β in normal and osteoarthritis articular cartilage,119 and it is
stimulates proteoglycan synthesis and limits cartilage dam- a molecular marker, along with type II collagen and FGF
age in inflammatory arthritis models, injection or adenovi- receptor 3 (FGFR3), for the capacity of adult articular chon-
rus-mediated delivery of TGF-β1 may result in side effects drocyte cultures to form stable cartilage in vivo.140 BMP-2,
in joint tissues, such as osteophyte formation, swelling, and BMP-7, and BMP-9 are able to oppose many of the detri-
synovial hyperplasia.259,260 Administration of agents that mental effects of IL-1 on chondrocyte metabolism in vitro
block TGF-β activity, such as the soluble form of TGF-βRII, and in vivo.269,273 BMP-7 can prevent retinoic acid–induced
inhibitory SMADs, or the physiologic antagonist, latency- dedifferentiation of articular chondrocytes.274 BMPs have
associated peptide-1, increases proteoglycan loss and cartilage pleiotropic effects in vivo, however, acting in a concentra-
damage in an experimental model of osteoarthritis.261,262 tion-dependent manner. While initiating chondrogenesis in
A more recent finding that TGF-β induces expression of the limb bud, they generally set the stage for bone morpho-
ADAMTS (a disintegrin and metalloproteinase [ADAM genesis (see Chapter 1). Several BMPs also are true morpho-
family] with thrombospondin-1 domains)-4 in primary gens for other tissues, such as kidney, eye, heart, and skin.
human chondrocytes and promotes the degradation of
aggrecan suggests that it may be involved in normal turn- RECEPTORS, SIGNALING MOLECULES,
over of proteoglycans in mature cartilage.263 Findings that AND ANTAGONISTS THAT MEDIATE
IL-1 differentially regulates inhibitory SMADs and tran- CHONDROCYTE RESPONSES TO GROWTH
scriptional mediators of TGF-β and BMP signaling,264 and AND DIFFERENTIATION FACTORS
decreases TGF-β signaling associated with the loss of a pro-
tective effect of TGF-β during osteoarthritis progression265 The major pathways activated by the growth and differ-
suggest that the balance of inhibitory and stimulatory mol- entiation factors discussed earlier involve members of the
ecules determines cartilage homeostasis. ERK1/2, p38 MAPK, and phosphatidylinositol-3′-kinase
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE 51

(PI-3K)/AKT pathways.275 As in other cell types, FGF

family members activate ERK1/2 and p38 kinases in chon-
drocytes. Specific inhibitors of these pathways block FGF- BMPs
2-induced and FGF-18-induced mitogenesis in chondrocytes Noggin
Extracellular matrix
and prevent FGF-2 induction of Sox9 in primary chondro- Collagens I and IV Chordin
cytes.246,251 The PI-3K pathway is required for the stimula- Heparan sulfate Dan
tion of proteoglycan synthesis by IGF-I in primary human
articular chondrocytes, whereas the ERK1/2 pathway acts as Cytoplasm BMPR-IA BMPR-IB
a negative regulator.276 P
P
P
SMAD-6
TGF-β and BMP family members transduce signals P
­thro­ugh the formation of heteromeric complexes of ligand- SMAD-7
specific  receptors,  which  have  serine-threonine kinase 
activity. The specificity of subsequent signals is determined
BMPR-II
mainly by the type I receptors. Type I and type II recep- SMAD-1 SMAD-5
tors are required for signal transduction (Fig. 3-5).266,277,278
Seven types of type I receptors, called activin receptor-like P
SMAD-1
P
SMAD-5
kinases (ALK), have been identified in mammals and have
similar structures. TGF-β interacts with the type II recep- + +

tor (TβRII), which recruits a TGF-β type I receptor (princi- SMAD-4 SMAD-4

pally TβRI) to form a heterotrimeric receptor complex. The

­constitutively active TβRII kinase phosphorylates TβRI
at serine and threonine residues. Three type I receptors,
BMP type IA (BMPR-IA or ALK-3), BMPR-IB (ALK-6),
and ALK-2, mediate BMP signaling. Although type I BMP
receptors are able to bind ligand in the absence of type II
BMP receptors, cooperativity has been shown in binding
assays. On ligand binding, analogous to TβRI and TβRII,
BMP type I receptors are phosphorylated by the BMP type
II receptors, which include activin (Act) RII, ActRIIB, and
T-ALK. Spatial and temporal differences in the distribution
of these receptors in different tissues can govern the response
patterns to different members of the TGF-β/BMP family.
Signaling by TGF-β and BMP is mediated by the canoni-
cal SMAD pathway, through phosphorylation of receptor-
­activated SMADs (R-SMADs), BMP-induced Smad 1, Smad
5, and Smad 8 for BMPs and TGF-β-induced Smad 2 and
Smad 3. The SMADs are related to the Drosophila ­mothers
against decapentaplegic and nematode SMA signaling mol-
ecules. These regulatory SMADs form complexes with the
common Smad 4 and translocate to the nucleus, where
they bind to SMAD elements in the promoters of target
genes.278 Other BMP-induced transcription factors include
JunB, JunD, and ID and DLX family members, suggesting
alternative pathways of signaling. TGF-β and BMPs also can
signal by activating TGF-β activated kinase 1 (TAK1),279
which interacts with MEKK1 and activates p38 and JNK
cascades, or by activating Ras/ERK1/2 or RhoA/ROCK sig-
naling.266,278 Findings of numerous studies suggest that the
differentiation of chondrocytes from mesenchymal precur-
sors is positively regulated by p38 kinase and negatively reg-
ulated by ERK1/2.266,280 ERK1/2 activation cross-interacts
with BMP-2-induced signaling to regulate chondrogenesis
in a positive manner, whereas p38 activity is essential for
the TGF-β induction of proteoglycan synthesis in articu-
lar chondrocytes.281,282 Cytoskeletal compartmentation of
SMAD signaling complexes may regulate the differentiation
of chondroprogenitor cells into chondrocytes.273
Inhibitory Smad 6 and Smad 7, which prevent phosphor-
ylation of the R-SMADs, also control BMP-induced and
TGF-β-induced activities. An additional SMAD-interacting
molecule, Tob1, negatively regulates signaling by seques-
tering R-SMADs and is an antiproliferative protein that
Nucleus
P P SMAD-6
SMAD-1 SMAD-5
SMAD-4 SMAD-4
SMAD-7
BMP
response genes
Figure 3-5  Bone morphogenetic protein (BMP) receptors and ­signaling
cascade. BMPs are dimeric ligands with a single interchain, disulfide bond. 
BMPs interact with type I and II BMP receptors (BMPR-I and BMPR-II).
BMPR-II phosphorylates BMPR-I and activates the serine/threonine 
kinase receptor. The BMP-RI protein serine/threonine kinase phosphory-
lates cytoplasmic signaling substrates Smad 1 or Smad 5. This phos-
phorylation is modulated and inhibited by inhibitory Smad 6 and Smad 7.
Phosphorylated Smad 1 and Smad 5 interact with a common co-Smad 4
and are translocated into the nucleus to initiate the transcription of BMP-
response genes. A Smad interacting protein (SIP) modulates binding of
the Smad 1/Smad 4 complex to DNA. The bioavailability of BMP for in-
teraction with cognate receptors depends on BMP binding proteins and
antagonists, such as noggin and chordin, and extracellular matrix com-
ponents, such as collagens I and IV and heparan sulfate. (From Reddi AH:
Role of morphogenetic proteins in skeletal tissue engineering and regenera-
tion. Nat Biotech 16:247-252, 1998.)
is downregulated in osteoarthritis cartilage.283 BMP
antagonists play important roles in spatial and temporal
regulation of BMP activities during skeletal development.
Originally discovered in Xenopus, they act as antagonists
by determining the bioavailability of BMPs for binding to
BMP receptors. The roles of noggin and chordin seem to
be crucial for determining boundaries during joint mor-
phogenesis. They display different spatial and temporal
patterns of expression, binding affinities, and susceptibil-
ity to proteinases that release BMP. BMPs bind to chordin
and noggin via cysteine-rich domains that are similar to
domains in the N-terminal propeptides of fibrillar procol-
lagens I, II, III, and V, which also bind BMPs and are sus-
ceptible to cleavage by MMPs.284 BMP may be released
from chordin by cleavage with MMPs or BMP-1/tolloid,
whereas noggin binds BMP with high affinity and cannot
be cleaved to release BMP.
 52 Goldring  |  Cartilage and Chondrocytes
Follistatin, gremlin, chordin, and chordin-like 2 are
upregulated in osteoarthritis cartilage.285-287 Follistatin,
which has been linked to inflammatory processes; grem-
lin, which is associated with hypertrophic phenotype; and
chordin appear at different stages of osteoarthritis and with
different topographic distribution. Because each antago-
nist binds preferentially to different BMPs, the differential
expression may serve as a feedback mechanism to balance
anabolic activities at different stages.
ROLE OF THE CHONDROCYTE
IN CARTILAGE PATHOLOGY
The chondrocyte, the unique cell type in mature cartilage,
maintains a stable equilibrium between the synthesis and
the degradation of matrix components. During aging and
joint diseases, such as RA and osteoarthritis, this equilib-
rium is disrupted, and the rate of loss of collagens and pro-
teoglycans from the matrix exceeds the rate of deposition
of newly synthesized molecules. The cartilage destruction
in osteoarthritis is believed to be chondrocyte mediated in
response to biomechanical insult, which may occur directly
or indirectly through the production of cytokines and car-
tilage matrix-degrading proteinases in cartilage and other
joint tissues (Fig. 3-6).
Cartilage destruction in RA occurs primarily in areas
contiguous with the proliferating synovial pannus as a result
of the release and activation of proteinases from the synovial
cells and, to some extent, at the cartilage surface exposed to
matrix-degrading enzymes from polymorphonuclear neutro-
phils in the synovial fluids. In addition to the direct action
of proteinases, the RA synovial tissues contribute indirectly
to cartilage loss by releasing cytokines and other mediators
that act on the chondrocytes to produce dysregulation of
chondrocyte function.288 Understanding of basic cellular
mechanisms regulating chondrocyte responses to inflamma-
tory cytokines has been inferred from numerous studies in
vitro using cultures of cartilage fragments or isolated chon-
drocytes and is supported by studies in experimental models
of inflammatory arthritis, such as collagen-induced arthritis
and antigen-induced arthritis in mice.289,290
CARTILAGE
IL-1
Chondrocytes
Figure 3-6  The role of chondro-
cyte-derived proteinases in ­ cartilage
­destruction in osteoarthritis. ­Although Active
studies in vitro and in vivo have shown aggrecanases
that the chondrocyte can respond di-
rectly to mechanical loading, to cata-
bolic cytokines such as interleukin-1
(IL-1) and tumor necrosis factor-α (−)
(TNF-α), and to cartilage breakdown TIMPs
products, the initiating signals and
their relative importance have not
been defined clearly. MMP, matrix MMP-14
metalloproteinase; TIMP, tissue inhibi-
tor of metalloproteinase; uPA, urinary Plasmin
plasminogen activator. (From Goldring uPA
MB: Osteoarthritis and cartilage: The (-)
role of cytokines. Curr Rheumatol Rep
2:459-465, 2000.) Plasminogen PA inhibitors
Direct analysis of cartilage or chondrocytes from osteo-
arthritis patients undergoing joint replacement has yielded
more information than that available from RA patients,
where cartilage damage is extensive. These studies indi-
cate that chondrocytes produce not only proinflamma-
tory cytokines, but also inhibitory and anabolic cytokines
that modulate the responses. The impact of cytokines on
chondrocyte function, particularly with respect to their
various roles in cartilage destruction, has been reviewed
extensively.2-4,87,351
CARTILAGE MATRIX-DEGRADING PROTEINASES
Chondrocytes synthesize and secrete MMPs in latent forms,
which are activated outside the cells via activation cascades.
An important cascade in cartilage is initiated by plasmin, the
product of plasminogen activator activity, which may be pro-
duced by the chondrocyte; plasmin activates latent strome-
lysin (MMP-3), an activator of latent collagenases. In early
studies, chondrocytes were among the first identified sources
of TIMP-1, and they are now known to synthesize additional
TIMPs. Chondrocytes are assumed to be a major source of the
TIMPs and MMPs, detected in synovial fluids, where they
reflect an adaptive response to the local imbalance caused by
increased production of active MMPs by chondrocytes and
other joint tissues. The collagenases 1, 2, and 3 (MMP-1,
MMP-8, and MMP-13); gelatinases (MMP-2 and MMP-9);
stromelysin-1 (MMP-3); membrane type I MMP (MT1)-
MMP (MMP-14); and the aggrecanases, ADAMTS-4
and ADAMTS-5, specifically degrade native collagens
and proteoglycans in cartilage matrix (Table 3-3) (see also
Chapter 7).300,301,314
MMPs, aggrecanases, and the cleavage fragments gen-
erated by them are localized in regions of cartilage deg-
radation110,291,292 and are detected in synovial fluids and
cartilage from osteoarthritis and RA patients.33,293 The
expression of MMP-13 in osteoarthritis and RA cartilage
and its ability to degrade type II collagen more effectively
suggest a major role for this enzyme in cartilage degradation.
Postnatal overexpression of constitutively active MMP-13
in cartilage in mice produces osteoarthritis-like changes in
SYNOVIUM
Genetic
Mechanical
Biochemical
Factors
Synovial
fibroblasts
IL-1
± TNF-α
Active
MMPs
Synovial
macrophages
Cartilage
Latent MMPs breakdown
products
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE 53

knee joints.294 Although elevated levels of MMPs in RA cartilage-­pannus junction and in deeper zones of cartilage
synovial fluids likely originate from the synovium, intrinsic matrix in some RA specimens.295,296 MMP-1 does not derive
chondrocyte-derived chondrolytic activity is present at the from the RA synovial pannus, but is produced by chondro-
cytes.297 MMP-10, similar to MMP-3, activates procollage-
Table 3-3  Chondrocyte Proteinases That Mediate nases and is produced by the synovium and chondrocytes in
­Degradation of Cartilage Matrix response to inflammatory cytokines.298 MMP-14, produced
Cartilage Matrix principally by the synovial tissue, is important for syno-
Proteinase Class Substrates Activity vial invasiveness, and antisense mRNA inhibition of this
Matrix Metalloproteinases membrane proteinase has been shown to reduce cartilage
Collagenase-1, Collagen II Fibrillar domain, 3/4
destruction.299
­collagenase-2, from N-terminus Several of the MMPs, including MMP-3, MMP-8,
­collagenase-3   MMP-14, MMP-19, and MMP-20, are capable of degrading
(MMP-1, MMP-8, proteoglycans. The members of the reprolysin-related pro-
MMP-13) teinases of the ADAM family, particularly ADAMTS-4 and
N-telopeptide ­ 
(MMP-13) ADAMTS-5, are now regarded as the principal mediators
Aggrecan core   Asn341-Phe342 IGD of aggrecan degradation.300-302 The activities of MMPs and
protein aggrecanases are complementary, however.303 Of the aggre-
Stromelysins-1   Aggrecan core   Asn341-Phe342 IGD canases, to date, only aggrecanase-2, ADAMTS-5, seems to
(MMP-3, MMP-10) protein
Collagens IX, XI Telopeptide region
be associated with increased susceptibility to osteoarthritis,
Link protein, as shown in Adamts5-deficient mice.304,305 TIMP-3, but
­fibronectin not TIMP-1, TIMP-2, or TIMP-4, is a potent inhibitor of
proMMPs, proTNF ADAMTS-4 and ADAMTS-5 in vitro.306,307
Gelatinases   Collagens II, XI Telopeptide or The cysteine proteinases, cathepsins B and L, and the
(MMP-2, MMP-9) ­denatured
­collagen chains aspartic proteinase, cathepsin D, are lysosomal enzymes
Proteoglycans, that may play a secondary role in cartilage degradation via
link protein intracellular digestion of products released by other protein-
MT-MMP-1,   Collagen II Telopeptide ases. Cathepsin B also may have a role in extracellular deg-
MT-MMP-2, MT- 
MMP-3, MT-MMP-4
radation of collagen telopeptides, collagens IX and XI, and
(MMP-14, MMP-15, aggrecan. Cathepsin K is expressed in synovial fibroblasts on
MMP-16, MMP-17) the cartilage surface at the pannus-cartilage junction and
Fibronectin, aggre- is upregulated by inflammatory cytokines.308 Among the
can, pro­MMP-2,   known cathepsins, cathepsin K is the only proteinase that is
proMMP-13
ProTNF capable of hydrolyzing type I and type II collagens at multi-
Matrilysin (MMP-7) Link protein ple sites within the triple-helical regions, and its requirement
Enamelysin   COMP, link   for acidic pH may be provided by the microenvironment
(MMP-20) protein between the synovial pannus and the cartilage.309
Aggrecanases MT1-MMP (MMP-14) also may serve as an activator of
(ADAMTS-1,   Aggrecan core   Glu373-Ala374,
other MMPs produced by chondrocytes. Other MMPs, includ-
ADAMTS-4,   ­protein IGD Glu1545-Gly1546, ing MMP-16 and MMP-28,310,311 and numerous ADAM/
ADAMTS-5) Glu1714-Gly1715, ADAMTS family members, including ADAM-17/TACE
Glu1819-Ala1820, (TNF-α converting enzyme),312 are expressed by chondro-
Glu1919-Leu1920 cytes, but their roles in cartilage have yet to be defined.313,314
Serine Identification of the precise roles of these proteinases and their
Plasminogen   Aggrecan,   Activation of endogenous inhibitors in chondrocyte-mediated cartilage deg­
activators   ­fibronectin,   ­plasminogen radation has provided the opportunity to develop targeted
(tPA, uPA) proMMPs gives rise to therapies that interfere with the activities of aggrecanases or
plasmin MMPs without disrupting physiologic homeostasis.300,301,315
Cathepsin G Aggrecan,  
collagen II,  
proMMPs BALANCE OF CYTOKINES IN CARTILAGE
Cysteine DESTRUCTION
Cathepsins B, K, L, S Collagens IX, XI Telopeptides (opti- Of the cytokines that affect cartilage metabolism, most are
mal pH 4.0-6.5)
Link protein; pleiotropic factors that were identified originally as immuno-
­aggrecan modulators, but were found to regulate cellular functions in
Aspartate
cells of mesenchymal origin. IL-1 and TNF-α not only stim-
ulate chondrocytes to synthesize cartilage matrix-degrading
Cathepsin D Phagocytosed ECM In lysosomes (opti- proteinases, but they also regulate matrix protein synthesis
components mal pH 3.0-6.0)
and cell proliferation. Considerable redundancy and over-
ADAMTS, a distintegrin and metalloproteinase with thrombospondin-1 lap in the biologic activities exist among the individual
­domains; COMP, cartilage oligomeric matrix protein; ECM, extracellular
­matrix; IGD, interglobular domain; MMP, matrix metalloproteinase; MT-MMP,
cytokines, and they do not act alone, but rather in synergy
membrane-type MMP; proMMP, proenzyme form of MMP; TNF, tumor or partnership with or in opposition to other cytokines via
­necrosis factor. cytokine networks. In addition to IL-1 and TNF-α, IL-17
 54 Goldring  |  Cartilage and Chondrocytes
and IL-18 have been characterized as catabolic cytokines,
and their actions may be modulated by inhibitory or ana-
bolic cytokines produced by the chondrocytes themselves
or by other cells in joint tissues (Table 3-4). Investigations
in vitro and in vivo have begun to sort out the complexities
of the cytokine networks and to determine how the balance
in normal homeostasis can be restored when it is disrupted
(Fig. 3-7). Examination of type II collagen–induced arthritis
and other types of induced arthritis in transgenic animals
with overexpressed or deleted genes encoding cytokines,
their receptors, or activators has provided further insights
into the roles of these factors in cartilage destruction.
Interleukin-1 and Tumor Necrosis Factor-α
IL-1 and TNF-α are the predominant catabolic cytokines
involved in the destruction of the articular cartilage.
The first recognition of IL-1 as a regulator of chondro-
cyte function stems largely from the early work of Fell
and ­ others,316,317 who identified a soluble factor, termed
catabolin, in supernatants of normal, noninflamed porcine
synovial fragment cultures that stimulated chondrocytes to
degrade the surrounding cartilage matrix. Similar activities
in ­ culture supernatants from mononuclear cells and rheu-
matoid synovium were attributed to IL-1,318,319 and the
catabolin isoforms were identified as IL-1α and IL-1β.320
Since those early findings, numerous studies in vitro and in
vivo indicate that IL-1 and TNF-α, originating primarily
from the inflamed synovium, are the predominant catabolic
cytokines involved in the destruction of the articular carti-
lage in RA.288-290
Table 3-4  Cytokines That Regulate Cartilage   receptor antagonists and in transgenic or knockout mouse
Destruction
Catabolic Interleukin (IL)-1
Tumor necrosis factor (TNF)-α
IL-17
IL-18
Modulatory IL-6
Leukemia inhibitory factor (LIF)
Oncostatin M
IL-11
Inhibitory IL-4
IL-10
IL-13
IL-1 receptor antagonist (IL-1Ra)
Figure 3-7  The cytokine balance in car-
tilage metabolism. The soluble mediators
toward the left of the balance promote
the loss of cartilage matrix. The mediators
on the right side prevent the synthesis
or actions of the catabolic cytokines and
prevent the loss of cartilage matrix. The
anabolic factors, including insulin-like
IL-1
growth factor I (IGF-I) and bone mor- TNF-α
phogenetic proteins (BMPs), and pros-
taglandin E2 (PGE2) maintain or promote
cartilage matrix synthesis. (Adapted from
Goldring MB: Osteoarthritis and cartilage: Cartilage
The role of cytokines. Curr Rheumatol Rep matrix
2:459-465, 2000.) degradation
The major events in osteoarthritis pathogenesis occur
within the cartilage itself, and there is evidence that the
chondrocytes participate in this destructive process not
only by responding to the cytokines released from other
joint tissues, but also by synthesizing them.321,322 They
may be exposed continuously to the autocrine and para-
crine effects of IL-1 and other inflammatory mediators at
high local concentrations. Chondrocytes in osteoarthritis
cartilage, especially those in clonal clusters, are positive for
IL-1 immunostaining and express IL-1β converting enzyme
(caspase-1) and type 1 IL-1 receptor (IL-1R1).323 IL-1 colo-
calizes with TNF-α, MMP-1, MMP-3, MMP-8, MMP-13,
and type II collagen cleavage epitopes in regions of matrix
depletion in osteoarthritis cartilage.110,292,324 The increased
sensitivity of osteoarthritis chondrocytes to IL-1 and TNF-α
may be associated with increased levels of IL-1R1 and p55
TNF-R at localized sites.325,326 Colocalization of these cyto-
kines, MMPs, and type II collagen cleavage epitopes also
has been reported in regions of matrix depletion in RA
­cartilage.132,327
Originally known as cachectin, TNF-α produces many
effects on chondrocytes in vitro that are similar to those
of IL-1, including stimulation of the production of matrix-
degrading proteinases and suppression of cartilage matrix
synthesis.321,322 Although IL-1 is 100-fold to 1000-fold
more potent on a molar basis than TNF-α, strong synergis-
tic effects occur at low concentrations of the two cytokines
together, eliciting more severe cartilage damage than injec-
tion of either cytokine alone.289 The concept that TNF-α
drives acute inflammation, whereas IL-1 has a pivotal role
in sustaining inflammation and cartilage erosion, has been
derived from work in animal models of RA using cyto-
kine-specific neutralizing antibodies, soluble receptors, or
models.289,290 In a surgically induced osteoarthritis model,
IL-1 knockout mice are protected against cartilage dam-
age.328 A more recent study showed that crossing arthritic
human TNF transgenic (hTNFtg) mice with IL-1α-­deficient
and IL-β-deficient mice protects against cartilage erosion
without affecting synovial inflammation.329
Cytokine Networks
IL-1 and TNF-α also can induce chondrocytes to produce
several other proinflammatory cytokines, including IL-6,
leukemia inhibitory factor, IL-17, and IL-18, and chemo-
kines.321,322,330 IL-6 seems to play a dual role by increasing
Cartilage
IGF-I matrix
BMPs synthesis
IL-4 IL-1Ra PGE2
IL-10 sTNF-R
IL-6 IL-13
LIF IL-8
IL-17 OSM
IL-18
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
the IL-1 receptor antagonist, soluble TNF receptor, and
TIMPs, while also enhancing immune cell function and
inflammation.311,331 The activity of IL-6 requires soluble IL-6
receptor to synergize with IL-1 to stimulate the expression
of MMPs and ADAMTS and to downregulate COL2A1
and aggrecan in cultured chondrocytes.332,333 IL-6 knockout
mice are more susceptible to cartilage degeneration during
aging,334 however, suggesting that this cytokine may play a
protective role in normal physiology.
Other members of the IL-6 family that act via recep-
tors that heterodimerize with gp130 also may serve modu-
latory roles. IL-11 shares several actions of IL-6, including
stimulation of TIMP production without affecting MMP
production by chondrocytes,332 and may inhibit cartilage
destruction.335 Leukemia inhibitory factor participates in a
positive feedback loop by increasing the production of IL-6
by chondrocytes. Oncostatin M (OSM), a product of macro-
phages and activated T cells, is a potent stimulator of chon-
drocyte production of MMPs and aggrecanases in synergism
with IL-1 or TNF-α.298,332,336 Direct evidence supporting a
role for OSM in contributing to cartilage loss in inflamma-
tory arthritis is provided by studies in animal models.337,338
IL-17 and IL-18 are potent catabolic factors that stimu-
late the production of IL-1β, MMPs, IL-6, inducible nitric
oxide synthase (iNOS), cyclooxygenase (COX)-2, and
microsomal PGE synthase-1 (mPGES-1) by human chon-
drocytes.339 IL-17 is produced by activated T helper type 1
(Th1), or CD4+, lymphocytes and binds to a receptor that is
not related to any known cytokine receptor family. IL-17A,
one of at least six family members, is primarily a product
of Th17 cells, a newly described subset of T cells, and is
a potent inducer of catabolic responses in chondrocytes by
itself or in synergy with other cytokines.340 IL-17 can drive
T cell–dependent erosive arthritis in TNF-deficient and
IL-1Ra knockout mice, and treatment of collagen-induced
arthritis or antigen-induced arthritis mice with neutralizing
IL-17 antibody effectively inhibits cartilage destruction in
those models of RA.341 A role for IL-17 in the promotion
of angiogenesis through induction of VEGF in osteoarthritis
chondrocytes and synovial fibroblasts has been proposed.217
IL-18 is produced by macrophages, its receptor shares
homology with IL-1RI, and it has effects similar to IL-1 in
human chondrocytes, but stimulates chondrocyte apopto-
sis.342,343 IL-18 deficiency or blockade with IL-18-neutralizing
antibody or IL-18-binding protein reduces cartilage destruc-
tion and inflammation, and IL-18 gene transfer promotes
IL-1-driven cartilage destruction in a TNF-α-independent
manner.344 Of the other members of the IL-1 family identi-
fied by DNA database searches, IL-1F8 seems to be capable
of stimulating IL-6, IL-8, and nitric oxide production by
human chondrocytes, but at 100-fold to 1000-fold higher
concentrations than IL-1.345 IL-32, a more recently discov-
ered cytokine that induces TNF-α, IL-1β, IL-6, and chemo-
kines and is expressed in the synovia of patients with RA,
contributes to TNF-α-dependent inflammation and carti-
lage proteoglycan loss.346
Inhibitory Cytokines
IL-4, IL-10, IL-13, and the naturally occurring IL-1Ra are clas-
sified as inhibitory cytokines because they decrease the pro-
duction and activities of the catabolic and proinflammatory
55
cytokines in chondrocytes in vitro and suppress cartilage
destruction in vivo (see Table 3-4).289,323,347,348 IL-4 and IL-10
inhibit cartilage-degrading proteinases and reverse some
effects of the catabolic cytokines in vitro, and together they
produce a synergistic suppression of cartilage destruction
in vivo. The efficacy of IL-4, IL-10, and IL-13 in retarding
cartilage damage may be related partly to their stimulatory
effects on IL-1Ra production,323,349 and their therapeutic
application has been proposed as a means of restoring the
cytokine balance in RA.350 IL-1Ra is capable of blocking
the actions of IL-1 if added at sufficiently high concentra-
tions in vitro and is among the first agents to be developed
for anticytokine therapy.288,351 IL-1Ra can be produced by
the same cells that secrete IL-1 and exists as at least three
isoforms, including an intracellular form.
Despite the capacity of IL-4 to inhibit the effects of
proinflammatory cytokines on chondrocyte function,352,353
differential effects have been observed in mice depending
on the model.354,355 Gene transfer of IL-10 in combination
with IL-1Ra inhibits cartilage destruction by a mechanism
involving activin, a TGF-β family member.356 IL-10 is
part of the response induced by immunomodulatory neu-
ropeptides that have been shown to inhibit inflammation
and cartilage and bone destruction by downregulating the
Th1-driven immune response and upregulating IL-10/TGF-
β–producing T regulatory lymphocytes.357 IL-13 decreases
the breakdown of collagen and proteoglycans by inhibit-
ing IL-1-induced and OSM-induced MMP-3 and MMP-13
expression.358 Local gene transfer of IL-13 inhibits chondro-
cyte death and MMP-mediated cartilage degradation despite
enhanced inflammation in the immune complex arthritis
model.359 The inhibitory cytokines may have direct effects
on cartilage metabolism and indirect effects by mediating
the production and actions of catabolic cytokines.
Other Mediators
The balance of mediators determining normal homeosta-
sis is complex, and modulation of their activities may pro-
duce positive or negative effects on chondrocyte function.
In addition to inducing the synthesis of MMPs and other
proteinases by chondrocytes, IL-1 and TNF-α upregulate
the production of nitric oxide via iNOS (or NOS2), and
prostaglandin (PG) E2 by stimulating the expression or
activities of COX-2, mPGES-1, and soluble phospholipase
A2. In the production of prostaglandins, mPGES-1, which
is induced by IL-1 in chondrocytes, is a major player.360,361
Although PGE2 and nitric oxide have been well character-
ized as proinflammatory mediators, there is evidence that
they also may be protective and play roles in chondrocyte
survival and responses to mechanical stress. COX-2 is
involved in the chondrocyte response to high shear stress,
associated with reduced antioxidant capacity and increased
apoptosis.362 The mechanisms of cross-talk between pros-
taglandins and nitric oxide in chondrocytes have been
reviewed.322
Another regulator is peroxisome proliferator-activated
receptor γ (PPARγ), which is activated by the endogenous
ligand 15-deoxy-∆12,14 PGJ2, or 15-deoxy-∆ 12,14. PPARγ
activation opposes the induction of COX-2, iNOS, MMPs,
and mPGES-1 and the suppression of aggrecan synthesis by
IL-1.339,363 Evidence indicates that PPARα agonists may
 56 Goldring  |  Cartilage and Chondrocytes
protect chondrocytes against IL-1-induced responses by
increasing the expression of IL-1Ra.364 The IL-1-induced
COX-2 response depends on the differentiated phenotype
of chondrocytes, and PGE2 opposes the effects of IL-1 by
stimulating type II collagen and inhibiting type I collagen
gene expression.365,366
Roles for nitric oxide as a mediator of other IL-1-induced
responses, including the inhibition of aggrecan synthesis,
enhancement of MMP activity, and reduction in IL-1Ra
synthesis, also have been suggested.367 Nitric oxide also may
increase chondrocyte susceptibility to injury by other oxi-
dants and contribute to the resistance to the anabolic effects
of IGF-I. Nitric oxide also has been implicated as an impor-
tant mediator in chondrocyte apoptosis. PGE2 may mediate
directly the induction of apoptosis by nitric oxide or sensitize
chondrocytes to nitric oxide–induced apoptosis.368 There is
evidence, however, that nitric oxide may inhibit cytokine
production or activity in chondrocytes. IL-1 seems to pro-
tect chondrocytes from CD95-induced apoptosis by a mech-
anism that is independent of IL-1-induced nitric oxide.
Novel mediators that affect chondrocyte metabolism
include the IL-1-induced suppressor of cytokine signaling 3,
which acts as a negative feedback regulator during IGF-I
desensitization in the absence of nitric oxide by inhibit-
ing insulin receptor substrate-1 phosphorylation.240 The
receptor for advanced glycation end products interacts
preferentially with S100A4, a member of the S100 family
of calcium-binding proteins, in chondrocytes and stimu­
lates MMP-13 production.369 The fibroblast activation
protein α, a membrane serine proteinase, which colocalizes
in synovium with MMP-1 and MMP-13 and is induced by
IL-1 and OSM in chondrocytes, may play a role in collagen
degradation.370,371 Many of these proteins may be activated
during the chondrocyte response to abnormal stimuli and
may serve as endogenous mediators of cellular responses to
stress and inflammation.
Adipokines, which were identified originally as products
of adipocytes, also have roles in cartilage metabolism.372
White adipose tissue is a major source of proinflammatory and
anti-inflammatory cytokines, including IL-1Ra and IL-10,373
and the dysregulated balance between leptin and other
adipokines, such as adiponectin, promotes destructive
processes during inflammation.374 Leptin expression is
enhanced during acute inflammation, correlating negatively
with inflammatory markers in RA sera,375 and may serve as
a link between the neuroendocrine and immune systems.376
The elevated expression of leptin in osteoarthritis cartilage
and in osteophytes and its capacity to stimulate IGF-I and
TGF-β1 synthesis suggest a role for this adipokine in ana-
bolic responses of chondrocytes.377 Leptin synergizes with
IL-1 or interferon-γ to increase nitric oxide production in
chondrocytes,378 and leptin deficiency attenuates inflamma-
tory processes in experimental arthritis.379
Chemokines
Chemokines, which are small heparin-binding cytokines
identified originally as chemotactic factors, are classified
as C, CX3C, or CC molecules, indicating the presence of
distinct N-terminal cysteine (C) residues. Chondrocytes,
when activated by IL-1 and TNF-α, express several chemo-
kines, including IL-8, monocyte chemoattractant protein
Table 3-5  Chemokines and Receptors  
in Chondrocytes
Functional Name Systematic Name Chemokine Receptor
GROα CXCL1 CXCR1, CXCR2
IL-8 CXCL8 CXCR1, CXCR2
MCP-1 CCL2 CCR2
MIP-1α CCL3 CCR1, CCR5
MIP-1β CCL4 CCR5
RANTES CCL5 CCR1, CCR3, CCR5
SDF-1 CXCL12 CXCR4
Groα, growth-related oncogene α; IL-8; interleukin-8; MCP-1, monocyte che-
moattractant protein-1; MIP-1, macrophage inhibitory protein-1; RANTES,
regulated on activation, normal T cell expressed and secreted; SDF-1, stromal-
derived growth factor-1.
Chemokines are classified according to the positions of the first two cys-
teines (C) of the four conserved N-terminal cysteines: CC chemokine ligand
(CCL), first two cysteines are adjacent; CXC chemokine ligand (CXCL), first
two cysteines are separated by amino acid X other than cysteine. CCR, CC
chemokine receptor; CXCR, CXC chemokine receptor.
(MCP)-1, MCP-4, macrophage inhibitory protein (MIP)-
1α, MIP-1β, RANTES (regulated on activation normal
T cell expressed and secreted), and growth-related onco-
gene (GRO) α, and the receptors that enable responses
to some of these chemokines, and may feedback regulate
synovial cell responses (Table 3-5).380,381 The first report of
­expression of functional CC and CXC chemokine receptors
(CCR and CXCR) on chondrocytes showed that interac-
tion of these receptors with their corresponding ligands,
MCP-1, RANTES, and GROα, resulted in upregulation of
MMP-3.380,382
Normal and osteoarthritis chondrocytes express the C-C
chemokines, MCP-1, MIP-1α, MIP-1β, and RANTES.
RANTES increases expression of its own receptor, CCR5.
MCP-1 and RANTES increase MMP-3 expression, inhibit
proteoglycan synthesis, and enhance proteoglycan release
from the chondrocytes. The RANTES receptors CCR3
and CCR5, but not CCR1, are expressed in normal carti-
lage, whereas all three receptors are expressed in osteoar-
thritis cartilage or after stimulation of normal chondrocytes
by IL-1β. RANTES induces the expression of iNOS, IL-6,
and MMP-1.
High levels of stromal cell–derived factor 1 are detected
in RA synovial fluids, and its receptor, CXCR4, is expressed
by chondrocytes, but not synovial fibroblasts, suggesting a
direct influence of this chemokine on cartilage damage.383
Microarray studies have elucidated many chemokines that
are inducible in chondrocytes by fibronectin fragments
and cytokines.184 Stromal cell–derived factor 1 and sev-
eral other cytokines also increase the synthesis of S100A,
N-acetyl-β-D-glucuronidase, cathepsin B, and MMPs by
chondrocytes and DNA synthesis, cell proliferation, and
PGE2 production.384,385 Osteoarthritis chondrocytes in
contact with autologous T lymphocytes produce enhanced
levels of MMP-1, MMP-3, MMP-13, and RANTES.386 In
addition to recruiting leukocytes to sites of inflammation in
arthritic joints and mediating synovial fibroblast responses
and actions, chemokines are capable of modulating chon-
drocyte functions that are associated with cartilage degra-
dation.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE 57

pathway is important for signaling by gp130 cytokines,

CYTOKINE SIGNALING PATHWAYS INVOLVED
IN CARTILAGE METABOLISM
Human articular chondrocytes are capable of expressing
receptors of the IL-1R/Toll-like receptor (TLR) superfamily,
including TLR1, TLR2, and TLR4. IL-1, TNF-α, peptido-
glycans, lipopolysaccharide, or fibronectin fragments induce
the expression of TLR2, associated with increased produc-
tion of MMPs, nitric oxide, PGE, and VEGF.387-389 Carti-
lage proteoglycan loss in streptococcal cell wall–induced
arthritis is predominantly dependent on TLR2 signaling.390
In immune complex–mediated arthritis, TLR4 regulates
cartilage destruction by IL-10-mediated upregulation of Fcγ
receptor expression and enhanced cytokine production.391
Although the receptors for IL-1 and TNF-α and associ-
ated adapter molecules are distinct, they share the capac-
ity to activate some of the same signaling pathways (Fig.
3-8).322,348,392,393 The major pathways induced by catabolic
cytokines involve signal transduction by the stress-activated
protein kinases JNKs and p38 kinases and the nuclear factor
κB (NFκB) and PI3K pathways. The JAK/STAT ­signaling
including IL-6 and OSM.394 Specific adapter molecules
involved in the pathways induced by TNF-α receptors,
which are members of the TNF receptor superfamily, are
different from the adapter molecules used by IL-1 signal-
ing pathways. The TNF receptor pathway uses TNF recep-
tor associated factor 2 (TRAF2), TRAF6, and the receptor
interacting protein kinase, whereas the IL-1 receptor path-
way uses TRAF6, IL-1 receptor-associated kinase, and evolu-
tionarily conserved signaling intermediate in Toll pathways
as adapter molecules. Signaling through TNF-RI associated
with TNF receptor–associated death domain receptor acti-
vates apoptosis, whereas TNF-RII signaling through TRAF2
activates JNK and NFκB.
In contrast to ERK-1 and ERK-2, p38 and JNK are
weakly activated by growth factors. Studies in chondrocytes
in vitro have shown that the p38 and JNK cascades mediate
the induction of proteinases and proinflammatory genes by
IL-1 and TNF-α.348 These pathways also may be activated in
chondrocytes by mechanical stress and cartilage matrix prod-
ucts via integrins and other receptor-­mediated events.395-397

IL-1 gp130
IL-1RAcP IL-1R1
JAK JAK
PI3K
TIR TIR
MyD88 MyD88 P P
St

3
DD DD

at
at

St
3

IRAK IRAK2

TRAF6
MEKKI RIP

AkT
MKK3/6 MKK4/7 NIK

p38 MAPK SAPK/JNK IKK1/2

P
IκB
IκB

P P P
Fo s ATF- 2 Jun P
Stat3
Stat3

P P NFκB
P ETS P

Nuclear Translocation
CBP
Sp1 B DNA Binding B
Basal Transcriptio n Target Gene Transactivation
Figure 3-8  Intracellular signaling pathways activated by interleukin-1 (IL-1) in chondrocytes. Binding of IL-1 to the type I IL-1 receptor (IL-1R1) leads
to the recruitment of the IL-1R accessory protein (IL-1RAcP). The cytoplasmic Toll/IL-1 receptor (TIR) domains of the receptor recruit the MyD88 via its
TIR, and the MyD88 death domain (DD) recruits the IL-1 receptor–associated kinases (IRAK and IRAK2) to the receptor complex before being rapidly
phosphorylated and degraded. The IRAKs mediate tumor necrosis factor receptor–associated factor 6 (TRAF6) oligomerization, initiating various pro-
tein kinase cascades, the major ones of which involve (1) the stress-activated protein kinases, p38 mitogen-activated protein kinase (MAPK), and c-Jun 
N-terminal kinase (JNK), which lead to activation of activator protein-1 (AP-1) (c-Fos/c-Jun), activating transcription factor-2 (ATF-2), and E Twenty Six
(ETS) factors, among other transcription factors, and (2) inhibitor of κB (IκB) kinases 1 and 2 (IKK-1 and IKK-2), which lead to activation of nuclear factor κB
(NFκB). TNF-α also stimulates these pathways, but via TRAF2 and TRAF5. Other signaling pathways also may influence the target gene responses, such
as the growth factor–induced or chemokine-induced phosphatidylinositol 3-kinase (PI3K) via the serine/threonine kinase, Akt/protein kinase B, and
the gp130 cytokine–induced janus activating kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. The responses of the target
genes depend on the presence of DNA sequences within the respective promoters that bind to the various transcription factors.
 58 Goldring  |  Cartilage and Chondrocytes
The upregulation of IL-1 and TNF-α expression via these
pathways suggests their involvement as secondary media-
tors in a feedback mechanism. At least four isoforms of p38
MAPK exist with different substrate specificities and differ-
ential effects on essential chondrocyte functions.
JNKs are serine threonine protein kinases that phosphor-
ylate Jun family members, components of AP-1 transcrip-
tion factors, and they exist as three JNK isoforms, JNK1,
JNK2, and JNK3, in humans. A potent JNK1/2 inhibitor,
SP600125, which blocks inflammation and joint damage in
animal models of RA, and other JNK isoform–specific inhib-
itors are useful tools for analyzing chondrocyte function in
vitro and in vivo. Activated JNK is detected in osteoarthri-
tis, but not in normal cartilage, and JNK inhibition attenu-
ates cytokine-induced chondrocyte responses.398,399 NFκB is
a “master switch” of the inflammatory cascade.400 NFκB is
actvated when the IκB kinases IKK-1 and IKK-2 phosphory-
late IκB, dissociating it from NFκB and permitting translo-
cation of active NFκB to the nucleus. NFκB mediates the
expression of cytokines and chemokines induced by fibro-
nectin fragments,184 and inhibition of DNA-binding activ-
ity of NFκB by agents that deplete polyamine blocks IL-1
and TNF-α without promoting chondrocyte apoptosis.401 In
addition to NFκB, transcription factors that are members of
the C/EBP, ETS, and AP-1 families are important for the
regulation of gene expression by IL-1 and TNF-α in chon-
drocytes.87
ROLE OF THE CHONDROCYTE
IN CARTILAGE REPAIR
AGING OF ARTICULAR CARTILAGE
It is important, but often difficult, to distinguish among
the effects of aging itself and diseases such as osteoarthri-
tis that become more common with increasing age.220,402
In both cases, biochemical alterations in matrix composi-
tion are reflected in changes in cartilage structure.403 The
thickness of articular cartilage, as shown by magnetic res-
onance imaging, decreases with increasing age.404 Fatigue
fracture of superficial collagen bundles and a heterogeneous
depletion of glycosaminoglycans at the periphery of joint
surfaces may contribute to the mild splitting and fraying
of superficial cartilage, which is termed fibrillation. If fibril-
lation progresses into deeper layers of cartilage, abnormal
multicellular clusters of chondrocytes that stain intensely
for glycosaminoglycans are found at the base of clefts. These
changes include decreased size and aggregation of aggrecan
and increased collagen denaturation, resulting in the loss of
compressive stiffness and tensile strength.9
Zonal differences in tensile strength and compressive
resistance are related to differences in matrix composition
and can be observed to change during the aging of adult
articular cartilage and in response to traumatic damage. The
territorial, or pericellular, matrix and the interterritorial
matrix differ in the amounts and types of matrix proteins. The
chondrocytes are normally surrounded by a 2-μm pericellular
matrix, composed of a highly branched filamentous network
of collagen VI tetramers, which serves as a scaffold for deco-
rin, biglycan, perlecan, and chondroadherin, which predom-
inate in this region, and hyaluronan, fibrilin-1, and PRELP.
The interterritorial region, in contrast, contains primarily
a collagen II/XI fibril network, which binds decorin, fibro-
modulin, collagen IX, and COMP, and large numbers of
intact aggrecan molecules attached via link protein to long
chains of hyaluronic acid. In the deep zone, the interter-
ritorial region most remote from the cells contains a larger
number of degraded aggrecan molecules that lack the G3
domain.
Proteoglycans in aged cartilage have a wide range of
sizes, with small forms resulting from low substitution of
glycosaminoglycan residues and shorter lengths compared
with glycosaminoglycans in young articular cartilage.9
Unsubstituted proteoglycan core proteins of aggrecan and
biglycan are detectable in articular cartilage from elderly
subjects. Hyaluronan content increases in aged cartilage,
but with a reduced mean chain length, and link protein
also seems to be fragmented. Collagen fibrils become thin-
ner with age and are less densely packed. Nonenzymatic
glycation results in the formation and accumulation of the
advanced-­glycation end product pentosidine in long-lived
proteins, including cartilage collagen and aggrecan.405 Such
biochemical changes may result partly from changes in
chondrocyte synthetic function and from increased suscep-
tibility of the matrix to degradation.406 Increased and more
extensive collagen degradation can be observed in cartilage
from older, healthy individuals, and similar to cartilage in
early osteoarthritis, the damage is concentrated closer to the
­articular surface and colocalizes with MMP-13 activity.110
(See Chapter 89 for a detailed discussion of the pathogen-
esis of osteoarthritis.)
AGING CHONDROCYTE
Chondrocyte function, including mitotic and synthetic
activity, deteriorates with age. Degradative changes are gen-
erally due to the actions of proteinases and are, at least par-
tially, the cumulative consequences of adverse conditions,
such as mechanic insults or inflammation, to which the
chondrocyte is exposed throughout life. Deficiencies in car-
tilage matrix proteins also may disrupt chondrocyte-matrix
interactions that are important to cell survival. The decline
in chondrocyte number also may be attributed to increased
cell death with age. Although programmed cell death, or
apoptosis, increases with age in adult rats and mice, this
may be due to skeletal growth that occurs throughout life
in these animals. In human adult cartilage, apoptotic cell
removal does not seem to be common, however.41 Repli-
cative senescence, detected as β-galactosidase activity and
decreased telomere length, has been proposed to contribute
to the age-related changes in the proliferative potential of
adult articular chondrocytes.407,408
TGF-β, FGFs, IGF-I, and other anabolic factors that sup-
port cartilage matrix biosynthesis are expressed at declining
levels with aging, or their activities are downregulated.220
The capacity of BMP-6 to stimulate proteoglycan synthesis
and the production of BMP-7 (OP-1) decline with age.223,270
Chondrocytes also show an age-related decline in the ana-
bolic response to IGF-I, possibly owing to increased synthesis
of IGFBP-3, which is itself antiproliferative. Chondrocytes
from elderly donors depend strongly on IGF-I and IGF-II for
survival.226 It has been proposed that the reduction in TGF-β
signaling in aging chondrocytes may be a factor in their
reduced capacity to repair cartilage.409
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
MARKERS OF CARTILAGE MATRIX DEGRADATION
AND TURNOVER
With increasing knowledge of the composition of the car-
tilage matrix, molecular markers in body fluids have been
identified for monitoring changes in cartilage metabolism
and for assessing joint damage in arthritis.33-35,410 Molecules
originating from the articular cartilage, including aggrecan
fragments, which contain chondroitin sulfate and keratan
sulfate; type II collagen fragments; collagen pyridinoline
cross-links; and COMP, are usually released as degradation
products as a result of catabolic processes. Specific mono-
clonal antibodies have been developed for analyzing osteo-
arthritis and RA body fluids for products of proteoglycan
or collagen degradation (catabolic epitopes) or synthesis
of newly synthesized matrix components (anabolic neo-
epitopes), which represent attempts to repair the damaged
matrix. Different monoclonal antibodies can distinguish
subtle biochemical differences in chondroitin sulfate or ker-
atan sulfate chains that result from degraded versus newly
synthesized proteoglycans. Such epitopes can be detected in
the synovial fluids and serum of patients with osteoarthritis
and RA, and the synovial fluid-to-serum ratio has been sug-
gested as a potential diagnostic indicator. The degradation of
aggrecan in cartilage has been characterized using antibod-
ies 846, 3B3(−), and 7D4, which detect chondroitin sulfate
neoepitopes; 5D4, which detects keratan sulfate epitopes;
and the VIDIPEN and NITEGE antibodies, which recog-
nize aggrecanase and MMP cleavage sites within the inter-
globular G1 domain of aggrecan (see Chapter 32).300,301
Similarly, the synthesis of type II collagen can be moni-
tored by measuring serum and synovial fluid levels of the
carboxyl-terminal propeptide, and urinary excretion of hydro­
xylysyl pyridinoline cross-links may indicate collagen degra-
dation.34,35 Specific antibodies that recognize epitopes on
denatured type II collagen at the collagenase cleavage site
are promising diagnostic reagents. These include the C2C
antibody (previously known as Col2-3/4C Long mono) that
has been used to detect cleavage of the triple helix of type
II collagen in experimental models and in osteoarthritis
and RA cartilage.110,327 The ratios of these markers to the
synthetic marker, CPII, are associated with a greater likeli-
hood of radiologic progression in osteoarthritis patients.36
These biomarker assays have been used as research tools and
are currently being developed and validated as diagnostic
tools for monitoring cartilage degradation or repair in osteo-
arthritis and RA patient populations and for assessment
of treatment (see Chapters 67 and 91). Although a single
marker may be insufficient, it may be possible eventually to
determine a combination of biomarkers that may discrimi-
nate between different stages of osteoarthritis in different
populations.
REPAIR OF ARTICULAR CARTILAGE
Articular cartilage has a poor capacity for regeneration, and
pharmacologic enhancement of cartilage repair would have
considerable potential in the treatment of arthritides and
intra-articular fractures. The extent of intrinsic repair of
a cartilage defect depends on the depth of the lesion and
whether the defect penetrates the subchondral bone plate.411
Repair of superficial defects occurs if the chondrocytes
59
remain viable. Owing to the avascularity of cartilage, it dif-
fers from most other tissues in its response to injury. The vas-
cular-dependent inflammatory and reparative phases of the
classic healing response are unavailable. Partial defects gen-
erally do not regenerate because resident chondrocytes can-
not migrate into the defect, and there is no vascular access
for progenitor cells. Deep cartilage defects with disruption
of the subchondral bone plate initiate vascular responses,
however, including bleeding, fibrin clot ­ formation, and
inflammation, which permit cell invasion from the blood
or underlying bone marrow. The lesion becomes filled by
granulation tissue, which is eventually replaced by fibro-
cartilage, but rarely by true hyaline cartilage. Current
procedures for cartilage repair include joint lavage, tissue
débridement, microfracture of the subchondral bone, and
the transplantation of autologous or allogeneic osteochon-
dral grafts, in addition to the ultimate therapy of total joint
replacement.411 These procedures may lead to the formation
of fibrous tissue, chondrocyte death, and further cartilage
degeneration and have variable success rates.
Transplantation of cultured autologous chondrocytes has
been used successfully to repair small, full-thickness lesions
in knee cartilage in young adults with sports injuries.412 Evi-
dence of successful repair has been shown by turnover and
remodeling of the initial fibrocartilaginous matrix formed
by the transplanted chondrocytes owing to enzyme degra-
dation and new synthesis of type II collagen.413 The donor
site, although not load bearing, may undergo significant
morbidity and osteoarthritic changes. A randomized, con-
trolled trial suggests little difference in efficacy compared
with microfracture of the subchondral bone.414
Major challenges for cartilage repair are the restoration
of the three-dimensional collagen structure and integra-
tion of the newly synthesized matrix with the resident tis-
sue.411,415 Novel approaches using autologous chondrocytes
genetically engineered ex vivo to express anabolic fac-
tors have been explored to promote differentiation before
implantation in the defect.225,416 The induction of the syn-
thesis of IGF-I, TGF-β, BMP-2, and BMP-7 by gene transfer
increases the synthesis of cartilage proteoglycans and col-
lagens in cultured chondrocytes.417 Many of these factors,
including BMP-2, BMP-4, BMP-6, BMP-7, BMP-9, TGF-β,
and CDMP-1, are able to induce chondrogenic differentia-
tion of mesenchymal progenitor cells in vitro.418,419 BMP-2
and BMP-7 (OP-1) are approved for multiple indications in
the area of bone fracture repair and spinal fusion and can
promote cartilage repair in various models of focal cartilage
defects. The introduction of BMPs into joints via in vivo or
ex vivo gene delivery or in injectable or implantable carriers
has been investigated for the repair of small defects in ani-
mal models.420 Findings in vitro using BMP-2, BMP-9, and
BMP-13 suggest, however, that they may serve as potent
anabolic factors for juvenile cartilage, which contains chon-
droprogenitors, but not for adult cartilage.421
Several studies have shown that injection of free TGF-β
or adenovirus-mediated delivery of TGF-β promotes fibrosis
and osteophyte formation, while stimulating proteoglycan
synthesis in cartilage. Based on these observations, local
application of molecules that block endogenous TGF-β sig­
nal­ing, such as the soluble form of TGF-βRII, inhibitory
SMADs, or the physiologic antagonist, latency-associated
peptide-1, have been proposed as a means for blocking
 60 Goldring  |  Cartilage and Chondrocytes
osteophyte formation.422 Gene transfer of combinations of  
anabolic factors and inhibitory cytokines combined with
cartilage engineering approaches may be a long-term goal
for repair of the extensive defects in RA or late osteoar-  
thritis patients and for the prevention of further damage.
The use of bone marrow–derived chondroprogenitor cells or
 
mesenchymal stem cells from other sources as gene delivery
vehicles to the site of cartilage damage is a promising strat-
egy for the future, although many challenges remain.  
SUMMARY AND CONCLUSION  
As the single cellular component in adult articular carti-  
lage, chondrocytes are responsible for maintaining the ECM
components in a low-turnover state. The composition and
organization of matrix macromolecules, unique to this tis-
sue, are determined during chondrocyte differentiation in
embryonic and postnatal development of cartilage. Adult
chondrocytes exist in a hypoxic environment within articu-
lar cartilage. They are inactive metabolically, partially as a
result of an absence of blood vessels and nerves, and display
a rounded morphology that reflects their quiescent state.
Chondrocyte culture models have been developed with the
aim of maintaining differentiated phenotypes, characterized
by the major collagen and proteoglycan constituents, type II
collagen and aggrecan. Chondrocytes interact with specific
ECM components via integrins, annexins, and CD44 on the
cell surface.
Studies in vitro and in vivo have shown that adult articu-
lar chondrocytes are capable of responding to biologic and
mechanical stimuli that are anabolic or catabolic. Anabolic
factors include members of the TGF-β/BMP family and IGF-I.
Catabolic factors include proinflammatory cytokines, such
as IL-1, TNF-α, IL-17, and IL-18, which stimulate the syn-
thesis of matrix-degrading proteinases, such as MMPs and
aggrecanases, and inhibit cartilage matrix-protein synthesis.
Many of the signaling pathways and transcription factors
that mediate the responses of chondrocytes to these factors
have been elucidated, but how they orchestrate the specific
chondrocyte functions is complex and not fully understood.
Under physiologic conditions, the adult articular chondro-
cyte maintains a stable equilibrium between the synthesis
and the degradation of matrix components. Adult chondro-
cytes have a poor capacity for mediating effective repair of
extensive cartilage lesions, and this capacity declines with
age. Age-related changes in chondrocyte function decrease
the ability of the cells to maintain the tissue, including
decreased synthetic activity, synthesis of smaller and less uni-
form aggrecan molecules and less functional link proteins,
and decreased responsiveness to anabolic growth factors.
Further understanding of how the adult articular chondro-
cyte functions within its unique environment would aid
in the development of rational strategies for maintaining
homeostasis and protecting against cartilage damage.
REFERENCES
  1. Benedek TG: A history of the understanding of cartilage. Osteoar-
thritis Cartilage 14:203-209, 2006.
  2. Otero M, Goldring MB: Cells of the synovium in rheumatoid arthri-
tis. Chandrocytes. Arthritis Res Ther, 9:220, 2007.
  3. Goldring MB, Goldring SR: Osteoarthritis. J Cell Physiol 213:
626-634, 2007.
4. Goldring MB: Update on the chondrocyte lineage and implica-
tions for cell therapy in osteoarthritis. In Sharma L, Berenbaum F
(eds): Osteoarthritis. A Companion to Rheumatology. Philadelphia,
Mosby, 2007, pp 53-76.
5. Goldring MB: The musculoskeletal system, B: Articular cartilage. In
Klippel JH, Crofford LJ, Stone JH, et al (eds): Primer on the Rheu-
matic Diseases. Atlanta, Arthritis Foundation, 2001, pp 10-16.
6. Poole AR: Cartilage in health and disease. In Koopman W (ed):
Arthritis and Allied Conditions: A Textbook of Rheumatology.
Philadelphia, Lippincott Williams & Wilkins, 2005, pp 223-269.
7. Stockwell RA: Metabolism of cartilage. In Hall BK (ed): Cartilage:
Structure, Function and Biochemistry, Vol 1. New York Academic
Press, 1983, pp 253-280.
8. Eyre DR, Weis MA, Wu JJ: Articular cartilage collagen: An irre-
placeable framework? Eur Cell Mater 12:57-63, 2006.
9. Dudhia J: Aggrecan, aging and assembly in articular cartilage. Cell
Mol Life Sci 62:2241-2256, 2005.
10. Poole AR, Pidoux I, Reiner A, et al: An immunoelectron microscope
study of the organization of proteoglycan monomer, link protein, and col-
lagen in the matrix of articular cartilage. J Cell Biol 93:921-937, 1982.
11. Hyllested JL, Veje K, Ostergaard K: Histochemical studies of the
extracellular matrix of human articular cartilage—a review. Osteo-
arthritis Cartilage 10:333-343, 2002.
12. Poole AR, Kojima T, Yasuda T, et al: Composition and structure of artic-
ular cartilage: A template for tissue repair. Clin Orthop 391:S26-S33,
2001.
13. Hunziker EB, Michel M, Studer D: Ultrastructure of adult human
articular cartilage matrix after cryotechnical processing. Microsc Res
Tech 37:271-284, 1997.
14. Stockwell RA: Inter-relationship of articular cartilage thickness and
cellularity. Ann Rheum Dis 31:424, 1972.
15. Wong M, Wuethrich P, Buschmann MD, et al: Chondrocyte biosyn-
thesis correlates with local tissue strain in statically compressed adult
articular cartilage. J Orthop Res 15:189-196, 1997.
16. Muir H, Bullough P, Maroudas A: The distribution of collagen in
human articular cartilage with some of its physiological implications.
J Bone Joint Surg Br 52:554-563, 1970.
17. Stanescu V, Stanescu R, Maroteaux P: [Differences in distribution
of type I and type II collagens in the superficial and intermediary
zones of articular cartilage]. C R Acad Sci Hebd Seances Acad Sci D
283:279-282, 1976.
18. Maroudas A, Muir H, Wingham J: The correlation of fixed negative
charge with glycosaminoglycan content of human articular cartilage.
Biochim Biophys Acta 177:492-500, 1969.
19. Franzen A, Inerot S, Hejderup SO, et al: Variations in the composi-
tion of bovine hip articular cartilage with distance from the articular
surface. Biochem J 195:535-543, 1981.
20. Ratcliffe A, Fryer PR, Hardingham TE: The distribution of aggregat-
ing proteoglycans in articular cartilage: Comparison of quantitative
immunoelectron microscopy with radioimmunoassay and biochemi-
cal analysis. J Histochem Cytochem 32:193-201, 1984.
21. Lane LB, Villacin A, Bullough PG: The vascularity and remodel-
ling of subchondrial bone and calcified cartilage in adult human
femoral and humeral heads: An age- and stress-related phenom-
enon. J Bone Joint Surg Br 59:272-278, 1977.
22. Kempson GE, Muir H, Swanson SA, et al: Correlations between stiff-
ness and the chemical constituents of cartilage on the human femoral
head. Biochim Biophys Acta 215:70-77, 1970.
23. Kempson GE, Muir H, Pollard C, et al: The tensile properties of
the cartilage of human femoral condyles related to the content of
collagen and glycosaminoglycans. Biochim Biophys Acta 297:456-
472, 1973.
24. Schmidt MB, Mow VC, Chun LE, et al: Effects of proteoglycan
extraction on the tensile behavior of articular cartilage. J Orthop Res
8:353-363, 1990.
25. Urban JP, Maroudas A, Bayliss MT, et al: Swelling pressures of
proteoglycans at the concentrations found in cartilaginous tissues.
Biorheology 16:447-464, 1979.
26. Lai WM, Hou JS, Mow VC: A triphasic theory for the swelling and
deformation behaviors of articular cartilage. J Biomech Eng 113:
245-258, 1991.
27. Savarirayan R, Rimoin DL: The skeletal dysplasias. Best Pract Res
Clin Endocrinol Metab 16:547-560, 2002.
28. Helminen HJ, Saamanen AM, Salminen H, et al: Transgenic mouse
models for studying the role of cartilage macromolecules in osteoar-
thritis. Rheumatology (Oxf) 41:848-856, 2002.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
29. Young MF: Mouse models of osteoarthritis provide new research
tools. Trends Pharmacol Sci 26:333-335, 2005.
30. Saamanen AM, Vuorio E: Generation and use of transgenic mice as
models of osteoarthritis. Methods Mol Med 101:1-23, 2004.
31. Ho MS, Tsang KY, Lo RL, et al: COL10A1 nonsense and frame-
shift mutations have a gain-of-function effect on the growth plate in
human and mouse metaphyseal chondrodysplasia type Schmid. Hum
Mol Genet 16:1201-1215, 2007.
32. Li Y, Xu L, Olsen BR: Lessons from genetic forms of osteoar-
thritis for the pathogenesis of the disease. Osteoarthritis Cartilage
15:1101-1105, 2007.
33. Goldring SR, Goldring MB: Rheumatoid arthritis and other inflam-
matory joint pathologies. In Seibel MJ, Robins SP, Bilezikian JP
(eds): Dynamics of Bone and Cartilage Metabolism: Principles
and Clinical Applications. San Diego, Academic Press, 2006, pp
843-869.
34. Charni-Ben Tabassi N, Garnero P: Monitoring cartilage turnover.
Curr Rheumatol Rep 9:16-24, 2007.
35. Rousseau JC, Delmas PD: Biological markers in osteoarthritis. Nat
Clin Pract Rheumatol 3:346-356, 2007.
36. Cahue S, Sharma L, Dunlop D, et al: The ratio of type II collagen
breakdown to synthesis and its relationship with the progression of
knee osteoarthritis. Osteoarthritis Cartilage 15:819-823, 2007.
37. von der Mark K: Structure, biosynthesis, and gene regulation of col-
lagens in cartilage and bone. In Seibel MJ, Robins SP, Bilezekian JP
(eds): Dynamics of Bone and Cartilage Metabolism: Principles and
Clinical Applications. San Diego, Academic Press, 2006, pp 3-40.
38. Bruckner P: Supramolecular structure of cartilage matrix. In Seibel
MJ, Robins SP, Bilezekian JP (eds): Dynamics of Bone and Cartilage
Metabolism: Principles and Clinical Applications. San Diego, Aca-
demic Press, 2006, pp 407-420.
39. Sandell LJ, Nalin AM, Reife RA: Alternative splice form of type II
procollagen mRNA (IIA) is predominant in skeletal precursors and
non-cartilaginous tissues during early mouse development. Dev Dyn
199:129-140, 1994.
40. Sandell LJ, Aigner T: Articular cartilage and changes in arthritis: An
introduction: Cell biology of osteoarthritis. Arthritis Res 3:107-113,
2001.
41. Aigner T, Haag J, Martin J, et al: Osteoarthritis: Aging of matrix and
cells—going for a remedy. Curr Drug Targets 8:325-331, 2007.
42. Vertel BM: The ins and outs of aggrecan. Trends Cell Biol 5:458-464,
1995.
43. Knudson CB, Knudson W: Cartilage proteoglycans. Semin Cell Dev
Biol 12:69-78, 2001.
44. Hardingham T: Proteoglycans and glycosaminoglycans. In Seibel
MJ, Robins SP, Bilezekian JP (eds): Dynamics of Bone and Cartilage
Metabolism: Principles and Clinical Applications. San Diego, Aca-
demic Press, 2006, pp 85-98.
45. Heinegard D, Lorenzo P, Saxne T: Noncollagenous proteins: Glyco-
proteins and related proteins. In Seibel MJ, Robins SP, Bilezekian JP
(eds): Dynamics of Bone and Cartilage Metabolism: Principles and
Clinical Applications. San Diego, Academic Press, 2006, pp 71-84.
46. Svensson L, Oldberg A, Heinegard D: Collagen binding proteins.
Osteoarthritis Cartilage 9(Suppl A):S23-S28, 2001.
47. Thur J, Rosenberg K, Nitsche DP, et al: Mutations in cartilage oligo-
meric matrix protein causing pseudoachondroplasia and multiple
epiphyseal dysplasia affect binding of calcium and collagen I, II, and
IX. J Biol Chem 276:6083-6092, 2001.
48. Zhang Y, Chen Q: Changes of matrilin forms during endochondral
ossification: Molecular basis of oligomeric assembly. J Biol Chem
275:32628-32634, 2000.
49. Segat D, Paulsson M, Smyth N: Matrilins: Structure, expression and
function. Osteoarthritis Cartilage 9(Suppl A):S29-S35, 2001.
50. Iwamoto M, Tamamura Y, Koyama E, et al: Transcription factor ERG
and joint and articular cartilage formation during mouse limb and
spine skeletogenesis. Dev Biol 305:40-51, 2007.
51. Ghert MA, Qi WN, Erickson HP, et al: Tenascin-C expression and
distribution in cultured human chondrocytes and chondrosarcoma
cells. J Orthop Res 20:834-841, 2002.
52. Lorenzo P, Bayliss MT, Heinegard D: A novel cartilage protein
(CILP) present in the mid-zone of human articular cartilage increases
with age. J Biol Chem 273:23463-23468, 1998.
53. Hirose J, Ryan LM, Masuda I: Up-regulated expression of cartilage
intermediate-layer protein and ANK in articular hyaline cartilage
from patients with calcium pyrophosphate dihydrate crystal deposi-
tion disease. Arthritis Rheum 46:3218-3229, 2002.
61
54. Lorenzo P, Aspberg A, Onnerfjord P, et al: Identification and char-
acterization of asporin: A novel member of the leucine-rich repeat
protein family closely related to decorin and biglycan. J Biol Chem
276:12201-12211, 2001.
55. Henry SP, Takanosu M, Boyd TC, et al: Expression pattern and gene
characterization of asporin: A newly discovered member of the leu-
cine-rich repeat protein family. J Biol Chem 276:12212-12221, 2001.
56. Kizawa H, Kou I, Iida A, et al: An aspartic acid repeat polymorphism
in asporin inhibits chondrogenesis and increases susceptibility to
osteoarthritis. Nat Genet 37:138-144, 2005.
57. Recklies AD, White C, Ling H: The chitinase 3-like protein human
cartilage glycoprotein 39 (HC-gp39) stimulates proliferation of
human connective-tissue cells and activates both extracellular sig-
nal-regulated kinase- and protein kinase B-mediated signalling path-
ways. Biochem J 365:119-126, 2002.
58. Ling H, Recklies AD: The chitinase 3-like protein human cartilage
glycoprotein 39 inhibits cellular responses to the inflammatory cyto-
kines interleukin-1 and tumour necrosis factor-alpha. Biochem J
380:651-659, 2004.
59. Recklies AD, Ling H, White C, et al: Inflammatory cytokines induce
production of chitinase-3-like protein 1 by articular chondrocytes. J
Biol Chem 280:41213-41221, 2005.
60. Sekine T, Masuko-Hongo K, Matsui T, et al: Recognition of YKL-39,
a human cartilage related protein, as a target antigen in patients with
rheumatoid arthritis. Ann Rheum Dis 60:49-54, 2001.
61. Knorr T, Obermayr F, Bartnik E, et al: YKL-39 (chitinase 3-like pro-
tein 2), but not YKL-40 (chitinase 3-like protein 1), is up regulated
in osteoarthritic chondrocytes. Ann Rheum Dis 62:995-998, 2003.
62. Stockwell RA, Meachim G: The chondrocytes. In Freeman MAR
(ed): Adult Articular Cartilage. Tunbridge Wells, England, Pitman
Medical, 1979, pp 69-144.
63. Aydelotte MB, Kuettner KE: Differences between sub-populations of
cultured bovine articular chondrocytes, I: Morphology and cartilage
matrix production. Connect Tissue Res 18:205-222, 1988.
64. Aydelotte MB, Greenhill RR, Kuettner KE: Differences between sub-
populations of cultured bovine articular chondrocytes, II: Proteogly-
can metabolism. Connect Tissue Res 18:223-234, 1988.
65. Quinn TM, Hunziker EB, Hauselmann HJ: Variation of cell and
matrix morphologies in articular cartilage among locations in the
adult human knee. Osteoarthritis Cartilage 13:672-678, 2005.
66. Bush PG, Hall AC: The volume and morphology of chondrocytes
within non-degenerate and degenerate human articular cartilage.
Osteoarthritis Cartilage 11:242-251, 2003.
67. Poole CA, Jensen CG, Snyder JA, et al: Confocal analysis of primary
cilia structure and colocalization with the Golgi apparatus in chondro-
cytes and aortic smooth muscle cells. Cell Biol Int 21:483-494, 1997.
68. Goldring MB, Tsuchimochi K, Ijiri K: The control of chondrogen-
esis. J Cell Biochem 97:33-44, 2006.
69. Hill TP, Spater D, Taketo MM, et al: Canonical Wnt/beta-catenin
signaling prevents osteoblasts from differentiating into chondrocytes.
Dev Cell 8:727-738, 2005.
70. Tamamura Y, Otani T, Kanatani N, et al: Developmental regula-
tion of Wnt/beta-catenin signals is required for growth plate assem-
bly, cartilage integrity, and endochondral ossification. J Biol Chem
280:19185-19195, 2005.
71. Day TF, Guo X, Garrett-Beal L, et al: Wnt/beta-catenin signaling in
mesenchymal progenitors controls osteoblast and chondrocyte differ-
entiation during vertebrate skeletogenesis. Dev Cell 8:739-750, 2005.
72. Lefebvre V, Smits P: Transcriptional control of chondrocyte fate and
differentiation. Birth Defects Res C Embryo Today 75:200-212, 2005.
73. Yoon BS, Ovchinnikov DA, Yoshii I, et al: Bmpr1a and Bmpr1b have
overlapping functions and are essential for chondrogenesis in vivo.
Proc Natl Acad Sci U S A 102:5062-5067, 2005.
74. Huang W, Lu N, Eberspaecher H, et al: A new long form of c-Maf
cooperates with Sox9 to activate the type II collagen gene. J Biol
Chem 277:50668-50675, 2002.
75. Huang Z, Xu H, Sandell L: Negative regulation of chondrocyte dif-
ferentiation by transcription factor AP-2alpha. J Bone Miner Res
19:245-255, 2004.
76. Davies SR, Sakano S, Zhu Y, et al: Distribution of the transcrip-
tion factors Sox9, AP-2, and [delta]EF1 in adult murine articular
and meniscal cartilage and growth plate. J Histochem Cytochem 50:
1059-1065, 2002.
77. Kronenberg HM: PTHrP and skeletal development. Ann N Y Acad
Sci 1068:1-13, 2006.
 62 Goldring  |  Cartilage and Chondrocytes
78. Karsenty G, Wagner EF: Reaching a genetic and molecular under-
standing of skeletal development. Dev Cell 2:389-406, 2002.
79. Colnot C: Cellular and molecular interactions regulating skeletogen-
esis. J Cell Biochem 95:688-697, 2005.
80. Komori T: Regulation of skeletal development by the Runx family of
transcription factors. J Cell Biochem 95:445-453, 2005.
81. Hassan MQ, Javed A, Morasso MI, et al: Dlx3 transcriptional regu-
lation of osteoblast differentiation: Temporal recruitment of Msx2,
Dlx3, and Dlx5 homeodomain proteins to chromatin of the osteocal-
cin gene. Mol Cell Biol 24:9248-9261, 2004.
82. Lengner CJ, Hassan MQ, Serra RW, et al: Nkx3.2-mediated repres-
sion of Runx2 promotes chondrogenic differentiation. J Biol Chem
280:15872-15879, 2005.
83. Bialek P, Kern B, Yang X, et al: A twist code determines the onset of
osteoblast differentiation. Dev Cell 6:423-435, 2004.
84. Wang W, Wang YG, Reginato AM, et al: Groucho homologue Grg5
interacts with the transcription factor Runx2-Cbfa1 and modulates its
activity during postnatal growth in mice. Dev Biol 270:364-381, 2004.
85. Vega RB, Matsuda K, Oh J, et al: Histone deacetylase 4 controls
chondrocyte hypertrophy during skeletogenesis. Cell 119:555-566,
2004.
86. Provot S, Schipani E: Molecular mechanisms of endochondral bone
development. Biochem Biophys Res Commun 328:658-665, 2005.
87. Goldring MB, Sandell LJ: Transcriptional control of chondrocyte
gene expression. In Buckwalter J, Lotz M, Stoltz JF (eds): OA,
Inflammation and Degradation: A Continuum. Amsterdam IOS
Press, 2007, pp 118-142.
88. Wilkins RJ, Browning JA, Ellory JC: Surviving in a matrix: Membrane
transport in articular chondrocytes. J Membr Biol 177:95-108, 2000.
89. Langelier E, Suetterlin R, Hoemann CD, et al: The chondrocyte
cytoskeleton in mature articular cartilage: Structure and distribution
of actin, tubulin, and vimentin filaments. J Histochem Cytochem
48:1307-1320, 2000.
90. Woods A, Wang G, Beier F: Regulation of chondrocyte differen-
tiation by the actin cytoskeleton and adhesive interactions. J Cell
Physiol 213:1-8, 2007.
91. Lee RB, Wilkins RJ, Razaq S, et al: The effect of mechanical stress on
cartilage energy metabolism. Biorheology 39:133-143, 2002.
92. Sengers BG, Heywood HK, Lee DA, et al: Nutrient utilization by
bovine articular chondrocytes: A combined experimental and theo-
retical approach. J Biomech Eng 127:758-766, 2005.
93. Shikhman AR, Brinson DC, Lotz MK: Distinct pathways regulate
facilitated glucose transport in human articular chondrocytes during
anabolic and catabolic responses. Am J Physiol Endocrinol Metab
286:E980-E985, 2004.
94. Mobasheri A, Richardson S, Mobasheri R, et al: Hypoxia inducible
factor-1 and facilitative glucose transporters GLUT1 and GLUT3:
Putative molecular components of the oxygen and glucose sens-
ing apparatus in articular chondrocytes. Histol Histopathol 20:
1327-1338, 2005.
95. Ruiz-Romero C, Lopez-Armada MJ, Blanco FJ: Proteomic charac-
terization of human normal articular chondrocytes: A novel tool for
the study of osteoarthritis and other rheumatic diseases. Proteomics
5:3048-3059, 2005.
96. Pufe T, Lemke A, Kurz B, et al: Mechanical overload induces VEGF
in cartilage discs via hypoxia-inducible factor. Am J Pathol 164:
185-192, 2004.
97. Lin C, McGough R, Aswad B, et al: Hypoxia induces HIF-1alpha
and VEGF expression in chondrosarcoma cells and chondrocytes. J
Orthop Res 22:1175-1181, 2004.
98. Grimshaw MJ, Mason RM: Modulation of bovine articular chondro-
cyte gene expression in vitro by oxygen tension. Osteoarthritis Car-
tilage 9:357-364, 2001.
99. Robins JC, Akeno N, Mukherjee A, et al: Hypoxia induces chon-
drocyte-specific gene expression in mesenchymal cells in association
with transcriptional activation of Sox9. Bone 37:313-322, 2005.
100. Pfander D, Cramer T, Schipani E, et al: HIF-1alpha controls extra-
cellular matrix synthesis by epiphyseal chondrocytes. J Cell Sci
116:1819-1826, 2003.
101. Coimbra IB, Jimenez SA, Hawkins DF, et al: Hypoxia inducible
factor-1 alpha expression in human normal and osteoarthritic chon-
drocytes. Osteoarthritis Cartilage 12:336-345, 2004.
102. Brucker PU, Izzo NJ, Chu CR: Tonic activation of hypoxia-­inducible
factor 1alpha in avascular articular cartilage and implications for
metabolic homeostasis. Arthritis Rheum 52:3181-3191, 2005.
103. Hofstaetter JG, Wunderlich L, Samuel RE, et al: Systemic hypoxia alters
gene expression levels of structural proteins and growth factors in knee
joint cartilage. Biochem Biophys Res Commun 330:386-394, 2005.
104. Jubb RW: Effect of hyperoxia on articular tissues in organ culture.
Ann Rheum Dis 38:279-286, 1979.
105. Martin G, Andriamanalijaona R, Grassel S, et al: Effect of hypoxia and
reoxygenation on gene expression and response to interleukin-1 in cul-
tured articular chondrocytes. Arthritis Rheum 50:3549-3560, 2004.
106. Yudoh K, Nakamura H, Masuko-Hongo K, et al: Catabolic stress
induces expression of hypoxia-inducible factor (HIF)-1alpha in artic-
ular chondrocytes: Involvement of HIF-1alpha in the pathogenesis of
osteoarthritis. Arthritis Res Ther 7:R904-R914, 2005.
107. Maroudas A, Palla G, Gilav E: Racemization of aspartic acid in
human articular cartilage. Connect Tissue Res 28:161-169, 1992.
108. Verzijl N, DeGroot J, Thorpe SR, et al: Effect of collagen turnover on
the accumulation of advanced glycation end products. J Biol Chem
275:39027-39031, 2000.
109. Maroudas A, Bayliss MT, Uchitel-Kaushansky N, et al: Aggrecan turn-
over in human articular cartilage: Use of aspartic acid racemization as a
marker of molecular age. Arch Biochem Biophys 350:61-71, 1998.
110. Wu W, Billinghurst RC, Pidoux I, et al: Sites of collagenase cleav-
age and denaturation of type II collagen in aging and osteoarthritic
articular cartilage and their relationship to the distribution of matrix
metalloproteinase 1 and matrix metalloproteinase 13. Arthritis
Rheum 46:2087-2094, 2002.
111. Pritzker KP, Gay S, Jimenez SA, et al: Osteoarthritis cartilage histo-
pathology: Grading and staging. Osteoarthritis Cartilage 14:13-29,
2006.
112. Poole AR, Guilak F, Abramson SB: Etiopathogenesis of osteoar-
thritis. In Moskowitz RW, Altman RW, Hochberg MC, et al (eds):
Osteoarthritis: Diagnosis and Medical/Surgical Management. Phila-
delphia, Lippincott Williams & Wilkins, 2007, pp 27-49.
113. Miosge N, Hartmann M, Maelicke C, et al: Expression of collagen
type I and type II in consecutive stages of human osteoarthritis. His-
tochem Cell Biol 122:229-236, 2004.
114. Tchetina EV, Squires G, Poole AR: Increased type II collagen degra-
dation and very early focal cartilage degeneration is associated with
upregulation of chondrocyte differentiation related genes in early
human articular cartilage lesions. J Rheumatol 32:876-886, 2005.
115. Bau B, Gebhard PM, Haag J, et al: Relative messenger RNA expression
profiling of collagenases and aggrecanases in human articular chondro-
cytes in vivo and in vitro. Arthritis Rheum 46:2648-2657, 2002.
116. Hermansson M, Sawaji Y, Bolton M, et al: Proteomic analysis of
articular cartilage shows increased type II collagen synthesis in osteo-
arthritis and expression of inhibin betaA (activin A), a regulatory
molecule for chondrocytes. J Biol Chem 279:43514-43521, 2004.
117. Aigner T, Fundel K, Saas J, et al: Large-scale gene expression profil-
ing reveals major pathogenetic pathways of cartilage degeneration in
osteoarthritis. Arthritis Rheum 54:3533-3544, 2006.
118. Fukui N, Zhu Y, Maloney WJ, et al: Stimulation of BMP-2 expres-
sion by pro-inflammatory cytokines IL-1 and TNF-α in normal and
osteoarthritic chondrocytes. J Bone Joint Surg Am 85A(Suppl 3):
59-66, 2003.
119. Nakase T, Miyaji T, Tomita T, et al: Localization of bone morpho-
genetic protein-2 in human osteoarthritic cartilage and osteophyte.
Osteoarthritis Cartilage 11:278-284, 2003.
120. Tchetina EV, Antoniou J, Tanzer M, et al: Transforming growth
factor-beta2 suppresses collagen cleavage in cultured human osteo-
arthritic cartilage, reduces expression of genes associated with chon-
drocyte hypertrophy and degradation, and increases prostaglandin
E(2) production. Am J Pathol 168:131-140, 2006.
121. Kolettas E, Muir HI, Barrett JC, et al: Chondrocyte phenotype and
cell survival are regulated by culture conditions and by specific cyto-
kines through the expression of Sox-9 transcription factor. Rheuma-
tology (Oxf) 40:1146-1156, 2001.
122. Thirion S, Berenbaum F: Culture and phenotyping of chondrocytes
in primary culture. Methods Mol Med 100:1-14, 2004.
123. Goldring MB: Human chondrocyte cultures as models of cartilage-
specific gene regulation. Methods Mol Med 107:69-95, 2005.
124. Salvat C, Pigenet A, Humbert L, et al: Immature murine articular
chondrocytes in primary culture: A new tool for investigating carti-
lage. Osteoarthritis Cartilage 13:243-249, 2005.
125. Poole AR: Honor Bridgett Fell, Ph.D., D.Sc. F.R.S., D.B.E., 1900-
1986. The scientist and her contributions. In Vitro Cell Dev Biol
25:450-453, 1989.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
126. Hascall VC, Handley CJ, McQuillan DJ, et al: The effect of serum on
biosynthesis of proteoglycans by bovine articular cartilage in culture.
Arch Biochem Biophys 224:206-223, 1983.
127. Campbell MA, Handley CJ, Hascall VC, et al: Turnover of proteo-
glycans in cultures of bovine articular cartilage. Arch Biochem Bio-
phys 234:275-289, 1984.
128. Goldring MB, Sandell LJ, Stephenson ML, et al: Immune interferon
suppresses levels of procollagen mRNA and type II collagen synthesis
in cultured human articular and costal chondrocytes. J Biol Chem
261:9049-9056, 1986.
129. Goldring MB, Krane SM: Modulation by recombinant interleu-
kin 1 of synthesis of types I and III collagens and associated pro-
collagen mRNA levels in cultured human cells. J Biol Chem 262:
16724-16729, 1987.
130. Schnabel M, Marlovits S, Eckhoff G, et al: Dedifferentiation-associ-
ated changes in morphology and gene expression in primary human
articular chondrocytes in cell culture. Osteoarthritis Cartilage 10:
62-70, 2002.
131. Hiraki Y, Mitsui K, Endo N, et al: Molecular cloning of human chon-
dromodulin-I, a cartilage-derived growth modulating factor, and its
expression in Chinese hamster ovary cells. Eur J Biochem 260:869-
878, 1999.
132. Tetlow LC, Woolley DE: Expression of vitamin D receptors and
matrix metalloproteinases in osteoarthritic cartilage and human
articular chondrocytes in vitro. Osteoarthritis Cartilage 9:423-431,
2001.
133. Grogan SP, Barbero A, Diaz-Romero J, et al: Identification of markers
to characterize and sort human articular chondrocytes with enhanced
in vitro chondrogenic capacity. Arthritis Rheum 56:586-595, 2007.
134. Barbero A, Grogan SP, Mainil-Varlet P, et al: Expansion on specific
substrates regulates the phenotype and differentiation capacity of
human articular chondrocytes. J Cell Biochem 98:1140-1149, 2006.
135. Thonar EJ, Buckwalter JA, Kuettner KE: Maturation-related dif-
ferences in the structure and composition of proteoglycans synthe-
sized by chondrocytes from bovine articular cartilage. J Biol Chem
261:2467-2474, 1986.
136. Chua KH, Aminuddin BS, Fuzina NH, et al: Insulin-transferrin-sele-
nium prevent human chondrocyte dedifferentiation and promote the
formation of high quality tissue engineered human hyaline cartilage.
Eur Cell Mater 9:58-67; discussion 67, 2005.
137. Lin Z, Willers C, Xu J, et al: The chondrocyte: Biology and clinical
application. Tissue Eng 12:1971-1984, 2006.
138. Yates KE, Allemann F, Glowacki J: Phenotypic analysis of bovine
chondrocytes cultured in 3D collagen sponges: Effect of serum substi-
tutes. Cell Tissue Bank 6:45-54, 2005.
139. Capito RM, Spector M: Effect of expansion medium on ex vivo gene
transfer and chondrogenesis in type II collagen-glycosaminoglycan
scaffolds in vitro. Osteoarthritis Cartilage 14:1203-1213, 2006.
140. Dell’Accio F, De Bari C, Luyten FP: Molecular markers predictive
of the capacity of expanded human articular chondrocytes to form
stable cartilage in vivo. Arthritis Rheum 44:1608-1619, 2001.
141. Kisiday JD, Kurz B, DiMicco MA, et al: Evaluation of medium sup-
plemented with insulin-transferrin-selenium for culture of primary
bovine calf chondrocytes in three-dimensional hydrogel scaffolds.
Tissue Eng 11:141-151, 2005.
142. Loeser RF, Chubinskaya S, Pacione C, et al: Basic fibroblast growth
factor inhibits the anabolic activity of insulin-like growth factor 1
and osteogenic protein 1 in adult human articular chondrocytes.
Arthritis Rheum 52:3910-3917, 2005.
143. Jenniskens YM, Koevoet W, de Bart AC, et al: Biochemical and
functional modulation of the cartilage collagen network by IGF1,
TGFbeta2 and FGF2. Osteoarthritis Cartilage 14:1136-1146, 2006.
144. Schulze-Tanzil G, de Souza P, Villegas Castrejon H, et al: Rediffer-
entiation of dedifferentiated human chondrocytes in high-density
cultures. Cell Tissue Res 308:371-379, 2002.
145. Tallheden T, Karlsson C, Brunner A, et al: Gene expression during
redifferentiation of human articular chondrocytes. Osteoarthritis
Cartilage 12:525-535, 2004.
146. Tew SR, Li Y, Pothacharoen P, et al: Retroviral transduction with
SOX9 enhances re-expression of the chondrocyte phenotype in pas-
saged osteoarthritic human articular chondrocytes. Osteoarthritis
Cartilage 13:80-89, 2005.
147. Horton WE Jr, Cleveland J, Rapp U, et al: An established rat cell
line expressing chondrocyte properties. Exp Cell Res 178:457-468,
1988.
63
148. Mallein-Gerin F, Olsen BR: Expression of simian virus 40 large T
(tumor) oncogene in chondrocytes induces cell proliferation with-
out loss of the differentiated phenotype. Proc Natl Acad Sci U S A
90:3289-3293, 1993.
149. Lefebvre V, Garofalo S, deCrombrugghe B: Type X collagen gene
expression in mouse chondrocytes immortalized by a tempera-
ture-sensitive simian virus 40 large tumor antigen. J Cell Biol 128:
239-245, 1995.
150. Mataga N, Tamura M, Yanai N, et al: Establishment of a novel chon-
drocyte-like cell line derived from transgenic mice harboring the
temperature-sensitive simian virus 40 large T-antigen. J Bone Min
Res 11:1646-1654, 1996.
151. Johnstone B, Barthel T, Yoo J: In vitro chondrogenesis with mam-
malian progenitor cells. In Rosier RV, Evans CH (eds): Molecular
Biology of Orthopaedics. Rosemont, Ill, American Academy of Ortho­
paedic Surgeons, 2003, pp 273-287.
152. Block JA, Inerot SE, Gitelis S, et al: Synthesis of chondrocytic keratan
sulphate-containing proteoglycans by human chondrosarcoma cells in
long-term cell culture. J Bone Joint Surg Am 73:647-658, 1991.
153. Takigawa M, Pan HO, Kinoshita A, et al: Establishment from a
human chondrosarcoma of a new immortal cell line with high
tumorigenicity in vivo, which is able to form proteoglycan-rich car-
tilage-like nodules and to respond to insulin in vitro. Int J Cancer
48:717-725, 1991.
154. Goldring MB: Culture of immortalized chondrocytes and their use as
models of chondrocyte function. Methods Mol Med 100:37-52, 2004.
155. Robbins JR, Thomas B, Tan L, et al: Immortalized human adult
articular chondrocytes maintain cartilage-specific phenotype and
responses to interleukin-1β. Arthritis Rheum 43:2189-2201, 2000.
156. Grigolo B, Roseti L, Neri S, et al: Human articular chondrocytes
immortalized by HPV-16 E6 and E7 genes: Maintenance of differ-
entiated phenotype under defined culture conditions. Osteoarthritis
Cartilage 10:879-889, 2002.
157. Piera-Velazquez S, Jimenez SA, Stokes D: Increased life span of
human osteoarthritic chondrocytes by exogenous expression of
telomerase. Arthritis Rheum 46:683-693, 2002.
158. Finger F, Schorle C, Zien A, et al: Molecular phenotyping of human
chondrocyte cell lines T/C-28a2, T/C-28a4, and C-28/I2. Arthritis
Rheum 48:3395-3403, 2003.
159. Gebauer M, Saas J, Sohler F, et al: Comparison of the chondrosar-
coma cell line SW1353 with primary human adult articular chon-
drocytes with regard to their gene expression profile and reactivity to
IL-1beta. Osteoarthritis Cartilage 13:697-708, 2005.
160. Helfrich MH, Horton MA: Integrins and other adhesion molecules.
In Seibel MJ, Robins SP, Bilezikian JP (eds): Dynamics of Bone and
Cartilage Metabolism: Principles and Clinical Applications. San
Diego, Academic Press, 2006, pp 129-151.
161. Leitinger B, Hohenester E: Mammalian collagen receptors. Matrix
Biol 26:146-155, 2007.
162. Mobasheri A, Carter SD, Martin-Vasallo P, et al: Integrins and
stretch activated ion channels: Putative components of functional
cell surface mechanoreceptors in articular chondrocytes. Cell Biol
Int 26:1-18, 2002.
163. Lucchinetti E, Bhargava MM, Torzilli PA: The effect of mechani-
cal load on integrin subunits alpha5 and beta1 in chondrocytes
from mature and immature cartilage explants. Cell Tissue Res 315:
385-391, 2004.
164. Millward-Sadler SJ, Salter DM: Integrin-dependent signal cascades in
chondrocyte mechanotransduction. Ann Biomed Eng 32:435-446, 2004.
165. Loeser RF: Integrins and cell signaling in chondrocytes. Biorheology
39:119-124, 2002.
166. Chen FH, Thomas AO, Hecht JT, et al: Cartilage oligomeric matrix
protein/thrombospondin 5 supports chondrocyte attachment through
interaction with integrins. J Biol Chem 280:32655-32661, 2005.
167. Loeser RF, Sadiev S, Tan L, et al: Integrin expression by primary and
immortalized human chondrocytes: Evidence of a differential role
for alpha1beta1 and alpha2beta1 integrins in mediating chondro-
cyte adhesion to types II and VI collagen. Osteoarthritis Cartilage
8:96-105, 2000.
168. Kapyla J, Jaalinoja J, Tulla M, et al: The fibril-associated collagen IX
provides a novel mechanism for cell adhesion to cartilaginous matrix.
J Biol Chem 279:51677-51687, 2004.
169. Enomoto-Iwamoto M, Iwamoto M, Nakashima K, et al: Involvement
of alpha5beta1 integrin in matrix interactions and proliferation of
chondrocytes. J Bone Miner Res 12:1124-1132, 1997.
 64 Goldring  |  Cartilage and Chondrocytes
170. Camper L, Holmvall K, Wangnerud C, et al: Distribution of the col-
lagen-binding integrin alpha10beta1 during mouse development.
Cell Tissue Res 306:107-116, 2001.
171. Garciadiego-Cazares D, Rosales C, Katoh M, et al: Coordination
of chondrocyte differentiation and joint formation by alpha5beta1
integrin in the developing appendicular skeleton. Development
131:4735-4742, 2004.
172. Bengtsson T, Aszodi A, Nicolae C, et al: Loss of alpha10beta1 inte-
grin expression leads to moderate dysfunction of growth plate chon-
drocytes. J Cell Sci 118:929-936, 2005.
173. Wang W, Kirsch T: Annexin V/beta5 integrin interactions regu-
late apoptosis of growth plate chondrocytes. J Biol Chem 281:
30848-30856, 2006.
174. Aszodi A, Hunziker EB, Brakebusch C, et al: Beta1 integrins regulate
chondrocyte rotation, G1 progression, and cytokinesis. Genes Dev
17:2465-2479, 2003.
175. Zemmyo M, Meharra EJ, Kuhn K, et al: Accelerated, aging-­dependent
development of osteoarthritis in alpha1 integrin-deficient mice.
Arthritis Rheum 48:2873-2880, 2003.
176. Shakibaei M, John T, De Souza P, et al: Signal transduction by beta1
integrin receptors in human chondrocytes in vitro: Collaboration
with the insulin-like growth factor-I receptor. Biochem J 342(Pt 3):
615-623, 1999.
177. Clancy RM, Rediske J, Tang X, et al: Outside-in signaling in the
chondrocyte: Nitric oxide disrupts fibronectin-induced assembly
of a subplasmalemmal actin/rho A/focal adhesion kinase signaling
complex. J Clin Invest 100:1789-1796, 1997.
178. Schneiderbauer MM, Dutton CM, Scully SP: Signaling “cross-talk”
between TGF-beta1 and ECM signals in chondrocytic cells. Cell Sig-
nal 16:1133-1140, 2004.
179. Homandberg GA, Costa V, Wen C: Fibronectin fragments active
in chondrocytic chondrolysis can be chemically cross-linked to
the alpha5 integrin receptor subunit. Osteoarthritis Cartilage 10:
938-949, 2002.
180. Forsyth CB, Pulai J, Loeser RF: Fibronectin fragments and blocking
antibodies to alpha2beta1 and alpha5beta1 integrins stimulate mito-
gen-activated protein kinase signaling and increase collagenase 3
(matrix metalloproteinase 13) production by human articular chon-
drocytes. Arthritis Rheum 46:2368-2376, 2002.
181. Yasuda T, Poole AR: A fibronectin fragment induces type II collagen
degradation by collagenase through an interleukin-1-mediated path-
way. Arthritis Rheum 46:138-148, 2002.
182. Millward-Sadler SJ, Khan NS, Bracher MG, et al: Roles for the
interleukin-4 receptor and associated JAK/STAT proteins in human
articular chondrocyte mechanotransduction. Osteoarthritis Cartilage
14:991-1001, 2006.
183. Attur MG, Dave MN, Clancy RM, et al: Functional genomic analy-
sis in arthritis-affected cartilage: Yin-yang regulation of inflammatory
mediators by alpha 5 beta 1 and alpha V beta 3 integrins. J Immunol
164:2684-2691, 2000.
184. Pulai JI, Chen H, Im HJ, et al: NF-kappa B mediates the stimulation
of cytokine and chemokine expression by human articular chondro-
cytes in response to fibronectin fragments. J Immunol 174:5781-
5788, 2005.
185. Del Carlo M, Schwartz D, Erickson EA, et al: Endogenous produc-
tion of reactive oxygen species is required for stimulation of human
articular chondrocyte matrix metalloproteinase production by
fibronectin fragments. Free Radic Biol Med 42:1350-1358, 2007.
186. Heino J: The collagen receptor integrins have distinct ligand recog-
nition and signaling functions. Matrix Biol 19:319-323, 2000.
187. Chowdhury TT, Appleby RN, Salter DM, et al: Integrin-mediated
mechanotransduction in IL-1 beta stimulated chondrocytes. Bio-
mech Model Mechanobiol 5:192-201, 2006.
188. Kurtis MS, Tu BP, Gaya OA, et al: Mechanisms of chondrocyte
adhesion to cartilage: Role of beta1-integrins, CD44, and annexin V.
J Orthop Res 19:1122-1130, 2001.
189. Knudson CB, Knudson W: Hyaluronan and CD44: Modulators of
chondrocyte metabolism. Clin Orthop 427:S152-S162, 2004.
190. Tibesku CO, Szuwart T, Ocken SA, et al: Expression of the matrix
receptor CD44v5 on chondrocytes changes with osteoarthritis:
An experimental investigation in the rabbit. Ann Rheum Dis 65:
105-108, 2006.
191. Ohno S, Im HJ, Knudson CB, et al: Hyaluronan oligosaccharides
induce matrix metalloproteinase 13 via transcriptional activation
of NFkappaB and p38 MAP kinase in articular chondrocytes. J Biol
Chem 281:17952-17960, 2006.
192. Yasuda T, Poole AR, Shimizu M, et al: Involvement of CD44 in
induction of matrix metalloproteinases by a COOH-terminal hepa-
rin-binding fragment of fibronectin in human articular cartilage in
culture. Arthritis Rheum 48:1271-1280, 2003.
193. Yasuda T, Kakinuma T, Julovi SM, et al: COOH-terminal hepa-
rin-binding fibronectin fragment induces nitric oxide production in
rheumatoid cartilage through CD44. Rheumatology (Oxf) 43:1116-
1120, 2004.
194. Embry Flory JJ, Fosang AJ, Knudson W: The accumulation of intra-
cellular ITEGE and DIPEN neoepitopes in bovine articular chondro-
cytes is mediated by CD44 internalization of hyaluronan. Arthritis
Rheum 54:443-454, 2006.
195. Stewart MC, Fosang AJ, Bai Y, et al: ADAMTS5-mediated aggre-
canolysis in murine epiphyseal chondrocyte cultures. Osteoarthritis
Cartilage 14:392-402, 2006.
196. von der Mark K, Mollenhauer J: Annexin V interactions with col-
lagen. Cell Mol Life Sci 53:539-545, 1997.
197. Brachvogel B, Welzel H, Moch H, et al: Sequential expression of
annexin A5 in the vasculature and skeletal elements during mouse
development. Mech Dev 109:389-393, 2001.
198. Kirsch T: Annexins—their role in cartilage mineralization. Front
Biosci 10:576-581, 2005.
199. Lucic D, Mollenhauer J, Kilpatrick KE, et al: N-telopeptide of type II
collagen interacts with annexin V on human chondrocytes. Connect
Tissue Res 44:225-239, 2003.
200. Salminen-Mankonen H, Saamanen AM, Jalkanen M, et al: Synde-
can-1 expression is upregulated in degenerating articular cartilage
in a transgenic mouse model for osteoarthritis. Scand J Rheumatol
34:469-474, 2005.
201. Pfander D, Swoboda B, Kirsch T: Expression of early and late dif-
ferentiation markers (proliferating cell nuclear antigen, syndecan-3,
annexin VI, and alkaline phosphatase) by human osteoarthritic
chondrocytes. Am J Pathol 159:1777-1783, 2001.
202. Xu L, Peng H, Wu D, et al: Activation of the discoidin domain
receptor 2 induces expression of matrix metalloproteinase 13 asso-
ciated with osteoarthritis in mice. J Biol Chem 280:548-555, 2005.
203. Leitinger B, Kwan AP: The discoidin domain receptor DDR2 is a
receptor for type X collagen. Matrix Biol 25:355-364, 2006.
204. Xu L, Peng H, Glasson S, et al: Increased expression of the colla-
gen receptor discoidin domain receptor 2 in articular cartilage as a
key event in the pathogenesis of osteoarthritis. Arthritis Rheum 56:
2663-2673, 2007.
205. Walsh DA, Bonnet CS, Turner EL, et al: Angiogenesis in the
synovium and at the osteochondral junction in osteoarthritis. Osteo-
arthritis Cartilage 15:743-751, 2007.
206. Moses MA, Wiederschain D, Wu I, et al: Troponin I is present in
human cartilage and inhibits angiogenesis. Proc Natl Acad Sci U S A
96:2645-2650, 1999.
207. Shukunami C, Hiraki Y: Role of cartilage-derived anti-angiogenic
factor, chondromodulin-I, during endochondral bone formation.
Osteoarthritis Cartilage 9(Suppl A):S91-S101, 2001.
208. O’Reilly MS, Boehm T, Shing Y, et al: Endostatin: An endogenous
inhibitor of angiogenesis and tumor growth. Cell 88:277-285, 1997.
209. Pufe T, Petersen WJ, Miosge N, et al: Endostatin/collagen XVIII—an
inhibitor of angiogenesis—is expressed in cartilage and fibrocartilage.
Matrix Biol 23:267-276, 2004.
210. Pfander D, Kortje D, Zimmermann R, et al: Vascular endothelial
growth factor in articular cartilage of healthy and osteoarthritic
human knee joints. Ann Rheum Dis 60:1070-1073, 2001.
211. Pufe T, Petersen W, Tillmann B, et al: The splice variants VEGF121
and VEGF189 of the angiogenic peptide vascular endothelial growth
factor are expressed in osteoarthritic cartilage. Arthritis Rheum
44:1082-1088, 2001.
212. Pufe T, Kurz B, Petersen W, et al: The influence of biomechanical
parameters on the expression of VEGF and endostatin in the bone
and joint system. Ann Anat 187:461-472, 2005.
213. Mentlein R, Pufe T: New functions of angiogenic peptides in osteo-
arthritis cartilage. Curr Rheum Rev 1:37-43, 2005.
214. Green DM, Noble PC, Ahuero JS, et al: Cellular events leading to
chondrocyte death after cartilage impact injury. Arthritis Rheum
54:1509-1517, 2006.
215. Fearon U, Griosios K, Fraser A, et al: Angiopoietins, growth factors,
and vascular morphology in early arthritis. J Rheumatol 30:260-268,
2003.
216. Szekanecz Z, Gaspar L, Koch AE: Angiogenesis in rheumatoid arthri-
tis. Front Biosci 10:1739-1753, 2005.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
217. Honorati MC, Neri S, Cattini L, et al: Interleukin-17, a regulator of
angiogenic factor release by synovial fibroblasts. Osteoarthritis Carti-
lage 14:345-352, 2006.
218. Fearon U, Mullan R, Markham T, et al: Oncostatin M induces angio-
genesis and cartilage degradation in rheumatoid arthritis synovial tissue
and human cartilage cocultures. Arthritis Rheum 54:3152-3162, 2006.
219. Mould AW, Tonks ID, Cahill MM, et al: Vegfb gene knockout mice
display reduced pathology and synovial angiogenesis in both antigen-
induced and collagen-induced models of arthritis. Arthritis Rheum
48:2660-2669, 2003.
220. Loeser RF: Molecular mechanisms of cartilage destruction: Mechan-
ics, inflammatory mediators, and aging collide. Arthritis Rheum
54:1357-1360, 2006.
221. Reddi AH: Cartilage morphogenetic proteins: Role in joint develop-
ment, homeostasis, regeneration. Ann Rheum Dis 62(Suppl 2):ii73-
ii78, 2003.
222. van der Kraan PM, Buma P, van Kuppevelt T, et al: Interaction of
chondrocytes, extracellular matrix and growth factors: Relevance
for articular cartilage tissue engineering. Osteoarthritis Cartilage
10:631-637, 2002.
223. Chubinskaya S, Kuettner KE: Regulation of osteogenic proteins by
chondrocytes. Int J Biochem Cell Biol 35:1323-1340, 2003.
224. Trippel SB: Growth factor inhibition: Potential role in the etiopatho-
genesis of osteoarthritis. Clin Orthop 427:S47-S52, 2004.
225. Lories RJ, Luyten FP: Bone morphogenetic protein signaling in
joint homeostasis and disease. Cytokine Growth Factor Rev 16:
287-298, 2005.
226. Loeser RF, Pacione CA, Chubinskaya S: The combination of insu-
lin-like growth factor 1 and osteogenic protein 1 promotes increased
survival of and matrix synthesis by normal and osteoarthritic human
articular chondrocytes. Arthritis Rheum 48:2188-2196, 2003.
227. Studer RK, Decker K, Melhem S, et al: Nitric oxide inhibition of
IGF-1 stimulated proteoglycan synthesis: Role of cGMP. J Orthop
Res 21:914-921, 2003.
228. De Ceuninck F, Caliez A, Dassencourt L, et al: Pharmacological
disruption of insulin-like growth factor 1 binding to IGF-binding
proteins restores anabolic responses in human osteoarthritic chon-
drocytes. Arthritis Res Ther 6:R393-R403, 2004.
229. Morales TI, Hunziker EB: Localization of insulin-like growth factor
binding protein-2 in chondrocytes of bovine articular cartilage. J
Orthop Res 21:290-295, 2003.
230. Morales TI: The insulin-like growth factor binding proteins in uncul-
tured human cartilage: Increases in insulin-like growth factor binding
protein 3 during osteoarthritis. Arthritis Rheum 46:2358-2367, 2002.
231. Iwanaga H, Matsumoto T, Enomoto H, et al: Enhanced expression of
insulin-like growth factor-binding proteins in human osteoarthritic
cartilage detected by immunohistochemistry and in situ hybridiza-
tion. Osteoarthritis Cartilage 13:439-448, 2005.
232. Whellams EJ, Maile LA, Fernihough JK, et al: Alterations in insulin-
like growth factor binding protein-3 proteolysis and complex forma-
tion in the arthritic joint. J Endocrinol 165:545-556, 2000.
233. Neidel J: Changes in systemic levels of insulin-like growth factors
and their binding proteins in patients with rheumatoid arthritis. Clin
Exp Rheumatol 19:81-84, 2001.
234. Tyler JA: Insulin-like growth factor 1 can decrease degradation and
promote synthesis of proteoglycan in cartilage exposed to cytokines.
Biochem J 260:543-548, 1989.
235. Olney RC, Wilson DM, Mohtai M, et al: Interleukin-1 and tumor
necrosis factor-alpha increase insulin-like growth factor-binding
protein-3 (IGFBP-3) production and IGFBP-3 protease activity in
human articular chondrocytes. J Endocrinol 146:279-286, 1995.
236. Clancy R: Nitric oxide alters chondrocyte function by disrupting cyto-
skeletal signaling complexes. Osteoarthritis Cartilage 7:399-400, 1999.
237. van den Berg WB, van de Loo F, Joosten LA, et al: Animal mod-
els of arthritis in NOS2-deficient mice. Osteoarthritis Cartilage 7:
413-415, 1999.
238. Johnson K, Jung A, Murphy A, et al: Mitochondrial oxidative phos-
phorylation is a downstream regulator of nitric oxide effects on
chondrocyte matrix synthesis and mineralization. Arthritis Rheum
43:1560-1570, 2000.
239. Loeser RF, Carlson CS, Del Carlo M, et al: Detection of nitrotyrosine
in aging and osteoarthritic cartilage: Correlation of oxidative damage
with the presence of interleukin-1beta and with chondrocyte resis-
tance to insulin-like growth factor 1. Arthritis Rheum 46:2349-2357,
2002.
65
240. Smeets RL, Veenbergen S, Arntz OJ, et al: A novel role for SOCS3
in cartilage destruction via induction of chondrocyte desensitization
towards IGF-1. Arthritis Rheum 54:1518-1528, 2006.
241. Ornitz DM: FGF signaling in the developing endochondral skeleton.
Cytokine Growth Factor Rev 16:205-213, 2005.
242. Veilleux N, Spector M: Effects of FGF-2 and IGF-1 on adult canine
articular chondrocytes in type II collagen-glycosaminoglycan scaf-
folds in vitro. Osteoarthritis Cartilage 13:278-286, 2005.
243. Sah RL, Chen AC, Grodzinsky AJ, et al: Differential effects of bFGF
and IGF-I on matrix metabolism in calf and adult bovine cartilage
explants. Arch Biochem Biophys 308:137-147, 1994.
244. Vincent T, Hermansson M, Bolton M, et al: Basic FGF mediates an
immediate response of articular cartilage to mechanical injury. Proc
Natl Acad Sci U S A 99:8259-8264, 2002.
245. Vincent TL, Hermansson MA, Hansen UN, et al: Basic fibroblast
growth factor mediates transduction of mechanical signals when
articular cartilage is loaded. Arthritis Rheum 50:526-533, 2004.
246. Murakami S, Kan M, McKeehan WL, et al: Up-regulation of the
chondrogenic Sox9 gene by fibroblast growth factors is mediated by
the mitogen-activated protein kinase pathway. Proc Natl Acad Sci
U S A 97:1113-1118, 2000.
247. Schaefer JF, Millham ML, de Crombrugghe B, et al: FGF signaling
antagonizes cytokine-mediated repression of Sox9 in SW1353 chon-
drosarcoma cells. Osteoarthritis Cartilage 11:233-241, 2003.
248. Weksler NB, Lunstrum GP, Reid ES, et al: Differential effects of
fibroblast growth factor (FGF) 9 and FGF2 on proliferation, differ-
entiation and terminal differentiation of chondrocytic cells in vitro.
Biochem J 342(Pt 3):677-682, 1999.
249. Ellsworth JL, Berry J, Bukowski T, et al: Fibroblast growth factor-18
is a trophic factor for mature chondrocytes and their progenitors.
Osteoarthritis Cartilage 10:308-320, 2002.
250. Au A, Polotsky A, Krzyminski K, et al: Evaluation of thermorev-
ersible polymers containing fibroblast growth factor 9 (FGF-9) for
chondrocyte culture. J Biomed Mater Res A 69:367-372, 2004.
251. Shimoaka T, Ogasawara T, Yonamine A, et al: Regulation of osteo-
blast, chondrocyte, and osteoclast functions by fibroblast growth fac-
tor (FGF)-18 in comparison with FGF-2 and FGF-10. J Biol Chem
277:7493-7500, 2002.
252. Moore EE, Bendele AM, Thompson DL, et al: Fibroblast growth fac-
tor-18 stimulates chondrogenesis and cartilage repair in a rat model
of injury-induced osteoarthritis. Osteoarthritis Cartilage 13:623-631,
2005.
253. Newman B, Gigout LI, Sudre L, et al: Coordinated expression of
matrix Gla protein is required during endochondral ossification for
chondrocyte survival. J Cell Biol 154:659-666, 2001.
254. Stheneur C, Dumontier MF, Guedes C, et al: Basic fibroblast growth
factor as a selective inducer of matrix Gla protein gene expression in
proliferative chondrocytes. Biochem J 369:63-70, 2003.
255. Reddi AH: Marshall R. Urist: A renaissance scientist and orthopae-
dic surgeon. J Bone Joint Surg Am 85A(Suppl 3):3-7, 2003.
256. Pogue R, Lyons K: BMP signaling in the cartilage growth plate. Curr
Top Dev Biol 76:1-48, 2006.
257. Grimaud E, Heymann D, Redini F: Recent advances in TGF-beta
effects on chondrocyte metabolism: Potential therapeutic roles
of TGF-beta in cartilage disorders. Cytokine Growth Factor Rev
13:241-257, 2002.
258. Takahashi N, Rieneck K, van der Kraan PM, et al: Elucidation of IL-
1/TGF-beta interactions in mouse chondrocyte cell line by genome-
wide gene expression. Osteoarthritis Cartilage 13:426-438, 2005.
259. Bakker AC, van de Loo FA, van Beuningen HM, et al: Overexpres-
sion of active TGF-beta-1 in the murine knee joint: evidence for
synovial-layer-dependent chondro-osteophyte formation. Osteoar-
thritis Cartilage 9:128-136, 2001.
260. Mi Z, Ghivizzani SC, Lechman ER, et al: Adverse effects of adenovi-
rus-mediated gene transfer of human transforming growth factor beta
1 into rabbit knees. Arthritis Res 5:R132-R139, 2003.
261. Scharstuhl A, Glansbeek HL, van Beuningen HM, et al: Inhibition
of endogenous TGF-beta during experimental osteoarthritis pre-
vents osteophyte formation and impairs cartilage repair. J Immunol
169:507-514, 2002.
262. Scharstuhl A, Vitters EL, van der Kraan PM, et al: Reduction of
osteophyte formation and synovial thickening by adenoviral overex-
pression of transforming growth factor beta/bone morphogenetic pro-
tein inhibitors during experimental osteoarthritis. Arthritis Rheum
48:3442-3451, 2003.
 66 Goldring  |  Cartilage and Chondrocytes
263. Moulharat N, Lesur C, Thomas M, et al: Effects of transforming
growth factor-beta on aggrecanase production and proteoglycan deg-
radation by human chondrocytes in vitro. Osteoarthritis Cartilage
12:296-305, 2004.
264. Kaiser M, Haag J, Soder S, et al: Bone morphogenetic protein
and transforming growth factor beta inhibitory Smads 6 and 7 are
expressed in human adult normal and osteoarthritic cartilage in vivo
and are differentially regulated in vitro by interleukin-1beta. Arthri-
tis Rheum 50:3535-3540, 2004.
265. Blaney Davidson EN, Vitters EL, van der Kraan PM, et al: Expres-
sion of TGF-beta and the TGF-beta signaling molecule SMAD-2P in
spontaneous and instability-induced osteoarthritis: Role in cartilage
degradation, chondrogenesis and osteophyte formation. Ann Rheum
Dis 65:1414-1421, 2006.
266. Yoon BS, Lyons KM: Multiple functions of BMPs in chondrogenesis.
J Cell Biochem 93:93-103, 2004.
267. Li SW, Sieron AL, Fertala A, et al: The C-proteinase that processes
procollagens to fibrillar collagens is identical to the protein previ-
ously identified as bone morphogenic protein-1. Proc Natl Acad Sci
U S A 93:5127-5130, 1996.
268. Gruber R, Mayer C, Bobacz K, et al: Effects of cartilage-derived mor-
phogenetic proteins and osteogenic protein-1 on osteochondrogenic
differentiation of periosteum-derived cells. Endocrinology 142:2087-
2094, 2001.
269. Majumdar MK, Wang E, Morris EA: BMP-2 and BMP-9 promotes
chondrogenic differentiation of human multipotential mesenchy-
mal cells and overcomes the inhibitory effect of IL-1. J Cell Physiol
189:275-284, 2001.
270. Bobacz K, Gruber R, Soleiman A, et al: Expression of bone mor-
phogenetic protein 6 in healthy and osteoarthritic human articular
chondrocytes and stimulation of matrix synthesis in vitro. Arthritis
Rheum 48:2501-2508, 2003.
271. Grunder T, Gaissmaier C, Fritz J, et al: Bone morphogenetic protein
(BMP)-2 enhances the expression of type II collagen and aggrecan in
chondrocytes embedded in alginate beads. Osteoarthritis Cartilage
12:559-567, 2004.
272. Bobacz K, Gruber R, Soleiman A, et al: Cartilage-derived morpho-
genetic protein-1 and -2 are endogenously expressed in healthy and
osteoarthritic human articular chondrocytes and stimulate matrix
synthesis. Osteoarthritis Cartilage 10:394-401, 2002.
273. Vinall RL, Lo SH, Reddi AH: Regulation of articular chondrocyte
phenotype by bone morphogenetic protein 7, interleukin 1, and cel-
lular context is dependent on the cytoskeleton. Exp Cell Res 272:32-
44, 2002.
274. Nishihara A, Fujii M, Sampath TK, et al: Bone morphogenetic pro-
tein signaling in articular chondrocyte differentiation. Biochem Bio-
phys Res Commun 301:617-622, 2003.
275. Beier F: Cell-cycle control and the cartilage growth plate. J Cell
Physiol 202:1-8, 2005.
276. Starkman BG, Cravero JD, Delcarlo M, et al: IGF-I stimulation of
proteoglycan synthesis by chondrocytes requires activation of the
PI 3-kinase pathway but not ERK MAPK. Biochem J 389:723-729,
2005.
277. Reddi AH: Interplay between bone morphogenetic proteins and cog-
nate binding proteins in bone and cartilage development: Noggin,
chordin and DAN. Arthritis Res 3:1-5, 2001.
278. Derynck R, Zhang YE: Smad-dependent and Smad-independent
pathways in TGF-beta family signalling. Nature 425:577-584, 2003.
279. Qiao B, Padilla SR, Benya PD: Transforming growth factor (TGF)-
beta-activated kinase 1 mimics and mediates TGF-beta-induced
stimulation of type II collagen synthesis in chondrocytes indepen-
dent of Col2a1 transcription and Smad3 signaling. J Biol Chem
280:17562-17571, 2005.
280. Bobick BE, Kulyk WM: The MEK-ERK signaling pathway is a nega-
tive regulator of cartilage-specific gene expression in embryonic limb
mesenchyme. J Biol Chem 279:4588-4595, 2004.
281. Seghatoleslami MR, Roman-Blas JA, Rainville AM, et al: Pro-
gression of chondrogenesis in C3H10T1/2 cells is associated with
prolonged and tight regulation of ERK1/2. J Cell Biochem 88:
1129-1144, 2003.
282. Studer RK, Bergman R, Stubbs T, et al: Chondrocyte response to
growth factors is modulated by p38 mitogen-activated protein kinase
inhibition. Arthritis Res Ther 6:R56-R64, 2004.
283. Gebauer M, Saas J, Haag J, et al: Repression of anti-proliferative factor
Tob1 in osteoarthritic cartilage. Arthritis Res Ther 7:R274-R284, 2005.
284. Zhu Y, McAlinden A, Sandell LJ: Type IIA procollagen in develop-
ment of the human intervertebral disc: Regulated expression of the
NH(2)-propeptide by enzymic processing reveals a unique develop-
mental pathway. Dev Dyn 220:350-362, 2001.
285. Tardif G, Pelletier JP, Hum D, et al: Differential regulation of the
bone morphogenic protein antagonist chordin in human normal and
osteoarthritic chondrocytes. Ann Rheum Dis 65:261-264, 2006.
286. Nakayama N, Han CY, Cam L, et al: A novel chordin-like BMP
inhibitor, CHL2, expressed preferentially in chondrocytes of devel-
oping cartilage and osteoarthritic joint cartilage. Development
131:229-240, 2004.
287. Kawabata D, Tanaka M, Fujii T, et al: Ameliorative effects of fol-
listatin-related protein/TSC-36/FSTL1 on joint inflammation in a
mouse model of arthritis. Arthritis Rheum 50:660-668, 2004.
288. Dayer JM: The process of identifying and understanding cytokines:
From basic studies to treating rheumatic diseases. Best Pract Res Clin
Rheumatol 18:31-45, 2004.
289. van den Berg WB, van Riel PL: Uncoupling of inflammation and
destruction in rheumatoid arthritis: Myth or reality? Arthritis Rheum
52:995-999, 2005.
290. van den Berg WB: Animal models of arthritis: What have we learned?
J Rheumatol Suppl 72:7-9, 2005.
291. Hembry RM, Bagga MR, Reynolds JJ, et al: Immunolocalisation
studies on six matrix metalloproteinases and their inhibitors, TIMP-
1 and TIMP-2, in synovia from patients with osteo- and rheumatoid
arthritis. Ann Rheum Dis 54:25-32, 1995.
292. Tetlow LC, Adlam DJ, Woolley DE: Matrix metalloproteinase and
proinflammatory cytokine production by chondrocytes of human
osteoarthritic cartilage. Arthritis Rheum 44:585-594, 2001.
293. Struglics A, Larsson S, Pratta MA, et al: Human osteoarthritis syno-
vial fluid and joint cartilage contain both aggrecanase- and matrix
metalloproteinase-generated aggrecan fragments. Osteoarthritis Car-
tilage 14:101-113, 2006.
294. Neuhold LA, Killar L, Zhao W, et al: Postnatal expression in hyaline
cartilage of constitutively active human collagenase-3 (MMP-13)
induces osteoarthritis in mice. J Clin Invest 107:35-44, 2001.
295. Tetlow LC, Woolley DE: Comparative immunolocalization studies of
collagenase 1 and collagenase 3 production in the rheumatoid lesion,
and by human chondrocytes and synoviocytes in vitro. Br J Rheuma-
tol 37:64-70, 1998.
296. Cunnane G, FitzGerald O, Hummel KM, et al: Synovial tissue prote-
ase gene expression and joint erosions in early rheumatoid arthritis.
Arthritis Rheum 44:1744-1753, 2001.
297. Ainola MM, Mandelin JA, Liljestrom MP, et al: Pannus invasion and
cartilage degradation in rheumatoid arthritis: Involvement of MMP-
3 and interleukin-1beta. Clin Exp Rheumatol 23:644-650, 2005.
298. Barksby HE, Milner JM, Patterson AM, et al: Matrix metallopro-
teinase 10 promotion of collagenolysis via procollagenase activation:
Implications for cartilage degradation in arthritis. Arthritis Rheum
54:3244-3253, 2006.
299. Rutkauskaite E, Volkmer D, Shigeyama Y, et al: Retroviral gene
transfer of an antisense construct against membrane type 1 matrix
metalloproteinase reduces the invasiveness of rheumatoid arthritis
synovial fibroblasts. Arthritis Rheum 52:2010-2014, 2005.
300. Arner EC: Aggrecanase-mediated cartilage degradation. Curr Opin
Pharmacol 2:322-329, 2002.
301. Nagase H, Kashiwagi M: Aggrecanases and cartilage matrix degra-
dation. Arthritis Res Ther 5:94-103, 2003.
302. Plaas A, Osborn B, Yoshihara Y, et al: Aggrecanolysis in human
osteoarthritis: Confocal localization and biochemical characteriza-
tion of ADAMTS5-hyaluronan complexes in articular cartilages.
Osteoarthritis Cartilage 15:719-734, 2007.
303. Sandy JD: A contentious issue finds some clarity: On the independent
and complementary roles of aggrecanase activity and MMP activity
in human joint aggrecanolysis. Osteoarthritis Cartilage 14:95-100,
2006.
304. Glasson SS, Askew R, Sheppard B, et al: Deletion of active
ADAMTS5 prevents cartilage degradation in a murine model of
osteoarthritis. Nature 434:644-648, 2005.
305. Stanton H, Rogerson FM, East CJ, et al: ADAMTS5 is the major
aggrecanase in mouse cartilage in vivo and in vitro. Nature 434:
648-652, 2005.
306. Kashiwagi M, Tortorella M, Nagase H, et al: TIMP-3 is a potent
inhibitor of aggrecanase 1 (ADAM-TS4) and aggrecanase 2 (ADAM-
TS5). J Biol Chem 276:12501-12504, 2001.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
307. Hashimoto G, Aoki T, Nakamura H, et al: Inhibition of ADAMTS4
(aggrecanase-1) by tissue inhibitors of metalloproteinases (TIMP-1,
2, 3 and 4). FEBS Lett 494:192-195, 2001.
308. Hou WS, Li W, Keyszer G, et al: Comparison of cathepsins K and S
expression within the rheumatoid and osteoarthritic synovium.
Arthritis Rheum 46:663-674, 2002.
309. Salminen-Mankonen HJ, Morko J, Vuorio E: Role of cathepsin K in
normal joints and in the development of arthritis. Curr Drug Targets
8:315-323, 2007.
310. Kevorkian L, Young DA, Darrah C, et al: Expression profiling of
metalloproteinases and their inhibitors in cartilage. Arthritis Rheum
50:131-141, 2004.
311. Cawston TE, Wilson AJ: Understanding the role of tissue degrading
enzymes and their inhibitors in development and disease. Best Pract
Res Clin Rheumatol 20:983-1002, 2006.
312. Overall CM, Blobel CP: In search of partners: Linking extracellular
proteases to substrates. Nat Rev Mol Cell Biol 8:245-257, 2007.
313. Burrage PS, Huntington JT, Sporn MB, et al: Regulation of matrix
metalloproteinase gene expression by a retinoid X receptor-specific
ligand. Arthritis Rheum 56:892-904, 2007.
314. Murphy G, Lee MH: What are the roles of metalloproteinases in carti-
lage and bone damage? Ann Rheum Dis 64(Suppl 4):iv44-iv47, 2005.
315. Baker AH, Edwards DR, Murphy G: Metalloproteinase inhibitors:
Biological actions and therapeutic opportunities. J Cell Sci 115:
3719-3727, 2002.
316. Fell HB, Jubb RW: The effect of synovial tissue on the breakdown
of articular cartilage in organ culture. Arthritis Rheum 20:1359-
1371, 1977.
317. Dingle JT, Saklatvala J, Hembry R, et al: A cartilage catabolic factor
from synovium. Biochem J 184:177-180, 1979.
318. Dayer J-M, Russell RGG, Krane SM: Collagenase production by
rheumatoid synovial cells: Stimulation by a human lymphocyte fac-
tor. Science 195:181-183, 1977.
319. Meats JE, McGuire MB, Russell RGG: Human synovium releases a
factor which stimulates chondrocyte production of PGE and plas-
minogen activator. Nature 286:891-892, 1980.
320. Saklatvala J, Pilsworth LM, Sarsfield SJ, et al: Pig catabolin is a form
of interleukin 1: Cartilage and bone resorb, fibroblasts make prosta-
glandin and collagenase, and thymocyte proliferation is augmented
in response to one protein. Biochem J 224:461-466, 1984.
321. Goldring SR, Goldring MB: The role of cytokines in cartilage matrix
degeneration in osteoarthritis. Clin Orthop 427:S27-S36, 2004.
322. Goldring MB, Berenbaum F: The regulation of chondrocyte function
by proinflammatory mediators: Prostaglandins and nitric oxide. Clin
Orthop 427:S37-S46, 2004.
323. Fernandes JC, Martel-Pelletier J, Pelletier JP: The role of cytokines
in osteoarthritis pathophysiology. Biorheology 39:237-246, 2002.
324. Attur MG, Dave M, Cipolletta C, et al: Reversal of autocrine and
paracrine effects of interleukin 1 (IL-1) in human arthritis by type
II IL-1 decoy receptor: Potential for pharmacological intervention. J
Biol Chem 275:40307-40315, 2000.
325. Westacott CI, Barakat AF, Wood L, et al: Tumor necrosis factor
alpha can contribute to focal loss of cartilage in osteoarthritis. Osteo-
arthritis Cartilage 8:213-221, 2000.
326. Barakat AF, Elson CJ, Westacott CI: Susceptibility to physiological
concentrations of IL-1beta varies in cartilage at different anatomical
locations on human osteoarthritic knee joints. Osteoarthritis Carti-
lage 10:264-269, 2002.
327. Fraser A, Fearon U, Billinghurst RC, et al: Turnover of type II col-
lagen and aggrecan in cartilage matrix at the onset of inflammatory
arthritis in humans: Relationship to mediators of systemic and local
inflammation. Arthritis Rheum 48:3085-3095, 2003.
328. Glasson SS: In vivo osteoarthritis target validation utilizing geneti-
cally-modified mice. Curr Drug Targets 8:367-376, 2007.
329. Zwerina J, Redlich K, Polzer K, et al: TNF-induced structural joint
damage is mediated by IL-1. Proc Natl Acad Sci U S A 104:11742-
11747, 2007.
330. Aida Y, Maeno M, Suzuki N, et al: The effect of IL-1beta on the
expression of inflammatory cytokines and their receptors in human
chondrocytes. Life Sci 79:764-771, 2006.
331. Connell L: McInnes IB: New cytokine targets in inflammatory rheu-
matic diseases. Best Pract Res Clin Rheumatol 20:865-878, 2006.
332. Rowan AD, Koshy PJ, Shingleton WD, et al: Synergistic effects of gly-
coprotein 130 binding cytokines in combination with interleukin-1 on
cartilage collagen breakdown. Arthritis Rheum 44:1620-1632, 2001.
67
333. Legendre F, Dudhia J, Pujol JP, et al: JAK/STAT but not ERK1/ERK2
pathway mediates interleukin (IL)-6/soluble IL-6R down-regulation
of type II collagen, aggrecan core, and link protein transcription in
articular chondrocytes: Association with a down-regulation of SOX9
expression. J Biol Chem 278:2903-2912, 2003.
334. de Hooge AS, van de Loo FA, Bennink MB, et al: Male IL-6 gene
knock out mice developed more advanced osteoarthritis upon aging.
Osteoarthritis Cartilage 13:66-73, 2005.
335. Sack U, Sehm B, Kahlenberg F, et al: Investigation of arthritic joint
destruction by a novel fibroblast-based model. Ann N Y Acad Sci
1051:291-298, 2005.
336. Hui W, Barksby HE, Young DA, et al: Oncostatin M in combination
with tumour necrosis factor {alpha} induces a chondrocyte membrane
associated aggrecanase that is distinct from ADAMTS aggrecanase-1
or -2. Ann Rheum Dis 64:1624-1632, 2005.
337. Rowan AD, Hui W, Cawston TE, et al: Adenoviral gene transfer of
interleukin-1 in combination with oncostatin M induces significant
joint damage in a murine model. Am J Pathol 162:1975-1984, 2003.
338. van de Loo FA, de Hooge AS, Smeets RL, et al: An inflammation-
inducible adenoviral expression system for local treatment of the
arthritic joint. Gene Ther 11:581-590, 2004.
339. Li X, Afif H, Cheng S, et al: Expression and regulation of microsomal
prostaglandin E synthase-1 in human osteoarthritic cartilage and
chondrocytes. J Rheumatol 32:887-895, 2005.
340. Lubberts E, Koenders MI, van den Berg WB: The role of T cell inter-
leukin-17 in conducting destructive arthritis: Lessons from animal
models. Arthritis Res Ther 7:29-37, 2005.
341. Koenders MI, Joosten LA, van den Berg WB: Potential new targets
in arthritis therapy: Interleukin (IL)-17 and its relation to tumour
necrosis factor and IL-1 in experimental arthritis. Ann Rheum Dis
65(Suppl 3):iii29-iii33, 2006.
342. Dai SM, Shan ZZ, Nishioka K, et al: Implication of interleukin 18
in production of matrix metalloproteinases in articular chondrocytes
in arthritis: Direct effect on chondrocytes may not be pivotal. Ann
Rheum Dis 64:735-742, 2005.
343. John T, Kohl B, Mobasheri A, et al: Interleukin-18 induces apoptosis in
human articular chondrocytes. Histol Histopathol 22:469-482, 2007.
344. Joosten LA, Smeets RL, Koenders MI, et al: Interleukin-18 pro-
motes joint inflammation and induces interleukin-1-driven cartilage
destruction. Am J Pathol 165:959-967, 2004.
345. Magne D, Palmer G, Barton JL, et al: The new IL-1 family member IL-
1F8 stimulates production of inflammatory mediators by synovial fibro-
blasts and articular chondrocytes. Arthritis Res Ther 8:R80, 2006.
346. Joosten LA, Netea MG, Kim SH, et al: IL-32, a proinflamma-
tory cytokine in rheumatoid arthritis. Proc Natl Acad Sci U S A
103:3298-3303, 2006.
347. Bessis N, Boissier MC: Novel pro-inflammatory interleukins: Poten-
tial therapeutic targets in rheumatoid arthritis. Joint Bone Spine
68:477-481, 2001.
348. Goldring MB: Update on the biology of the chondrocyte and new
approaches to treating cartilage diseases. Best Pract Res Clin Rheu-
matol 20:1003-1025, 2006.
349. Palmer G, Guerne PA, Mezin F, et al: Production of interleukin-1
receptor antagonist by human articular chondrocytes. Arthritis Res
4:226-231, 2002.
350. van de Loo FA, Geurts J, van den Berg WB: Gene therapy works in
animal models of rheumatoid arthritis. . .so what! Curr Rheumatol
Rep 8:386-393, 2006.
351. Arend WP, Goldring MB: The development of anti-cytokine thera-
peutics for rheumatic diseases. Arthritis Rheum 58:5102-5109,
2008.
352. Chowdhury TT, Bader DL, Lee DA: Anti-inflammatory effects of
IL-4 and dynamic compression in IL-1beta stimulated chondrocytes.
Biochem Biophys Res Commun 339:241-247, 2006.
353. Schuerwegh AJ, Dombrecht EJ, Stevens WJ, et al: Influence of
pro-inflammatory (IL-1 alpha, IL-6, TNF-alpha, IFN-gamma) and
anti-inflammatory (IL-4) cytokines on chondrocyte function. Osteo-
arthritis Cartilage 11:681-687, 2003.
354. Ho SH, Hahn W, Lee HJ, et al: Protection against collagen-
induced arthritis by electrotransfer of an expression plasmid for
the interleukin-4. Biochem Biophys Res Commun 321:759-766,
2004.
355. Nandakumar KS, Holmdahl R: Arthritis induced with cartilage-
specific antibodies is IL-4-dependent. Eur J Immunol 36:1608-1618,
2006.
 68 Goldring  |  Cartilage and Chondrocytes
356. Neumann E, Judex M, Kullmann F, et al: Inhibition of cartilage
destruction by double gene transfer of IL-1Ra and IL-10 involves the
activin pathway. Gene Ther 9:1508-1519, 2002.
357. Gonzalez-Rey E, Chorny A, Varela N, et al: Therapeutic effect of uro-
cortin on collagen-induced arthritis by down-regulation of inflamma-
tory and Th1 responses and induction of regulatory T cells. Arthritis
Rheum 56:531-543, 2007.
358. Cleaver CS, Rowan AD, Cawston TE: Interleukin 13 blocks the
release of collagen from bovine nasal cartilage treated with proin-
flammatory cytokines. Ann Rheum Dis 60:150-157, 2001.
359. Nabbe KC, van Lent PL, Holthuysen AE, et al: Local IL-13 gene
transfer prior to immune-complex arthritis inhibits chondrocyte
death and matrix-metalloproteinase-mediated cartilage matrix deg-
radation despite enhanced joint inflammation. Arthritis Res Ther
7:R392-R401, 2005.
360. Masuko-Hongo K, Berenbaum F, Humbert L, et al: Up-regulation
of microsomal prostaglandin E synthase 1 in osteoarthritic human
cartilage: Critical roles of the ERK-1/2 and p38 signaling pathways.
Arthritis Rheum 50:2829-2838, 2004.
361. Whiteman M, Spencer JP, Zhu YZ, et al: Peroxynitrite-modified col-
lagen-II induces p38/ERK and NF-kappaB-dependent synthesis of
prostaglandin E2 and nitric oxide in chondrogenically differentiated
mesenchymal progenitor cells. Osteoarthritis Cartilage 14:460-470,
2006.
362. Healy ZR, Lee NH, Gao X, et al: Divergent responses of chondro-
cytes and endothelial cells to shear stress: Cross-talk among COX-2,
the phase 2 response, and apoptosis. Proc Natl Acad Sci U S A
102:14010-14015, 2005.
363. Cheng S, Afif H, Martel-Pelletier J, et al: Activation of peroxisome
proliferator-activated receptor gamma inhibits interleukin-1beta-
induced membrane-associated prostaglandin E2 synthase-1 expres-
sion in human synovial fibroblasts by interfering with Egr-1. J Biol
Chem 279:22057-22065, 2004.
364. Francois M, Richette P, Tsagris L, et al: Activation of the peroxisome
proliferator-activated receptor alpha pathway potentiates interleu-
kin-1 receptor antagonist production in cytokine-treated chondro-
cytes. Arthritis Rheum 54:1233-1245, 2006.
365. Thomas B, Thirion S, Humbert L, et al: Differentiation regulates
interleukin-1β-induced cyclo-oxygenase-2 in human articular chon-
drocytes: Role of p38 mitogen-activated kinase. Biochem J 362:
367-373, 2002.
366. Riquet RB, Lai W-FT, Birkhead JR, et al: Suppression of type I col-
lagen gene expression by prostaglandins in fibroblasts is mediated at
the transcriptional level. Mol Med 6:705-719, 2000.
367. Abramson SB, Attur M, Amin AR, et al: Nitric oxide and inflamma-
tory mediators in the perpetuation of osteoarthritis. Curr Rheumatol
Rep 3:535-541, 2001.
368. Kuhn K, D’Lima DD, Hashimoto S, et al: Cell death in cartilage.
Osteoarthritis Cartilage 12:1-16, 2004.
369. Yammani RR, Carlson CS, Bresnick AR, et al: Increase in produc-
tion of matrix metalloproteinase 13 by human articular chondrocytes
due to stimulation with S100A4: Role of the receptor for advanced
glycation end products. Arthritis Rheum 54:2901-2911, 2006.
370. Bauer S, Jendro MC, Wadle A, et al: Fibroblast activation protein is
expressed by rheumatoid myofibroblast-like synoviocytes. Arthritis
Res Ther 8:R171, 2006.
371. Milner JM, Kevorkian L, Young DA, et al: Fibroblast activation protein
alpha is expressed by chondrocytes following a pro-inflammatory stimu-
lus and is elevated in osteoarthritis. Arthritis Res Ther 8:R23, 2006.
372. Loeser RF: Systemic and local regulation of articular cartilage metab-
olism: Where does leptin fit in the puzzle? Arthritis Rheum 48:3009-
3012, 2003.
373. Dayer JM, Chicheportiche R, Juge-Aubry C, et al: Adipose tissue has
anti-inflammatory properties: Focus on IL-1 receptor antagonist (IL-
1Ra). Ann N Y Acad Sci 1069:444-453, 2006.
374. Lago F, Dieguez C, Gomez-Reino JJ, et al: Adipokines as emerging
mediators of immune response and inflammation. Nat Clin Pract
Rheumatol 12:716-724, 2007.
375. Popa C, Netea MG, Radstake TR, et al: Markers of inflammation are
negatively correlated with serum leptin in rheumatoid arthritis. Ann
Rheum Dis 64:1195-1198, 2005.
376. Otero M, Lago R, Gomez R, et al: Towards a pro-inflammatory and
immunomodulatory emerging role of leptin. Rheumatology (Oxf)
45:944-950, 2006.
377. Dumond H, Presle N, Terlain B, et al: Evidence for a key role of
leptin in osteoarthritis. Arthritis Rheum 48:3118-3129, 2003.
378. Otero M, Lago R, Lago F, et al: Leptin, from fat to inflammation: Old
questions and new insights. FEBS Lett 579:295-301, 2005.
379. Palmer G, Aurrand-Lions M, Contassot E, et al: Indirect effects of
leptin receptor deficiency on lymphocyte populations and immune
response in db/db mice. J Immunol 177:2899-2907, 2006.
380. Borzi RM, Mazzetti I, Marcu KB, et al: Chemokines in cartilage deg-
radation. Clin Orthop 427:S53-S61, 2004.
381. Iwamoto T, Okamoto H, Iikuni N, et al: Monocyte chemoattrac-
tant protein-4 (MCP-4)/CCL13 is highly expressed in cartilage from
patients with rheumatoid arthritis. Rheumatology (Oxf) 45:421-424,
2006.
382. Goldring MB, Goldring SR: Role of cytokines and chemokines in
cartilage and bone destruction in arthritis. Curr Opin Orthopaed
13:351-362, 2002.
383. Kanbe K, Takemura T, Takeuchi K, et al: Synovectomy reduces stro-
mal-cell-derived factor-1 (SDF-1) which is involved in the destruc-
tion of cartilage in osteoarthritis and rheumatoid arthritis. J Bone
Joint Surg Br 86:296-300, 2004.
384. Mazzetti I, Magagnoli G, Paoletti S, et al: A role for chemokines
in the induction of chondrocyte phenotype modulation. Arthritis
Rheum 50:112-122, 2004.
385. Masuko-Hongo K, Sato T, Nishioka K: Chemokines differentially
induce matrix metalloproteinase-3 and prostaglandin E2 in human
articular chondrocytes. Clin Exp Rheumatol 23:57-62, 2005.
386. Nakamura H, Tanaka M, Masuko-Hongo K, et al: Enhanced produc-
tion of MMP-1, MMP-3, MMP-13, and RANTES by interaction of
chondrocytes with autologous T cells. Rheumatol Int 26:984-990,
2006.
387. Su SL, Tsai CD, Lee CH, et al: Expression and regulation of Toll-like
receptor 2 by IL-1beta and fibronectin fragments in human articular
chondrocytes. Osteoarthritis Cartilage 13:879-886, 2005.
388. Kim HA, Cho ML, Choi HY, et al: The catabolic pathway mediated
by Toll-like receptors in human osteoarthritic chondrocytes. Arthri-
tis Rheum 54:2152-2163, 2006.
389. Varoga D, Paulsen F, Mentlein R, et al: TLR-2-mediated induction of
vascular endothelial growth factor (VEGF) in cartilage in septic joint
disease. J Pathol 210:315-324, 2006.
390. Joosten LA, Koenders MI, Smeets RL, et al: Toll-like receptor 2
pathway drives streptococcal cell wall-induced joint inflamma-
tion: Critical role of myeloid differentiation factor 88. J Immunol
171:6145-6153, 2003.
391. van Lent PL, Blom AB, Grevers L, et al: Toll-like receptor 4 induced
FcgammaR expression potentiates early onset of joint inflammation
and cartilage destruction during immune complex arthritis: Toll-like
receptor 4 largely regulates FcgammaR expression by interleukin 10.
Ann Rheum Dis 66:334-340, 2007.
392. Berenbaum F: Signaling transduction: Target in osteoarthritis. Curr
Opin Rheumatol 16:616-622, 2004.
393. Malemud CJ: Protein kinases in chondrocyte signaling and osteoar-
thritis. Clin Orthop 427:S145-S151, 2004.
394. Katoh M: STAT3-induced WNT5A signaling loop in embryonic
stem cells, adult normal tissues, chronic persistent inflammation,
rheumatoid arthritis and cancer. Int J Mol Med 19:273-278, 2007.
395. Fanning PJ, Emkey G, Smith RJ, et al: Mechanical regulation of
mitogen-activated protein kinase signaling in articular cartilage. J
Biol Chem 278:50940-50948, 2003.
396. Fitzgerald JB, Jin M, Dean D, et al: Mechanical compression of car-
tilage explants induces multiple time-dependent gene expression
patterns and involves intracellular calcium and cyclic AMP. J Biol
Chem 279:19502-19511, 2004.
397. Agarwal S, Deschner J, Long P, et al: Role of NF-kappaB transcrip-
tion factors in antiinflammatory and proinflammatory actions of
mechanical signals. Arthritis Rheum 50:3541-3548, 2004.
398. Ahmed S, Rahman A, Hasnain A, et al: Phenyl N-tert-butylnitrone
down-regulates interleukin-1 beta-stimulated matrix metalloproteinase-
13 gene expression in human chondrocytes: Suppression of c-Jun
NH2-terminal kinase, p38-mitogen-activated protein kinase and
activating protein-1. J Pharmacol Exp Ther 305:981-988, 2003.
399. Loeser RF, Forsyth CB, Samarel AM, et al: Fibronectin fragment acti-
vation of proline-rich tyrosine kinase PYK2 mediates integrin signals
regulating collagenase-3 expression by human chondrocytes through
a protein kinase C-dependent pathway. J Biol Chem 278:24577-
24585, 2003.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
400. Firestein GS: NF-kappaB: Holy Grail for rheumatoid arthritis?
Arthritis Rheum 50:2381-2386, 2004.
401. Facchini A, Borzi RM, Marcu KB, et al: Polyamine depletion inhibits
NF-kappaB binding to DNA and interleukin-8 production in human
chondrocytes stimulated by tumor necrosis factor-alpha. J Cell
Physiol 204:956-963, 2005.
402. Carrington JL: Aging bone and cartilage: Cross-cutting issues. Bio-
chem Biophys Res Commun 328:700-708, 2005.
403. Hough AJ: Pathology of osteoarthritis. In Seibel MJ, Robins SP,
Bilezekian JP (eds): Dynamics of Bone and Cartilage Metabolism:
Principles and Clinical Applications. San Diego, Academic Press,
2006, pp 51-72.
404. Burstein D, Gray ML: Is MRI fulfilling its promise for molecu-
lar imaging of cartilage in arthritis? Osteoarthritis Cartilage 14:
1087-1090, 2006.
405. Verzijl N, Bank RA, TeKoppele JM, et al: AGEing and osteoarthritis:
A different perspective. Curr Opin Rheumatol 15:616-622, 2003.
406. Hollander AP, Pidoux I, Reiner A, et al: Damage to type II collagen
in aging and osteoarthritis starts at the articular surface, originates
around chondrocytes, and extends into the cartilage with progressive
degeneration. J Clin Invest 96:2859-2869, 1995.
407. Martin JA, Buckwalter JA: Aging, articular cartilage chondrocyte
senescence and osteoarthritis. Biogerontology 3:257-264, 2002.
408. Price JS, Waters JG, Darrah C, et al: The role of chondrocyte senes-
cence in osteoarthritis. Aging Cell 1:57-65, 2002.
409. Blaney Davidson EN, Scharstuhl A, Vitters EL, et al: Reduced trans-
forming growth factor-beta signaling in cartilage of old mice: Role in
impaired repair capacity. Arthritis Res Ther 7:R1338-R1347, 2005.
410. Poole AR: Can serum biomarker assays measure the progression
of cartilage degeneration in osteoarthritis? Arthritis Rheum 46:
2549-2552, 2002.
411. Hunziker EB: Articular cartilage repair: Basic science and clinical
progress: A review of the current status and prospects. Osteoar-
thritis Cartilage 10:432-463, 2002.
69
412. Brittberg M, Lindahl A, Nilsson A, et al: Treatment of deep cartilage
defects in the knee with autologous chondrocyte transplantation.
N Engl J Med 331:889-895, 1994.
413. Roberts S, Hollander AP, Caterson B, et al: Matrix turnover in
human cartilage repair tissue in autologous chondrocyte implanta-
tion. Arthritis Rheum 44:2586-2598, 2001.
414. Knutsen G, Engebretsen L, Ludvigsen TC, et al: Autologous
chondrocyte implantation compared with microfracture in the
knee: A randomized trial. J Bone Joint Surg Am 86A:455-464,
2004.
415. Kuo CK, Li WJ, Mauck RL, et al: Cartilage tissue engineering: Its
potential and uses. Curr Opin Rheumatol 18:64-73, 2006.
416. Evans CH: Novel biological approaches to the intra-articular treat-
ment of osteoarthritis. BioDrugs 19:355-362, 2005.
417. Gooch KJ, Blunk T, Courter DL, et al: Bone morphogenetic pro-
teins-2, -12, and -13 modulate in vitro development of engineered
cartilage. Tissue Eng 8:591-601, 2002.
418. Huang JI, Kazmi N, Durbhakula MM, et al: Chondrogenic potential
of progenitor cells derived from human bone marrow and adipose
tissue: A patient-matched comparison. J Orthop Res 23:1383-1389,
2005.
419. Palmer GD, Steinert A, Pascher A, et al: Gene-induced chondro-
genesis of primary mesenchymal stem cells in vitro. Mol Ther 12:
219-228, 2005.
420. Seeherman H, Wozney JM: Delivery of bone morphogenetic proteins
for orthopedic tissue regeneration. Cytokine Growth Factor Rev
16:329-345, 2005.
421. Hills RL, Belanger LM, Morris EA: Bone morphogenetic protein 9 is
a potent anabolic factor for juvenile bovine cartilage, but not adult
cartilage. J Orthop Res 23:611-617, 2005.
422. Blaney Davidson EN, van der Kraan PM, van den Berg WB:
TGF-beta and osteoarthritis. Osteoarthritis Cartilage 15:597-
604, 2007.
 4 Biology, Physiology,
KEY POINTS
Bone function is structural and metabolic.
Cellular remodeling of bone requires the orchestrated action
of the following:
Osteoblasts—bone-forming cells
Osteoclasts—bone-resorbing cells
Osteocytes—bone-sensing cells
Skeletal development is controlled by multiple genes, muta-
tions in which lead to disease.
Skeletal homeostasis involves chemical and biophysical
signals, as follows:
Chemical factors largely regulate metabolic function
Mechanical factors largely regulate structural function
Bone is an extremely complex tissue that regulates its mass
and architecture to meet two crucial and competing respon-
sibilities, one structural and the other metabolic. First, the
skeleton provides a sophisticated framework for the body: It
protects vital organs and facilitates locomotion. Second, it
serves as a mineral reservoir containing 99% of the body’s
total calcium and 85% of its phosphorus. This dual responsi-
bility creates conflicting goals and competing stimuli in the
catabolic and anabolic regulation of skeletal tissues. Meta-
bolic aberrations, such as hypocalcemia, hyperparathyroid-
ism, renal failure, and aging, can cause impairment in the
structural integrity of the skeleton, whereas regulatory con-
trols struggle to maintain calcium homeostasis. The skeleton
perceives another set of regulatory controls over its structural
morphology and adapts to meet altered functional demand.
This structural adaptation can proceed apace, ignoring poten-
tial consequences of rapid fluctuations in serum calcium lev-
els (e.g., renal lithiasis, hypercalcemia). Despite what seem
to be independent sets of regulatory controls for metabolic
and structural needs of the organism, however, skeletal integ-
rity and the viability of the organism are ­critically dependent
on maintaining an intricate balance between them.
The balance between structural and metabolic respon-
sibilities is achieved via the complex and tightly regulated
­cellular processes of formation and resorption of bone tissue.
­Beginning at the level of the cell, this ­ chapter discusses
local and ­systemic factors that influence bone turnover and
growth of the skeleton, the composition and mineralization
of the matrix, the architecture and ­ material properties of
the tissue, and the adaptive capacity of the skeleton. This
multilevel, multidisciplinary overview is ­intended to provide
the reader with an appreciation of not only the ­complexity
of bone, but also its success in ­ meeting its wide-ranging
responsibilities.
and Morphology
of Bone
Janet E. Rubin  �  Clinton T. Rubin
CELLULAR BASIS OF BONE REMODELING
The constant modeling and remodeling of skeletal tissue
is necessary during growth and for the repair of skeletal
defects caused by fracture and inflammation. Remodel-
ing also is essential for the rapid mobilization of mineral
required for metabolic homeostasis and for the fine adjust-
ment of skeletal mass and morphology needed to achieve
an effective and efficient supporting structure. The skel-
eton’s capacity to adapt to changes in its functional and
metabolic milieu is achieved through its sophisticated
network of osteoregulatory cells. These cells—osteoblasts,
osteocytes, and osteoclasts—mediate the remodeling bal-
ance of the skeleton and provide the cellular machinery
necessary for the maintenance of calcium homeostasis in
the extracellular fluid.
OSTEOBLASTS
Function
The primary function of the osteoblast is to synthesize and
mineralize the extracellular matrix. Osteoblasts are inter-
connected to one another via extended cell processes that
communicate via gap junctions,1 establishing a single,
continuous blanket of cells on the bone surface (Fig. 4-1).
The cytoplastic elements of the plump, cuboidal osteo-
blast include abundant endoplasmic reticulum with cis-
ternae, a well-developed Golgi body, and numerous free
ribosomes that are responsible for the basophilia seen in
sections stained with hematoxylin and eosin. These fea-
tures help to distinguish osteoblasts from the thin, flat
mesenchymal precursors (preosteoblasts) that are found
on bone surfaces. Together, these two cell types are known
as bone-lining cells. Morphologically, the tight canopy
of the lining cells enables the selective isolation of the
mineralized surface from the extracellular milieu, which
is crucial for the site-specific control of mineralization or
resorption.2
In forming bone, the osteoblast secretes an extracellular
matrix termed osteoid (see section on composition of bone
matrix), which is subsequently mineralized. Although it
is unclear whether mineralization itself necessitates the
­presence of the osteoblast (see Chapter 1), this cell’s
­synthesis of a panel of proteins, including type I collagen,
several ­ noncollagenous proteins, and proteoglycans, is
critical to the appropriate orchestration of the remo­deling
response (Table 4-1). Many of these proteins, whether
­secreted or ­ displayed by osteoblasts, have signaling roles
in bone remodeling, such as the role of osteocalcin to
recruit ­ osteoclasts,3 the ­ initiation of collagen breakdown
71
 72 RUBIN  |  Biology, Physiology, and Morphology of Bone
Figure 4-1  Polyhedral osteoblasts lying on the surface of newly formed
bone matrix. (Courtesy of Dr. B. Boothroyd.)
by pla­sminogen activator,4 and the signal for osteoclast
recruitment by expression of receptor activator of nuclear
factor к B (NFкB) ligand (RANKL).5 Other proteins, such
as osteopontin and bone sialoprotein, are crucial com-
ponents of osteoid, strongly binding ionic calcium and
se­rving as cell attachment factors.6
The functional life of the osteoblast ranges in differ-
ent species from 3 days in young rabbits7 to 8 weeks in
humans.8 A typical active osteoblast in humans produces a
seam of osteoid about 15 μm thick, at a rate of 0.5 to 1.5
μm/day,9 suggesting the average osteoblast life in humans to
be 15 days. Although osteoid seams can exceed 70 mm,10
such seam widths also can indicate a disruption or flaw in
the mineralization process (e.g., rickets or osteomalacia).
Several reports suggest that a contraction of the life span
of the osteoblast might underlie pathophysiologic condi-
tions. In the presence of glucocorticoids, an increased rate
of ­programmed cell death, or apoptosis, is seen, which may
­account for the decrease in bone quality recognized in
­patients with ­glucocorticoid excess.11
Differentiation
Studies of the molecular mechanisms involved in
o­ steoblast maturation have revealed a complex set of
interacting factors. A specific helix-loop-helix nuclear
factor termed Runx2/cbfa1 directs entry of the pluripo-
tent mesenchymal stem cell into the osteoblast lineage
in vitro12 and in vivo, where heterozygous loss of Runx2
in several human kindreds causes cleidocranial dysplasia.
Osteoblast expression of osteopontin and type I collagen
is delayed, with resultant late ossification of the clavicle.13
This condition, defined by short stature and hypoplasia
of the clavicles,14 predicted a similar phenotype generated
by targeted di­sruption of the Runx2 gene in mice.15 In the
transgenic Runx2 knockout mouse, the cartilaginous skel-
eton never ossifies, however, emphasizing how necessary
Runx2 is to the normal skeleton. Since the de­scription of
Table 4-1  Composition of Bone Osteoid
Collagen
  Type I, some types III, V, XI, XIII
Proteoglycans
  Biglycan, decorin, hyaluronan
Glycoproteins
  Osteonectin, bone sialoprotein, osteopontin, thrombospondin,
fibronectin, γ-carboxyglutamic acid–containing proteins (osteo-
calcin, matrix gla protein)
Enzymes
  Alkaline phosphatase, collagenase, cysteine proteinases, plasmin-
ogen activator, tissue inhibitor of metalloproteinases
Growth factors
  Fibroblast growth factor, insulin-like growth factor, transforming
growth factor, bone morphogenetic protein, ­α2HS-glycoprotein
Proteolipids
the Runx2-null transgenic mouse, many other genes, such
as Lrp5 and osterix, have been shown to be important to
osteoblast differentiation and function, with proof-of-
concept in transgenic animals.
Transgenic Models Inform Osteoblast
Pathophysiology
Although this chapter cannot document all the genes
necessary for osteoblast function, a few, besides Runx2,
warrant mention. A second transcription factor, osterix,
was discovered while scientists attempted to learn more
about the role bone morphogenetic proteins (BMPs) play
in osteoblast function. Although osterix seems to func-
tion distal to Runx2, its absence precludes finding either
osteoblasts or bone tissue in osterix-null transgenic
animals.16 The cartilaginous skeleton in the absence of
osterix is normal.
Scientists seeking to understand human kindreds with
very high bone mineral densities uncovered the involvement
of the Wnt pathway in osteoblast function and perhaps dif-
ferentiation. These kindreds turned out to have mutations
of the Lrp5 protein, which inhibits signaling by the develop-
mental protein Wnt.17,18 Knockout of Lrp5 in mice caused a
decrease in osteoblast proliferation and osteopenia.19 Because
soluble mediators can interact with the Wnt signaling path-
way, manipulation of this pathway would surely offer means
of controlling osteoblast function in the future.
Identification of the sclerostin gene arose through study
of human genetics, in this case a group of diseases consistent
with increased bone mass, including van Buchem’s disease20
and sclerosteosis.21 Both diseases are associated with muta-
tions in sclerostin that alter its ability to bind members of
the family of BMPs, leading to their overactivity,22 and seem
to bind Lrp5/6 receptors, preventing activation of the Wnt
pathway.23
Other genes have been linked to abnormalities in mes-
enchymal condensation and differentiation, including pheno-
types with extra digits. One of these is synpolydactyly, which
involves an abnormal HOXD-13 gene.24 Similar to other mem-
bers of the Hox gene family, HOXD-13 is involved in skeletal
patterning. Fibrodysplasia ossificans progressiva is a disabling
disorder characterized by a gradual and continuous heterotopic
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
HC
OL
Figure 4-2  Ground section of bone photographed in normal light
showing empty osteocyte lacunae (OL) connected by fine, darkly stained
canaliculi. The central haversian canal (HC) is seen in the upper left-hand
corner; the reversal line (*) marking the junction between two adjacent
osteons and the cement line (arrows) separating adjacent lamellae also
can be seen.
ossification of soft tissues. This autosomal-dominant disorder
is associated with misexpression of the growth factor BMP-425
and has been shown to be due to a mutation in the ACVR1
gene, which encodes an activin receptor.26 The process by
which the mutation in ACVR1 leads to the exoskeleton seen
in this disease is unknown.
OSTEOCYTES
As mineralization proceeds, the osteoblast may become
engulfed in its own calcifying osteoid matrix, becoming
the osteocyte.27 Osteocytes are connected to each other
via cytoplasmic extensions that pass through a network
of catacombs radiating outward from the central vascular
canal (Fig. 4-2). The volume of bone occupied by this syn-
cytium is approximately 5% for the canalicular network
and 2% for the lacunar spaces.28 These interconnecting
canaliculi are ideal pathways for chemical, electric, and
stress-generated fluid communication through the dense
bone matrix.29 The processes themselves are intercon-
nected by gap junctions composed of the protein connexin
43, which facilitates the integration of many of these regu-
latory messages.30 The surface area of the lacunar and can-
alicular system is at least 250 m2/L of calcified bone matrix
and communicates with a submicroscopic, interfibrillar
space, representing 35,000 μm2/mm3. Exchange of miner-
als, nutrients, and chemical and physical stimuli through
this enormous network is facilitated, which is critical, con-
sidering the need for rapid mobilization of minerals in the
homeostatic control of the skeleton. Also, this cell syncy-
tium deteriorates markedly with increasing age and may
contribute to the age-related insensitivity of bone ­ tissue
to chemical and physical signals.
Although osteoblasts and osteoclasts are largely respon-
sible for executing the appropriate modeling-remodeling
responses, given the time required to recruit these cells
in the adult skeleton, it is likely that the extensive net-
work of resident cells, the osteocytes and bone-lining cells,
actually orchestrate the adaptive response. Past work has
provided strong evidence that the osteocyte is capable of
73
rapidly ­responding to changes in the bone’s chemical and
mechanical environment. Mechanical strain increases
osteocyte levels of glucose-6-phosphate dehydrogenase
activity31 and c-fos mRNA levels within 60 minutes of
loading, evidence that osteocytes are intimately—and rap-
idly—involved in the osteogenic response to mechanical
signals.32 Physiologic levels of mechanical strain reduce
the rate of osteocyte apoptosis, suggesting that matrix
deformation affects cell survival.33 When bone is subject to
disuse, osteocytes are capable of expressing collagenase,34
emphasizing that this cell has the ability to regulate the
recruitment of bone-forming and bone-resorbing cells and
to modulate its own immediate microenvironment.
OSTEOCLASTS
Function
The osteoclast, functioning as a bone-specific macrophage,
is found wherever bone mineral is being removed. This
large, multinucleated cell migrates on the bone surface, cre-
ating irregular, scalloped cavities termed Howship’s lacunae
(Fig. 4-3). An activated osteoclast can travel 100 μm/day,
resorbing a cavity 300 μm in diameter and excavating
200,000 μm3 of bone. The reclamation of this volume of bone
requires 7 to 10 generations of working osteoblasts to fill the
resorption space.35 An absence of osteoclasts or a population
of dysfunctional osteoclasts leads to osteopetrosis, or marble
bone disease, which can be fatal in childhood secondary to
pancytopenias caused by decreased marrow space.36
Through the interactions of systemic and local factors,
a cellular symbiosis exists in which an integrated “activa-
tion, resorption, reversal, and formation” occurs, reflecting
an intricate control of osteoblasts and osteoclasts.37 To ini-
tiate this sequence, osteoclasts are activated by chemical
or physical signals that recruit them to specific remodeling
areas. When activated and attached to the matrix, they
begin the resorption cycle. When resorption is completed,
new bone formation begins within Howship’s cavity, a pro-
cess that leaves a boundary designated in histologic sections
as a reversal line.38 This “pocket” of new bone is known as a
bone structural unit.39 A similar line, called a cement line, is
seen between adjacent lamellae of bone (see Fig. 4-2).
Mature osteoclasts possess several unique ultrastructural
characteristics that allow their very active functional phe-
notype (Fig. 4-4). These include abundant pleomorphic
mitochondria, vacuoles, and lysosomes, which store the
multiple secretory products necessary for the digestion of
bone substance.40 The apical membrane of the osteoclast is
tightly sealed to the calcified matrix, creating a clear zone
that has no cellular organelles, but is rich in contractile pro-
teins. Toward the center of the cell, the membrane becomes
deeply folded, creating the characteristic ruffled ­ border.41
Under the ruffled border is the resorption pit, where the
osteoclast secretes lysosomal enzymes and generates an acid
environment by secreting protons through a vacuolar pro-
ton pump.42 Protons are generated for the pump through
the actions of carbonic anhydrase II, an enzyme subtype
also associated with vacuolar proton pumps in the renal
cortex and the gastric parietal cell. Deficiencies in carbonic
anhydrase II lead to a mild form of osteopetrosis.43 The
bicarbonate resulting from this activity is transported via a
 74 RUBIN  |  Biology, Physiology, and Morphology of Bone
A
B arises out of the macrophage lineage of hematopoietic stem
Figure 4-3  A, Paragon-stained mineralized section with large, mul-
tinucleated osteoclasts lying along the pale-staining bone surface. B,
Microradiograph of the same area showing Howship’s lacunae in the
areas of osteoclastic resorption. (From Jowsey J, Gordan G: In Bourne GH
[ed]: The Biochemistry and Physiology of Bone, 2nd ed, Vol III. New York,
Academic Press, 1972, pp 201-238.)
chloride-­bicarbonate exchanger on the marrow-facing side
of the osteoclast. The osteoclast’s highly polarized nature
allows unidirectional secretion of protons into the resorption
bay at its basal membrane and transport of resorbed prod-
ucts out of its apical region.44 Protons accumulate within the
­confined subosteoclastic space, reducing the pH to a level
sufficient to dissolve the mineral phase of the matrix (pH of
2 to 4) and activate osteoclastic hydrolytic enzymes.
Osteoclast acid secretion can be inhibited through regu-
lation of calmodulin kinase. Tamoxifen, an antiestrogen that
prevents postmenopausal bone loss, can directly inhibit this
kinase and prevent osteoclast acidification.45 The remain-
ing organic matrix is subsequently dissolved by lysosomal
enzymes (e.g., cathepsin B), leaving the ­osteoclast’s signa-
ture scalloped resorption cavity.46 As the excavation pro-
ceeds, the ionized calcium levels just beyond the clear zone
increase from a base level of 1 to 2 mM to 20 mM.47 Other
clinically useful modulators of osteoclast development and
function include the bisphosphonates, which are widely
used to prevent osteoporotic fractures.48 Bisphosphonates
are incorporated into bone mineral in place of phosphate
and prevent osteoclast recruitment and activity, increasing
bone mineral density by slowing resorption.49
Differentiation
Osteoclast precursors arise from hematopoietic stem cells
in the bone marrow.50 The processes controlling osteoclast
differentiation can be reduced, at least in tissue culture, to
Calcitonin receptors
Mitochondria
cAMP
HCO3 H2O + CO2
Cl Carbonic anhydrase II
Clear zone H+ Proton pump
Ruffled border
H+ Lysosomal
enzymes
Figure 4-4  The osteoclast is a polarized cell that adheres to bone,
generating a subosteoclastic space where bone resorption occurs. Into
this space, the osteoclast pumps hydrogen ions and lysosomal enzymes.
The protons that create the acid pH in the resorption space derive from
the action of carbonic anhydrase II on bicarbonate, which has entered 
the cell via a chloride bicarbonate exchanger on the marrow side of the
cell. Calcitonin receptors distinguish this cell, as does the characteristic
ruffled border seen on electron micrographs.
three basic elements: the osteoclast precursor cell, which
cells; macrophage colony-stimulating factor (M-CSF),
which is necessary for the ­differentiation, proliferation, and
survival of cells of macrophage lineage; and RANKL.51,52
Loss of M-CSF action engenders deficiencies of osteoclasts
and macrophages: The osteopetrotic op/op mouse is defi-
cient in M-CSF.53 Further progression into the osteoclast
lineage with appearance of functional osteoclast charac-
teristics, such as calcitonin receptor and carbonic anhy-
drase II expression, requires other local stimuli, including
the physical presence of bone stromal or osteoblast cells54-56
that display RANKL on their membranes.51,52 In the
absence of RANKL, normal osteoclastogenesis does not
progress, as in the osteopetrotic RANKL-null transgenic
mouse.57 A secreted form of RANKL also suffices for osteo-
clastogenesis and can cause bone turnover when secreted
by activated T lymphocytes in rheumatic joints.58 RANKL
binding can be prevented by competition with a soluble
decoy receptor, osteoprotegerin, and overexpression of
osteoprotegerin leads to an inhibition of osteoclastogen-
esis and decreased bone turnover.59 Human deficiency of
osteoprotegerin is now known to be responsible for juve-
nile Paget’s disease, a familial disorder characterized by a
large head and expanded and bowed extremities that are
osteoporotic.60
The list of factors that influence the progression of
osteoclast differentiation grows steadily each year.
­Osteoactive systemic factors, such as 1,25-dihydroxyvita-
min D, parathyroid hormone (PTH), and tumor ­necrosis
factor, enhance the development of osteoclasts from
­hematopoietic progenitor cells in the presence of stromal
elements from bone.55 Each of these three factors induces
the expression of RANKL by bone cells. Cytokines, such
as interleukin-6 and interleukin-11, also influence osteo-
clast development and may have particular significance
in pathogenic states, such as postmenopausal osteoporo-
sis61 and Paget’s disease.62
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
Transgenic Models Inform Osteoclast
Pathophysiology
Many other cofactors participating in osteoclast lineage
­selection have been discovered as a result of gene dysfunction
or knockout experiments. Knockout of the murine ­tyrosine
kinase c-fos gene inhibits the divergence of cells from macro-
phage lineage into that of osteoclasts, despite normal levels
of M-CSF and RANKL.63 Knockout of the transcription
factor PU.1 inhibits early osteoclast precursor development
by disallowing entry into the macrophage lineage,64 and the
abnormal function of the helix-loop-helix factor MITF inhib-
its late differentiation of osteoclasts.65 Knockout of signaling
cascades necessary for RANKL ­ action (i.e., NFκB) also
results in deficient osteoclast differentiation.66 Upregulation
of signal cascades that enhance ­osteoclastic differentiation
and function should increase bone turnover, as is evidenced
by knockout of the src homology 2–containing inositol-5′-
phosphatase (SHIP). The SHIP-null transgenic mouse has
increased numbers of osteoclast precursors and overactive
osteoclasts, resulting in an osteoporotic skeleton.67
The unexpected finding of osteopetrosis after homolo-
gous knockout of the ubiquitous src protein kinase in the
mouse68 has precipitated study of the roles of osteoclast
attachment and movement. The src-deficient mouse has
osteoclasts, but these cells are unable to resorb bone. The
src tyrosine kinase is important in the downstream signal-
ing required for ­ osteoclast function at the bone surface.69
Scientists also have begun to examine the motility of the
osteoclast and its ability to transcytose resorbed materials
liberated from the mineralized matrix.44 The motility of the
osteoclast is under the control of many factors, including
factors recognized by its polarized basal surface connected
to the mineral and matrix by an integrin such as vitronec-
tin.70 As might be predicted, knockout of the β3 integrin
subunit leads to dysfunction of osteoclasts and osteosclero-
sis in transgenic mice.71 The list of deletion knockouts that
have human homologues continues to grow, as charted in
numerous more recent reviews.72
DEVELOPMENT OF THE SKELETON
GROWTH
Most of the bones of the skeleton appear first in the ­embryo
as cartilaginous models, which are later resorbed and
replaced by bone tissue. The cartilage template, or anlage,
is formed by the condensation of mesenchymal cells in the
developing limb bud. Although the shape of the anlage
­resembles that of the adult bone and seems to be genetically
determined, the bone that replaces the cartilage template is
greatly influenced by physical factors (e.g., weight bearing,
muscle pull) and is constantly modeled and remodeled as
these forces change according to the dicta of Wolff’s law.73
The cartilage anlage, or template, expands and elon-
gates by interstitial growth, in which chondrocytes divide,
enlarge, and surround themselves with the new matrix. At
about the same time, cells in the connective tissues sur-
rounding the anlage (perichondrium) begin to lay down
bone tissue and form a collar of bone around the center of
the cartilage model. At the time it is formed, the anlage
is pierced by a capillary that begins the process of matrix
75
e­ xcavation and allows entry of osteoblast precursors that
replace ­cartilage with bone. This process of vascular inva-
sion of the cartilage ­model, followed by bone deposition, is
referred to as ­enchondral ossification. The process continues
until the entire shaft of the anlage is replaced by marrow
and bone, confining the growth process of chondrocyte mul-
tiplication to the epiphyseal ends of the bone, away from
the primary center of ossification. As the epiphysis swells,
the central chondrocytes find themselves too remote from
the blood supply to survive solely by means of diffusion.
Cartilage canals facilitate the diffusion of nutrients and pro-
vide conduits for subsequent capillary ingrowth. The hyper-
trophic ­ chondrocytes survive and elaborate angiotrophic
substances.
Parathyroid hormone–related peptide (PTHrp) has
emerged as one of the most important factors ­ controlling
the complex process of embryologic bone growth and
­maturation. PTHrp, which has long been known to be the
most common causative factor in neoplastic hypercalcemia,
binds to the PTH receptor.74 During fetal development of
the cartilaginous anlage, the product of the Indian ­hedgehog
(Ihh) gene is released by hypertrophic chondrocytes in the
growth plate. The Ihh protein induces PTHrp release by
the perichondrium at the end of the long bones.75 PTHrp
binds to receptors in the proliferating chondrocytes, causing
a delay of their maturation and allowing further elongation
of the anlage before entry of osteoblasts and ossification. In
the absence of PTHrp, the developing bone ossifies prema-
turely and ends up foreshortened. The interplay of Ihh and
PTHrp, which has such profound effects during embryologic
development, may have a subtler role in the adult skeleton
involved in the modulation of osteoblast response to the
continual need for bone maintenance.
Although growth in length occurs at the growth plate,
growth in diameter occurs by the centrifugal proliferation
of cartilage cells along the groove of Ranvier,76 an anatomic
structure bordered outwardly by a continuation of the fibrous
periosteum and inwardly by the physeal cartilage. When
swelling of the cartilaginous anlage first begins, a condensed
layer of mesenchyme develops around it as a membrane of
cells and collagen, termed perichondrium. As the cartilage is
replaced by bone, this membrane is termed periosteum. In
the growing skeleton, periosteum is clearly divided into an
inner cellular layer and an outer fibrous layer, which merges
gradually into the surrounding muscle. Muscles originate
from the periosteum. Collagen bundles can be traced flow-
ing from tendon and ligament through the periosteum to
anchor directly into the bone via Sharpey’s fibers.77
Along with the genetic regulation discussed previously,
there are epigenetic regulatory mechanisms, including con-
trol by mechanical stresses as proposed by Roux.78 General
considerations of how mechanical stress might control skel-
etal growth abound, including the proposal of Carter and
coworkers.79 They proposed that intermittently applied shear
stresses promote enchondral ossification, and that intermit-
tent hydrostatic compression inhibits cartilage degeneration
and ossification. The contribution of mechanical stresses to
bone growth is not yet understood.
Rosenberg and colleagues80 characterized the changes that
occur during ossification of the anlage, including the depo-
sition of a 35,000-kD molecular weight protein (probably
osteocalcin) and the modification of existing proteoglycans.
 76 RUBIN  |  Biology, Physiology, and Morphology of Bone
Mineral deposition begins in the matrix vesicles located in
the columns between the last hypertrophic chondrocytes on
the extreme metaphyseal end of the physis.81 Invasion by
osteoblasts and osteoclasts results in the resorption of this
woven bone and its replacement by mature lamellar bone.82
The hydroxyapatite formed during primary ossification is a
rough crystal whose imperfections may render it more vul-
nerable to resorption,83 which also implies the facility with
which woven bone is removed.
Numerous cell-secreted factors are important in the
regulation of growth and the cascade of processes l­eading
to adequate mineralization of bone by osteoblasts. Urist’s
identification of a factor in demineralized bone powder
that initiated bone formation has led to the cloning and
sequencing of at least seven BMPs belonging to the trans-
forming growth factor (TGF)-β family of growth poten-
tiators.84 These polypeptide factors seem to be important
regulators of bone growth and repair through their ab­ility
to activate migration of mesenchymal cells and induce
osteoblastic differentiation followed by proliferation.85
TGF-β itself is abundant in bone matrix. In vitro cell work
has shown that TGF-β has positive and negative effects
on osteoblast proliferation, differentiation, and matrix
s­ynthesis.86-88 TGF-β is released during the process of osteo-
clastic bone resorption89 and may initiate the formative
part of the bone-remodeling unit, the “coupling” of forma-
tion to resorption.90 The differential effects of the various
BMPs to induce cartilage and bone formation in vivo are
being examined to determine their potential to accelerate
the healing of complex fractures and to improve the rate
of allograft and autograft successes.91 BMP-4 initiates the
sequential gene expression of osteopontin and osteocalcin
in chondrocytes and osteoblasts.92 BMP-6 may be involved
in the earliest part of the differentiation of osteoblast
cells.93
Osteoblasts also produce numerous growth factors and
cytokines that are crucial to the process of bone formation,
remodeling, and repair (Fig. 4-5). Many of these soluble
products also are released by circulating macrophages and
lymphocytes. Insulin-like growth factor-I, or somatomedin
C, directly stimulates osteoblast replication and function94
and may increase remodeling by inducing RANKL.95 Many
of these regulatory compounds are discussed in the section
on bone remodeling.
CFU-GM
Figure 4-5  Osteoclasts and osteoblasts interact
through cytokines released into the bone micromilieu. CFU-M
Macrophages secrete macrophage colony-stimulating
factor (M-CSF), various interleukins (IL), and tumor ne-
crosis factor (TNF), all of which promote osteoclast dif-
ferentiation from hematopoietic stem cells, from the
colony-forming unit for granulocyte-macrophages
(CFU-GM) and the CFU for macrophage (CFU-M) to
Osteoclast
terminal osteoclast phenotype. Osteoblasts interact
by secreting factors that affect osteoclasts, including  TNF-α
M-CSF and transforming growth factor (TGF)-β, and
factors affecting bone mineralization and progression
of their own phenotype, such as insulin-like growth
factors (IGFs) and basic fibroblast growth factors
(FGFs).
GENETIC ABNORMALITIES
Many genetic diseases have predominant skeletal
­phenotypes, including at least 150 osteochondrodysplasias.
The fact that dysfunction of many single genes is likely to
cause skeletal manifestations arises from the great num-
ber of genes involved in the complex processes of skeletal
development, growth, and remodeling. Numerous genes not
pr­eviously suspected to be associated with skeletal physiol-
ogy generate abnormal skeletal phenotypes when deleted
from transgenic mice. Examples are the ubiquitous c-src
tyrosine kinase68 and the intranuclear factor c-fos.63 Dele-
tion of either gene leads to osteopetrosis. The association of
genes with processes crucial to normal skeletal physiology
is a growing area of research; only a few striking examples
are offered here.
A receptor for fibroblast growth factor has been implicated
in achondroplasia, the most common form of chondroplasia
in humans. This disease is characterized by short-limbed
dwarfism combined with a large head. Gene abnormalities
in multiple subjects consist of point mutations in the fibro-
blast growth factor receptor-3,24 which result in a consti-
tutively active receptor,96 indicating that fibroblast growth
factor limits the expansion of the growth plate. Another
gene regulating elongation of the growth plate is PTHrp.
Interruption of PTHrp action causes a shortening of long
bone.97 In comparison, increasing PTHrp action, as seen in
the constitutively activated PTHrp receptor associated with
Jansen’s metaphyseal chondrodysplasia, causes metaphyseal
abnormalities.98
Alterations in either the organic (e.g., collagen) or the
inorganic (e.g., hydroxyapatite) matrix components bring
about changes in the bone structure, some of which are indi-
rect, and some of which can be catastrophic. An entire spec-
trum of bone diseases, including osteogenesis imperfecta, is
discussed in Chapter 92.
With even this briefest of presentations, the reader should
be aware of the virtual explosion of the knowledge base with
regard to the genes controlling skeletal growth and homeo-
stasis. Targeted disruption of genes in animals, genetic stud-
ies of human kindreds with abnormal skeletal phenotypes,
and the sequencing of the human genome are expected to
lead rapidly to a fuller understanding of the many heritable
diseases affecting the skeleton.
Macrophage
BMP
M-CS-F
TGF-β
IGFs
M-CSF FGFs Osteoblast
IL-6
IL-11
Preosteoblast
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
SYSTEMIC REGULATION OF BONE
REMODELING
CALCIUM METABOLISM
Calcium is crucial to many processes in the body, including
most signaling cascades and synaptic signaling, creating a
requirement for tight control of systemic calcium concen-
trations. As stated at the beginning of this chapter, the skel-
eton is the reservoir for calcium, and bone remodeling is one
of the processes crucial to calcium regulation. Hormones
involved in calcium homeostasis have profound effects on
the skeleton.
Parathyroid Hormone
The primary function of PTH is to maintain serum ion-
ized calcium levels within a narrow physiologic range. PTH
secretion increases as calcium levels decrease to less than 8
mg/dL, controlled by decreased binding of the calcium ion
to the calcium receptor on the parathyroid cell.99 When a
serum calcium value greater than 10 mg/dL accompanies a
high level of serum PTH, a diagnosis of hyperparathyroid-
ism can be made.100 Other circulating factors, such as vita-
min D, tumor necrosis factor-α, and prostaglandins, also are
involved in regulating calcium balance.
By stimulating osteoclast activity and bone resorption,
PTH provides calcium and phosphate for mineral homeo-
stasis. Membrane receptors for PTH on osteoblasts stimulate
substantial morphologic and metabolic changes in these
cells and subsequently in osteoclasts.101 The distal effects
of PTH occur by increases in levels of intracellular cyclic
adenosine monophosphate (cAMP) and mobilization of
cytosolic calcium ion.102 Although PTH receptors probably
do not exist on osteoclasts,103 these cells are activated within
minutes of osteoblast exposure to PTH. PTH also targets
renal 1α-hydroxylase, which is the final step in the activa-
tion of vitamin D. PTH contributes to maintaining serum
calcium levels by direct and indirect interactions with the
skeleton.
Vitamin D
Vitamin D has effects on bone remodeling and calcium
homeostasis. A 10-minute exposure of the skin to ultra-
violet light from the sun causes endogenous production of
sufficient quantities of vitamin D3 from 7-dehydrocholes-
terol to allow normal calcium balance. Vitamin D3 is first
hydroxylated to 25-hydroxyvitamin D in the liver, followed
by 25-hydroxyvitamin D hydroxylation in the kidney. Often
referred to as calcitriol, the active metabolite, 1,25-dihy-
droxyvitamin D, stimulates intestinal absorption of cal-
cium, providing the mineral necessary to build bone. An
absence of vitamin D action, such as in vitamin D–resistant
rickets secondary to mutations in the vitamin D receptor104
or in pseudovitamin D rickets resulting from an abnormal-
ity of 1α-hydroxylase,105 leads to hypocalcemia and severe
rachitic bone disease. Although adequate levels of 1,25-dihy-
droxyvitamin D are necessary to provide calcium for bone
formation, supraphysiologic levels induce bone resorption
by stimulating the differentiation of osteoclast precursors.106
This occurs through the effect of 1,25-dihydroxyvitamin D
77
of increasing bone stromal cell production of M-CSF107 and
RANKL expression.108
Besides the role of vitamin D in ensuring calcium
­acquisition from the intestine, vitamin D has subtle effects
on the formation and resorption of bone.109 Allelic vari-
ants of the vitamin D receptor may have subtle effects on
bone density, but this is controversial and may be subject to
et­hnicity.110-113 Full understanding of the role of vitamin D
in skeletal development requires continued epidemiologic
and basic studies.
Calcitonin and Other Calcium-Regulating Factors
Calcitonin is a calcium regulatory hormone that works
­independently of PTH and vitamin D and possesses a potent
capacity to modulate serum calcium and phosphate levels.114
Similar to the control of PTH secretion from parathyroid
cells, serum calcium levels regulate the secretion of calcito-
nin from parafollicular or C cells of the thyroid. In contrast
to PTH, which is secreted in inverse proportion to serum
calcium levels, calcium ion directly stimulates the secre-
tion of calcitonin115 by its binding to the calcium receptor
on the parafollicular C cells.116 The binding of calcitonin
to ­ receptors on osteoclasts117 causes a marked decrease in
osteoclast metabolic activity along with cell retraction.118,119
For these reasons, calcitonin has been used successfully in
the acute treatment of humoral hypercalcemia of malig-
nancy and in treatment of Paget’s disease. The use of calci-
tonin in the treatment of hyperresorptive osteoporosis also
is salutary.120 The response to calcitonin is transitory, how-
ever, because it downregulates its cognate receptor within
hours.121
Osteoblasts also secrete prostaglandin E2 (PGE2) in
response to hormonal or mechanical stimuli. PGE2 can
stimulate bone formation and resorption and serve as a che-
motactic factor for osteoclasts.122 Although PGE2 and PTH
stimulate the same second messengers in osteoblasts, PGE2
directly affects osteoclasts through its ubiquitous receptor.
PGE2 stimulates several second-messenger responses in bone
cells, including an increase in cytosolic calcium, ­ elevated
cAMP production, and activation of the phosphatidyl-
inositol pathway.123 In the anabolic response to loading,
prostaglandins may be particularly important because pros-
taglandin levels increase during loading and can reproduce
some specific responses of load when given in vitro.124
MATURATION: SEX STEROIDS
The rapid decline of skeletal mass after menopause
­underscores the crucial regulatory role of sex steroids in bone
cell metabolism. The expression of estrogen receptors125,126
and androgen receptors127 within osteoblasts ­ designates
bone cells as targets of these sex steroids. Elevated estro-
gen levels increase osteoblast proliferation128 and attenuate
the osteoblast response to PTH.129 In addition, estrogens
increase osteoblastic collagen gene expression28 and ­insulin-
like growth factor-II production130 and may even directly
regulate the production of osteoclastic lysosomal enzymes.131
It also has been recognized that interleukin-6 can stimulate
osteoclastic bone resorption only in the estrogen-deficient
state.61 Estrogen has been shown to have effects on the
RANKL-osteoprotegerin equilibrium. Estrogen deficiency,
 78 RUBIN  |  Biology, Physiology, and Morphology of Bone
although not changing RANKL expression on osteoclast
precursors, blunts the intracellular signaling stimulated by
RANKL binding, essentially decreasing osteoclastogen-
esis.132 Also, estrogen has been shown to increase osteopro-
tegerin in the circulation, diminishing RANKL effect by
preventing its access to potential osteoclasts.133
Further appreciation of estrogen action has emerged
with evidence of effects on the male skeleton. A unique
male patient shown to be unresponsive to estrogen because
of an abnormal estrogen receptor never fused his epiphyses,
despite an intact androgen axis.134 A second patient, still
growing taller at age 30 years, was found to lack estrogen
action in bone, in this case through the complete absence
of the cytochrome P-450 aromatase necessary to convert
androgen to estradiol.135 This patient was noted to have
extremely low bone mineral density. Treatment with low
doses of estradiol not only caused epiphyseal fusion, but
also increased bone mineral density by nearly 10% over the
first year of treatment, with a continued increase during
the subsequent 3 years.136 These concepts have led work-
ers in osteology to promote a “unifying model” suggesting
that bone mineral density in men and women depends
on su­fficient estrogen levels, and that androgen may have
completely separate, unknown effects on skeletal growth
and mass.137
REGULATION OF BONE MINERAL DENSITY
Humans attain a mature skeleton by age 30 years, when
bone mineral density peaks. The peak bone mineral density
is largely controlled by hereditary factors, which are not
well understood.138,139 After this peak, long-lived individuals
are subject to a continual decline in mineral status, increas-
ing skeletal susceptibility to fracture. With much attention
directed toward osteoporosis in the last several decades,
clinicians have recognized that some pathologic conditions
can prevent the achievement of peak bone mass, and other
conditions can promote the normal decline in bone min-
eral density after maturity. Although a detailed discussion
of this complex and thoroughly studied process is beyond
the scope of this chapter, several points can be made. First,
as suggested previously, normal sex steroids are required to
attain peak bone mass, as shown by the failure of young
hypogonadal adults to reach normal peak bone mineral
density.140 Deficiency of either estrogen or testosterone
after attaining adult bone mass increases the rate of mineral
loss, largely through increased resorption.141 Insulin-like
growth factor-I, a critical factor for bone development and
mineralization, seems to enhance normal mineralization,142
but also may promote bone remodeling by decreasing
osteoprotegerin and increasing RANKL.95 Certain medi-
cal conditions are associated with increased resorption and
decreased bone formation, such as excess glucocorticoids—
either iatrogenic or in Cushing’s disease.143 Increased loss
of bone mineral density also is associated with any coex-
isting illness,144 undoubtedly due to multifactorial causes,
including the inevitable decline in exercise that parallels
aging, which stimulates bone loss secondary to decreased
function.145,146
The Wnt pathway has been implicated in osteoblast
­function and bone mineral content, an extension of
the many roles that Wnt signaling has in development
and ­ maintenance of multiple tissues. Two kindreds with
increased bone mineral density with abnormalities in Lrp5
protein, which leads to enhanced Wnt signaling, have been
described so far.17,18 On the distaff side, loss-of-function
mutations in human Lrp5 cause the osteoporosis-pseudogli-
oma syndrome, where bone density is reduced.147 A growing
literature suggests that Wnt signaling regulates osteogenesis
at points from the mesenchymal stem cell all the way toward
mineralization.148
BIOPHYSICAL STIMULI
Working systemically or locally, chemical factors, such
as sex steroids, vitamin D, M-CSF, and TGF-β, play vital
roles in the regulation of skeletal modeling and remodel-
ing. There is increasing evidence that these “mediators of
change” may be orchestrated by local factors that are not
chemical in origin. One of the most potent influences of
the development and maintenance of the skeleton are by-
products of function (during use), referred to as biophysical
factors (i.e., mechanical-electric, discussed in detail at the
end of this chapter). Whether it is an increase in bone mass
as a result of exercise or work habits, or a decrease in bone
mass resulting from bed rest, cast immobilization, or space
flight, physical factors are key to determining the ultimate
structural success of the skeleton. It is essential to keep the
role of chemical mediators in perspective and acknowledge
physical factors as a crucial control mechanism in the regu-
lation of tissue differentiation, growth, repair, and remod-
eling.149 Although the molecular mechanisms are not well
understood, studies proving an effect of biophysical stimuli
on bone microstructure and mineral density are increasing
in number.150,151 Physical stimuli as potent determinants of
skeletal morphology have been postulated as primary regu-
lators of chondro-osseous morphogenesis,152 although the
specific mechanical parameters that control the process
have not yet been determined. Nevertheless, more recent
studies have documented the potential efficacy of bio-
physical stimuli in improving bone quantity and quality;
departing from the premise that larger signals are better,
extremely small mechanical strains (<10 microstrain) were
shown to be strongly anabolic to trabecular bone, indicat-
ing the potential for a nonpharmacologic intervention for
osteoporosis.153
COMPOSITION OF BONE MATRIX
Bone is composed of inorganic mineral (70% of weight),
organic matrix and cells (25%), and water (5%). Before its
calcification, newly synthesized bone matrix is essentially
completely organic and is termed osteoid. Collagen is the
predominant organic component in bone, accounting for
approximately 94% of the unmineralized matrix (see Table
4-1). Other noncollagenous proteins unique to bone are
found in osteoid, accounting for approximately 4% of its
weight. These include glycoproteins and phosphoproteins,
such as osteonectin154 and sialoproteins, which are predomi-
nantly osteopontin,155 osteocalcin,156 and BMP.157 Extracts of
bone also include enzymes, hormones, growth factors, and
other metabolites essential for bone metabolism.158 Bone
cells, for all their responsibility to mineral and structural
homeostasis, constitute only 2% of the organic tissue.159
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
COLLAGEN AND PROTEOGLYCANS
The major protein secretory product of the osteoblast is
collagen, whose complex primary, secondary, and tertiary
structure lends strength to bone and allows the seeding of
hydroxyapatite crystals from a supersaturated extracellular
fluid. Collagen is a crucial element of the skeleton, and
abnormalities in its structure and regulation lead to severe
skeletal phenotypes.
Osteoblast formation of collagen is typified by cross-link-
ing of lysine and proline residues to form procollagen ­trimers.
The processed collagen is organized into parallel fiber sheets
of tropocollagen.160 Within each sheet, or lamella, the fibers
lie parallel to each other,161 whereas the fibril orientation
on adjacent lamellae runs in directions distinct from this
axis (Fig. 4-6), contributing to the strength of bone, as in
the structure of plywood. This structure also facilitates the
seeding of hydroxyapatite crystals from a supersaturated
extracellular fluid.
Bone collagen is primarily type I and resembles other
type I collagens found in skin and tendon. The basic unit of
bone collagen, the tropocollagen molecule, is a triple ­helix
of three polypeptide α chains, each with ­ approximately
1000 amino acids.162 By stabilization of these soluble
molecules with cross-links of hydroxylysine and lysine,
the bone ­ collagen fibrils become insoluble.163 Type I col-
lagen differs from the type II collagen of cartilage in sev-
eral salient aspects. Each of the three α chains of type II
­collagen is ­ identical to the others, although their amino
acid ­composition differs from that of any of the three (one
Figure 4-6  Ground section of bone photographed with polarized light
showing the concentric lamellar structure of the basic unit of mature
bone, the osteon. The central vascular canal (empty in this preparation)
is surrounded by multiple lamellae of bone. The adjacent lamellae are
composed of collagen bundles with differing orientations, giving rise to
alternating light and dark bands in polarized light.
79
pair, one unique) α chains of the type I tropocollagen of
bone.164 Compared with the chains of type I collagen, the
chains of type II collagen contain much more glycosylated
hydroxylysine, making cartilage all the more resistant to
degradation by collagenase.
The triple helix of type I collagen forms a linear molecule
approximately 300 nm long.165 Each molecule is aligned par-
allel to the next, producing a collagen fibril. Within the col-
lagen fibril, gaps called hole zones exist between the end of
one molecule and the beginning of the next. It is thought
that noncollagenous proteins reside in these spaces, which
chemotactically attract and initiate the mineralization pro-
cess.159 The fibrils are grouped further in bundles to form the
collagen fiber.
Proteoglycans constitute the principal noncollagenous
protein in the mineralized matrix. Bone proteoglycan is
similar in structure to the proteoglycan found in carti-
lage, consisting of a thin protein core with multiple cova-
lently bound glycosaminoglycan chains.166 Although the
role of these proteins in bone has not been determined, it
has been proposed that they may store information after
functional activity, serving as a form of strain memory.167
These molecules deform rapidly in response to load, but
re-establish their original relative orientation after unload-
ing. Although speculative, this matrix cell interaction
may serve as a signal ­transduction mechanism to transfer
mechanical information from the matrix to the entombed
osteocytes.
OSTEOCALCIN
Also present in the matrix are substantial quantities of
o­ steocalcin, a protein found almost exclusively in bone
and that constitutes 1% to 2% of the total bone protein.
Osteocalcin synthesis occurs in the osteoblasts during the
carboxylation of glutamate, a vitamin K–dependent reac-
tion.168 1,25-Dihydroxyvitamin D enhances the synthesis
of this noncollagenous protein.169 The osteocalcin knock-
out mouse, which required that three gene isoforms be
deleted to achieve the unique phenotype, is characterized
by a late-onset increase in bone mineral density, suggest-
ing that an absence of the osteocalcin signal for activation
of osteoclasts results in a thicker, less remodeled, bone.12
Osteocalcin also seems to have a role in regulating mineral
properties.170
HYDROXYAPATITE
Bone mineral is generically referred to as hydroxyapatite
(Ca10[PO4]6[OH]2), a platelike crystal 20 to 80 nm long and
2 to 5 nm thick. Bone hydroxyapatite differs from naturally
occurring apatite because it contains numerous impurities,
including sodium, fluorine, strontium, lead, and radium.
It is smaller in size than natural apatites (10 nm versus 40
nm) and more reactive and soluble because of its less-per-
fect atomic arrangement.171 The nucleation sites of bone
mineral may not be the plates of hydroxyapatite, but the
more energetically favorable crystal spicules, such as amor-
phous ca­lcium phosphate and octacalcium phosphate.172 It is
believed these unstable precursors are formed first and gradu-
ally transformed to the more crystalline hydroxyapatite.173
Mineral exchange is facilitated by the enormous surface area
 80 RUBIN  |  Biology, Physiology, and Morphology of Bone
of ­ amorphous ­ calcium phosphate, including its hydration
shell. This is reflected in the greater avidity of new bone for
“bone-seeking” isotopes (e.g., technetium, fluorine, stron-
tium). The bone mineral continues to mature throughout an
individual’s lifetime,174 becoming more and more “perfect”
and exposing less surface area for a given volume of mineral.
BONE MARKERS
Physicians have sampled serum for products released
during bone remodeling as surrogates for histology and
­visualization of bone architecture. Although no one serum
marker has been found to predict adequately disease or
response to th­erapy, panels of markers can be used to su­ggest
the p­resence of osteoblast or osteoclast activity. Alkaline
phosphatase, in long use, is still an inexpensive marker
of increased osteoblast function and is only slightly less
ac­curate than bone-specific alkaline phosphatase. Osteo-
calcin also reflects bone formation activity.175 The presence
of active osteoclasts in bone can be measured with serum
and urine collagen breakdown products, such as N-telopep-
tides176 and pyridinolines177 (see Chapter 32). Successful
treatment of high-bone-turnover states should be accompa-
nied by decreases in these markers.178
MORPHOMETRIC ASSESSMENT OF BONE DENSITY
AND STRUCTURE
The “gold standard” of measurement of bone mineral den-
sity is dual-energy x-ray absorptiometry. The density units
are given in grams per centimeter squared, as opposed to
cubed, and they are indicative more of an apparent—rather
than real—density because the two-dimensional rendering
does not account fully for the bone size.179 In contrast, quan-
titative computed tomography (CT) measurement is a true
volumetric measure of bone mineral density, but it is used
infrequently because of the necessity of technical expertise
and cost. Quantitative CT has been used more recently to
show differential effects of PTH on trabecular compared
with cortical bone.180 Ultimately, to determine bone’s true
physical properties, assays such as transcutaneous ultra-
sound,181 magnetic resonance imaging, quantitative CT, and
real-time micro-CT182 may provide more specific informa-
tion regarding the quantity and the quality of the bone.
MINERALIZATION OF BONE TISSUE
The mineralization of bone begins 10 to 15 days after the
organic osteoid matrix has been laid down.183 At this point,
mineral increases almost immediately to 70% of the ultimate
content, whereas deposition of the final 30% takes several
months.184 The process of mineralization is extremely com-
plex and temporally dynamic.185 There is emerging evidence
that hydroxyapatite deposition and seeding of mineraliza-
tion are strongly interdependent on cartilage-derived and
bone-derived macromolecules, such as osteonectin, phos-
phoproteins, and proteolipids. The initial sites of calcium
phosphate nucleation in growing bone, fracture callus, and
calcifying cartilage appear to be not at the bone surface, but
on the processes of matrix vesicles.186 Matrix vesicles are
small, round, extracellular lipid-bilaminar bound organelles
that bud from hypertrophic chondrocytes or osteoblasts
undergoing the process of apoptosis187 and from cell pro-
cesses originating from the plasma membrane.188 There is a
definite polarity to the vesicles, with mineralization occur-
ring in a predictable and organized way adjacent to the req-
uisite phosphatases on the inner leaflet of the membrane.189
The matrix vesicles contain alkaline phosphatase, adenosine
triphosphatase, inorganic pyrophosphatase, 5′-nucleotidase,
and adenosine triphosphate–pyrophosphohydrolase190 in
addition to phospholipids (especially phosphatidyl serine),
which have a strong affinity for calcium ions.191 These ions
are believed to accumulate in the matrix vesicle because of
their affinity for the phospholipids and because of a mem-
brane-bound calcium pump. At a point of supersaturation,
nucleation of the mineral begins.192
In retrospect, the question of whether mineralization
is an active or passive process remains to be answered.
H­artgerink and colleagues193 have created nanofibers that
assemble in structures reminiscent of the osteoid matrix.
These asse­mbled nanofibers are able to direct mineralization
of hydroxyapatite in the absence of cells. The ost­eoblast may
enable passive mineralization entirely through pr­oduction
of the extracellular matrix.
NUCLEATION
Alkaline phosphatase, a biosynthetic product of osteoblasts,
is present in very high concentrations during develop-
ment and osteoid production.194 The regulatory role of this
disulfide-linked dimer is unknown, but its presence may
increase local concentrations of phosphate and facilitate
hydroxyapatite deposition.195 Increasing the concentration
of phosphate in the micromilieu exceeds the local solubility
product and catalyzes deposition along the inner leaflet of
the vesicle.
After this accretion, the destruction of the membrane has
been attributed to an increasing concentration of lysophos-
pholipids within the matrix vesicles, which suggests that
they are programmed to self-destruct.196 After dissolution
of the matrix vesicle membrane, the hydroxyapatite crys-
tals are exposed to the extravesicular environment, where
additional mineral accretes to the newly formed crystal.197
The crystal is believed to move chemotactically toward
and bind preferentially at the hole zones between collagen
fibrils, precipitated by the nesting osteonectin198 and fibro-
nectin.199 Mineralization proceeds and extends over the
collagen matrix, with the long axis of the hydroxyapatite
crystal parallel to the collagen fiber. The arrangement of the
collagen matrix that is synthesized during osteoblast activity
ultimately determines the orientation of the bone mineral
crystal.161
In the extravesicular milieu, glycosaminoglycans inhibit
the calcification process by modulating the advancing
mineral front.200 It may be just these proteoglycan macro-
molecules, found in high concentrations in noncalcifying
collagenous structures such as ligament, tendon, and skin,
that may prevent mineral deposition.201 Other theories for
the lack of calcification of dense connective tissues include
the tighter packing of their collagen fibrils; limited access
of phosphate ions to the interfibrillar nucleation sites; and
the existence of crystallization inhibitors, such as pyro-
phosphate, present in synovial fluid, plasma, and urine at
concentrations sufficient to prevent deposition of calcium
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
carbonates.202 When the concentration of these inhibitors
reaches a threshold, mineralization is halted, leaving a thin
layer of osteoid between the lining cells and the mineral-
ization front. This establishes the syncytium, or cellular
canopy, which must be retracted to expose the mineral and
reinitiate the remodeling cycle.
TURNOVER
In undecalcified ground sections, microradiography shows
subtle differences in the calcium content of the bone tissue,
allowing separation into “old” and “new” bone on the basis
of contrast intensity (Fig. 4-7). Young osteons, in the process
of formation, have large central vascular canals that nar-
row with infilling, showing progressively less mineralization
toward the center. This contrasts sharply with the active
tunneling process of resorption, in which case the inside
rim of the osteon appears equally mineralized as the outer
rings. Static remodeling parameters, such as the osteoid seam
width, the number of resorptive events, and the number of
formative events, can be inferred from these morphologic
characteristics. By using double-fluorescent labels (e.g.,
tetracyclines) administered at known intervals, the clini-
cian can determine dynamic parameters of bone remodeling
(i.e., rates of turnover, infilling, and formation). Static and
dynamic histomorphometric studies, quantified by means of
biopsy specimens harvested from such areas as the iliac crest,
are extremely powerful means of evaluating the systemic
state of the skeleton.203
The complex, composite nature of bone achieved through
the secretion of the collagen matrix and its subsequent min-
eralization is a product of the synergistic interrelationships
of the cell types, the systemic regulators of calcium metab-
olism, and the matrix-bound proteins that locally control
Figure 4-7  Microradiograph of cortical bone showing osteons in vari-
ous degrees of mineralization with numerous interstitial fragments (*).
81
bone remodeling. The product of this sophisticated and
interdependent process is an extremely successful tissue that
serves as a structural organ and as a mineral reservoir. Many
substitutes for bone, derived from calcium phosphate and
hydroxyapatite mixtures (e.g., coral), have been used suc-
cessfully in the clinic as bone substitutes in allografts or as
an osteoconductive surface on implants.
ARCHITECTURE OF BONE
Macroscopic Organization
Bones are remarkably well suited for their structural role. At
the gross level, as hollow tubes they derive maximal strength
from minimal weight.204 At the next-lower structural level,
cortical and cancellous morphology is strategically arranged
to distribute functional stresses evenly.205 Lower still, the
arrangement of the collagen within the cancellous or corti-
cal bone, combined with the two-phase composite matrix
of the collagen and mineral, provides tensile and com-
pressive strength.206 The ultimate tensile strength of bone
approaches that of cast iron, and its capacity to absorb and
release energy is twice that of oak, yet the weight of bone is
only one third that of steel. While proving a resilient and
resistant material, this tissue’s capacity to remodel, adapt,
and repair itself—without leaving a scar—is what identifies
bone as the ultimate biomaterial. Martin and colleagues207
provide an excellent review of the architecture of bone. The
structural success of skeletal morphology can be examined
at a series of levels, as follows:
1. Gross anatomy and functional responsibility
2. Ultrastructural morphology (cortical or cancellous)
3. Microscopic organization (lamellar or woven)
4. Mineralization process (enchondral or intramem-
branous)
At the gross structural level, each bone has diverse
and distinct morphologic features. Regardless of func-
tion, each bone is composed of dense cortical tissue
(e.g., diaphyseal shaft) and cancellous tissue, such as the
trabecular cascades found in the neck of the femur or
the metaphysis of the proximal tibia (Fig. 4-8). At the
microscopic level, two types of bone are identified: the
disorganized, hypercellular woven bone and the highly
organized, relatively hypocellular lamellar bone. Essen-
tially, all bone tissue can be described by one of these two
morphologic patterns, whether mature, growing, patho-
logic, or healing.
Woven bone is a product of rapid bone formation.
Architecturally, it has an irregular, disorganized pattern of
collagen orientation and osteocyte distribution (Fig. 4-9).
Although woven bone is characteristic of embryonic and
fetal development, it also is found in the healthy adult skel-
eton at li­gament and tendon insertions and in specific dis-
ease states, such as Paget’s disease, osteogenic sarcoma, and
metastases. Under less severe pathologic conditions (fracture
callus, inflammatory responses, stress fractures), woven bone
is usually reabsorbed and replaced by lamellar bone within
a few weeks of its deposition.208 Mechanical ­ stimulation,
if potent enough, can even cause a rapid production of
woven bone, which ultimately remodels into dense lamellar
bone.209 Many consider woven bone an aberrant response.
That it is laid down so quickly and is so readily remodeled
 82 RUBIN  |  Biology, Physiology, and Morphology of Bone
Figure 4-8  A, Macerated preparation of the human
knee showing the trabecular structure that supports the
flared articular surface. B, Radiograph of the specimen A B
shown in A.
Figure 4-9  Outer cortex of bone showing the results of rapid periosteal
bone formation producing woven bone (arrows), followed by the slower
formation of primary osteons surrounding blood vessels.
distinguishes this type of bone, however, as a wise strategy in
accommodating new, intense structural challenges.
Lamellar, or mature, bone can be packed tightly to
form the dense cortex of a bone, or it can be organized
as the trabecular struts in cancellous bone. In contrast to
the random and disorganized structure of woven bone, the
lamellar appearance of this mature bone is the product of
highly organized mineralized plates. In cancellous bone,
the ­ lamellae run parallel to the trabeculae. In cortical
bone, several ­patterns occur. The predominant one is that
found in ­osteons, which are composed of small, concentric
lamellar cylinders surrounding a central vascular channel,
similar to the rings in a tree trunk (Fig. 4-10). Osteons
are typically 200 to 300 μm in diameter, consisting of six
or seven concentric osteocyte rings, which are co­mposed
of 20 lamellar plates.210 Canaliculi in lamellar bone are
c­onsistent in diameter and orientation and, in total, con-
tain fewer osteocytes per unit volume than woven bone
(20,000 cells/μm3 versus 80,000 cells/μm3).
During growth in diameter, new bone must be added
appositionally. After the formation of the periosteal cuff
around the primary center of ossification, bone increases
its diameter by one of two methods. During rapid growth,
spicules of new woven bone are formed perpendicular to
the surface, which allows maximal radial expansion with
minimal material. The gaps between the spicules are sub-
sequently filled in and consolidated by the formation of
lamellar bone. In this process, individual periosteal blood
vessels that lie in the valley of the spicules are surrounded
by the encroaching new lamellar formation, creating pri-
mary osteons parallel to the long axis of the bone. An
osteon that has formed de novo, as in the woven bone
consolidation process just described, is known as a primary
osteon. If this occurs intracortically by a resorptive process
to replace preexisting bone tissue, it is referred to as a sec-
ondary osteon, or haversian system, and constitutes the
bulk of adult human bone.211
When slow diametric growth occurs, seams of new
bone are laid appositionally on the existing surface. As dis-
cussed previously, the birefringent pattern of circumferen-
tial ­lamellae and single osteons is believed to arise through
directional changes between collagen bundles in layers,
maximizing strength in many different planes. Although
the collagen bundles within each of these lamellar plates are
highly oriented, individual fibers often traverse interlamel-
lar spaces. Such a composite integration increases the indi-
vidual osteon’s resistance to external loads and the effective
strength of the bone structure.212
An alternative theory for the polarized light ­birefringence
of an osteon is based on fiber-rich, fiber-poor lamellar rings,
in which the thinner (1- to 2-μm) plates contain a greater
degree of glycosaminoglycans (ground substance) than the
adjacent 5- to 7-μm, collagen-rich layers.213 These glycos-
aminoglycans are thought to be continuous from the “thin”
lamellar plate, through the cement line, to interdigitate with
the ground substance of the “thick” plate. This architecture,
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
Mature haversian system (2 osteon)
1 Osteon
Circumferential
lamellae
Woven bone
Resorption cavity
(osteoclastic and
osteoblastic activity)
Periosteal
capillary and vein
Periosteum
Haversian canal
with a true continuity between plates, would produce an
­increase in the stiffness of each osteon or each circumfer-
ential plate. The morphology of lamellar bone may prove to
be some combination of these two postulations, maximizing
the stiffness of the material (continuity of ground substance)
and its toughness (integration of collagen layers).
CORTICAL DRIFTS: FLEXURAL NEUTRALIZATION
If periosteal surface modeling were to occur in the absence
of endosteal resorption, the overall thickness of the cortex
would increase with increasing age, leaving too much bone
and too little marrow space. This occurrence is avoided
by coordinating the increasing periosteal diameter with
a concomitant increase in the diameter of the endosteal
envelope, which is achieved through resorption at this
inner surface.214 Although these rapid surface drifts dimin-
ish in the mature skeleton, they increase again in elderly
individuals. In the aged skeleton, the rate of surface ero-
sion of the endosteal surface exceeds the formation rate of
the periosteum, resulting in a net decrease in total bone
mass. This age-related expansion of the cortex establishes a
biomechanical compensatory mechanism, however, by the
concurrent increase in the cross-sectional moments of iner-
tia, which results in an increased capacity of the bone to
resist bending loads.215
METAPHYSEAL RESHAPING
During growth in length, the changes in periosteal and
­endosteal compartments are directed toward periosteal
­resorption accompanied by endosteal extension. As the
metaphysis and diaphysis elongate, resorption at the peri-
osteal surface must be closely coordinated with deposition
83
Lamellar bone
(in trabecula of
cancellous bone)
Forming osteon
Endosteal
capillary and vein
Volkmann’s canal
Osteoclast
Osteoid Cutting
Osteoblast cone
Capillary loop
Figure 4-10  Architectural organization
of cortical bone.
at the endosteum, a process known as metaphyseal reshap-
ing.216 Under these circumstances, some of the cortical
bone within the epiphyseal shell is spared and subsequently
becomes a component of the cancellous structure within the
metaphysis. Where fragments of osteons and new lamel-
lae and abundant cement lines are seen, the ultrastructural
organization of trabeculae reflects their cortical origin.
HAVERSIAN REMODELING
Bone remodeling is a process of “real-time” tissue
r­ eplacement. In places, trabeculae are covered with
­osteoblasts making new bone; in others, osteoclasts are
eroding the surface. This process of resorption and forma-
tion can rapidly alter the orientation of the trabeculae to
accommodate changes in the manner of loading or shifts
in alignment secondary to disease or fracture.217 To remodel
cortical bone so that intracortical damage or dead tissue can
be replaced, the cortex first must be resorbed from within,
creating a surface for apposition. When haversian systems
replace or remodel existing bone, this is not achieved
through the identical pathway of the original osteon. These
secondary osteons can persist directly through an existing
arrangement of osteons and circumferential lamellae, leav-
ing only remnants of these preexisting structures. These
fragments of ­ lamellar or woven bone are termed intersti-
tial lamellae. Because the structural integrity of the bone
must be preserved during this remodeling, the replacement
process requires close integration between resorption and
formation. The cutting cone of the osteoclast must be fol-
lowed rapidly by capillary invasion and the simultaneous
intrusion of a population of osteoblasts infilling the lamel-
lar rings of the secondary osteon. As discussed previously,
matrix-bound proteins, such as TGF-β, which are released
 84 RUBIN  |  Biology, Physiology, and Morphology of Bone
by the resorption process, may be crucial to integration of
this coupling process.
Although levels of intracortical remodeling may be ele-
vated by changes in the organism’s nutritional status (e.g.,
calcium deficiency218), endocrine imbalance (e.g., hyperpara-
thyroidism, menopause219), or even aging (e.g., osteopenia220),
one of the most potent stimuli for remodeling is a change
in the level of physical activity.221 If physical demands are
altered (e.g., changing activity), or if the manner in which
the bone is loaded is changed (e.g., distribution of strain or
loading rate), the bone remodels internally to adapt to the
new demands.222 Evidence of this osteonal turnover has been
shown in rabbits, in which a 150% increase in the number of
labeled secondary osteons occurred in the subchondral plate
of the proximal tibia subjected to repetitive loads.223
Not only does loading activate modeling and remodel-
ing, but also by-products of loading are believed to influence
morphology. One of the strongest correlates to elevated
intracortical turnover is an increased strain rate.224 Another
alternative to strain magnitude, strain frequency, is a potent
determinant of bone morphology.225 This sensitivity to dis-
crete components of the biophysical milieu opens several
distinct avenues for the treatment of musculoskeletal disor-
ders, including induced electric fields226; low-intensity ultra-
sound227; and low-magnitude, high-frequency mechanical
stimulation.228
BLOOD SUPPLY OF BONE
Bone is extremely vascular and requires approximately 10%
of the cardiac output.229 Blood supply to the cortical diaphy-
sis is derived from the nutrient artery and the periosteal ves-
sels. In the metaphyseal ends of the bone, where metabolism
is most active, the periosteal vessels are large and abundant
and are also referred to as metaphyseal arteries, although
they are entirely analogous to the periosteal capillaries. The
third set of vessels, the epiphyseal arteries, supplies the sub-
articular ends of the bones and assumes special importance
because of the growth process in this area and the vulner-
ability of these vessels to injury.
During infancy and adolescence, the epiphyseal plate
serves as a barrier separating the epiphysis from the metaph-
ysis. Although a few vessels crossing the plate have been
described, it is widely accepted that there is no effective cir-
culation across the plate. Essentially, the epiphyses have an
isolated blood supply via the epiphyseal arteries, but those
few vessels do present a potential route for the spread of
infection or tumor from the metaphysis into the epiphysis.
In most joints, there are abundant soft tissue attachments to
the epiphyses (muscles, ligaments, capsule) so that numer-
ous vessels supply the bone through these attachments (Fig.
4-11). In a few locations, such as the proximal femur, the
entire epiphysis may be intra-articular and may be covered
by articular cartilage. Because neither the articular nor the
growth cartilage is penetrated by vessels, the few epiphy-
seal arteries must pass alongside the growth plate, covered
by a thin layer of periosteum, to perforate the epiphysis.230
This route of blood supply is extremely vulnerable to trauma
(fractures through the growth plate), increased intra-articu-
lar pressure (joint infections or bleeding into the joint), or
idiopathic interruption (Legg-Calvé-Perthes disease in chil-
dren, avascular necrosis in adults).
Epiphyseal vessels arborize within the bony nucleus to sup-
ply the marrow, the cancellous bone, the dividing chondrocytes
in the microepiphyseal plates in the depths of the articular car-
tilage, and the growth plate itself. Because of this, interruption
of the vessels leads to cessation of longitudinal growth and dia-
metric growth of the epiphysis and joint surface.
Within the cortex of bone, capillaries travel primarily in
the longitudinal direction within haversian canals. Occa-
sional branching is seen, and lateral communication with
the periosteal vessels through Volkmann’s canals provides
collateral circulation. The usual haversian system is 100 mm
or less in diameter. Individual osteocytes are not more than
50 mm from their blood supply. The rich system of cana-
liculi radiating out from the central canal enhances micro-
circulation to the most distant osteocytes.
MECHANICAL PROPERTIES OF BONE
Even considering the elaborate cell kinetics, mineralization
process, and morphology of bone, its success as a structure
is ultimately a product of bone’s mechanical properties: how
stiff it is, how resilient it is to fatigue, and how effectively it
withstands the extremes of physical activity. The skeleton’s
structural success can be jeopardized by genetic disorders
A B
Articular cartilage
Periosteum
Growth plate
Epiphyseal area
Vein
Artery
Bone plate
Germinal
Proliferative
Growth
Hypertrophic plate
(calcified
cartilage)
Vascular
invasion
Vein
Artery
C
Metaphyseal area
Figure 4-11  Epiphyseal blood supply of growing bone. A, The blood
supply of most secondary centers of ossification is abundant by virtue of
the numerous soft tissue attachments. B, Certain secondary centers, such
as the proximal femur, are devoid of soft tissue attachments, and the
blood supply follows a tenuous route through the joint, where it is liable
to injury. C, The blood supply to the growth plate showing the contribu-
tion of the epiphyseal artery to the germinal portion of the growth plate.
(From Sledge CB: Biology and development of the growth plate. In Cave EF
[ed]: Trauma Management. Chicago, Year Book Medical Publishers, 1974.)
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
such as osteogenesis imperfecta, metabolic diseases such as
Paget’s disease, or even the bone loss that parallels the aging
process (i.e., osteopenia). To appreciate the structural risks
that accompany metabolic bone diseases, several interde-
pendent concepts of bone’s biomechanical properties must
be considered.231
The mechanical strength of fully mature osteonal
bone is greater than that of immature bone, which is
composed of circumferential lamellae and a few osteons
that may be only partially mineralized.232 Values for the
mechanical properties of individual osteons range from
a tensile elastic modulus of 12 GPa and 114 MPa ulti-
mate tensile strength for a fully mature, mineralized
osteon to less than half that modulus and only 75% of
the ultimate tensile strength for a younger, less miner-
alized osteon. For normal tensile or compressive load-
ing, the stiffness of the material, or elastic modulus,
shows human haversian bone to be about 17 GPa in
the longitudinal direction, 11.5 GPa in the transverse
direction, and 3.3 GPa in shear.233 The degree of
­mineralization (young bone) or porosity (old bone) com-
promises the stiffness of the bone and reduces the elastic
modulus. The “effective” modulus of the bone can com-
pensate for decreased stiffness, however, by changes in
morphology (e.g., periosteal expansion).
STRENGTH
A major contributor to the strength of bone is derived
through its composite nature of haversian, circumferen-
tial, and interstitial lamellae that work synergistically
to avoid yield, or ultimate strain. Strain, a dimensionless
unit of change in length divided by its original length, is
used in bone physiology as 10−6 strain, or microstrain. The
yield strain of bone, or the degree of deformation reached
at which the bone does not elastically recover, is approxi-
mately 7000 microstrain. A 0.7% change in length causes
irreversible damage to the tissue. Ultimate strain in bone,
or the degree of deformation at which the material actually
fractures, is 15,000 microstrain.234
An analogy of a bundle of straws versus a solid stick illus-
trates how a composite structure, such as bone, can prove
more successful in resisting loads by avoiding yield and the
ultimate strain of the material. This analogy is often put into
practice with the use of multistranded wire rather than sin-
gle-stranded wire. During flexion, each individual strand slips
relative to its neighbor rather than straining, minimizing the
generation of potentially damaging levels of strain. In the same
manner, individual lamellae “slip” relative to adjacent lamel-
lae, dissipating energy and minimizing strain levels within
the ­material, allowing the entire system to react in a more
elastic manner, rather than sustain brittle failure or ultimate
fracture.
TOUGHNESS
As an organ, bone needs to be stiff (to resist deformation)
and tough (to prevent crack propagation). There is a com-
promise, however, between these two objectives because
they are attained through a balance of the composite of the
resiliency to crack propagation provided by collagen and the
resiliency to deformation provided by mineral. Comparatively
85
small changes in the mineral content of bone tissue can have
substantial effects on its properties as a material, as shown by
Curry235 in his determination of the mechanical properties of
diverse types of bone. By comparing the bovine femur, the
deer antler, and the whale tympanic bulla, Curry235 illustrated
that as the morphologic responsibility of the skeletal element
changed, so did its mineral content. In the extreme, the min-
eral content ranged from 86% in the bulla, which requires
high acoustic impedance, to 59% in the antler, which must
be resilient to high-impact loads. The consequence of this
high mineral content is revealed by comparing the relative
work-to-fracture ratio of these bones. The work-to-fracture
ratio of the bulla is only 3% that of the antler.
The material properties of the appendicular skeleton
remain remarkably consistent through a wide range of
animals.236 Over an animal mass range of 0.09 to 700 kg,
the bending strength of the bones relegated to traditional
load-bearing responsibilities remains approximately 200 to
250 MPa, with an elastic modulus consistently approaching
20 GPa. To adapt to changes in the physical demands placed
on it, it seems that the appendicular skeleton responds not
by changing its material properties, but by altering its shape
and morphology.237 This is achieved by functionally regu-
lated alterations in bone mass and architecture.
STRUCTURAL ADAPTATION IN BONE
Bone tissue has the capacity to adapt to its functional envi-
ronment such that its morphology is “optimized” for its
mechanical demand. The concept proposed in 1892—that
the course and balance of bone remodeling can be affected
by mechanical function—is one of the oldest in modern
medicine and is widely referred to as Wolff’s law.73 But what
component of the functional environment is osteoregulat-
ing, and what is the structural objective of bone morphol-
ogy? Strains measured during functional activity should
indicate what the architecture of the skeleton is trying to
amplify or suppress. Loads can be sustained with the smallest
strains if they are applied axially. The axial component of
functional activity is responsible, however, for only a small
percentage of the total strain measured at the bone surface.
The femur, humerus, radius, ulna, and tibia all show that
greater than 80% of the measured strain is caused by bend-
ing moments.238 As the neutral axis of strain typically passes
across the marrow cavity, a significant portion of the tissue
is subjected to tension.239 Although bending moments can-
not be extinguished, their effect would be minimized if the
bone’s longitudinal curvature were oriented such that the
moment created by curvature counteracted the moments
externally imposed by activity. Long bone curvature does not
appear to be directed toward the neutralization of bending,
and in some cases this curvature is oriented such that bend-
ing is increased.240 Perhaps bone curvature, a morphologic
modification attributable to functional loading,241 acts to
accentuate bone strain, rather than cancel it.
Similarity of Peak Strain Magnitudes in Functionally
Loaded Bone
Contrary to normal interpretation of Wolff’s law, it seems
that minimizing strain is not the ultimate goal of adapta-
tion; instead, skeletal morphology strives to generate a
 86 RUBIN  |  Biology, Physiology, and Morphology of Bone
certain type of strain. What kind of strain is morphology
trying to achieve? Although vertebrate design and function
are diverse, at the level of small volumes of tissue all loads
and bending moments resolve into strain. Peak strain mag-
nitudes measured in adult species, including horse, human,
lizard, sheep, goat, goose, pig, macaque, turkey, sunfish, and
dog, are remarkably similar, ranging from 2000 to 3500
microstrain. This relationship has been called dynamic
strain similarity and suggests that skeletal morphology and
locomotion character combine to elicit a specific and per-
haps beneficial level of strain.242 The interspecies similar-
ity in strain magnitudes is strong evidence for the existence
of a common strain-sensitive cellular population within
the skeletal tissues of each of these animals. It also suggests
the existence of a generic cellular mechanism that strives
toward a common, strain-determined structural goal that is
desired by and beneficial to the bone cell population.
Strain-Regulated Adaptation
Although the nature of this structure-function relationship
is only poorly understood, it has been proposed that bone
remodeling is continually influenced by the level and dis-
tribution of the functional strains within the bone.243 One
striking example of the skeleton’s capacity to adapt to its
functional environment has been shown in professional
tennis players. By comparing the humeral mass of the rac-
quet arm to the side that simply throws the ball into the air,
Jones and colleagues244 observed a 35% increase in men and
a 28% increase in women in the cortical thickness of the
more active humerus.
The converse also can be shown; immobilization and bed
rest can cause negatively balanced bone remodeling locally
or within the entire skeleton. Healthy men restricted to
complete bed rest for 36 weeks showed a total body calcium
loss averaging 4.2%. Bone mineral content measurements of
the calcaneus showed a mean decrease of 34%. In one case,
45% of bone mineral was lost,245 showing that at specific
weight-bearing sites the negatively balanced bone remodel-
ing stimulated by diminished demand can be severe.
Attempts to identify the aspects of the skeleton’s func-
tional milieu that are responsible for generating and con-
trolling this adaptive response have shown that alterations
in bone mass, turnover, and internal replacement are sen-
sitive to changes in the magnitude,246 distribution,247 and
rate of strain224 generated within the bone tissue. A load-
ing regimen must be dynamic. Static loads do not influ-
ence bone morphology,248 but the full osteogenic potential
of bone is achieved after only an extremely short exposure
to this stimulus.249 The potency of the stimulus is propor-
tional to the magnitude of the strain.246 As strain levels that
are acceptable in one location induce adaptive remodel-
ing in others, each region of each bone may be genetically
programmed to accept a particular amount and pattern of
intermittent strain as normal. Deviation from this optimal
strain environment stimulates changes in the bone’s remod-
eling balance, resulting in adaptive increases or decreases
in its mass.
It is unclear whether a discrepancy in strain is detected at
the level of each individual osteocyte, whether the cell has
the ability to manipulate the structural milieu of its adjacent
space, or whether the osteocyte network somehow spatially
integrates the load information across the cortex.34,250 Bone
mass is substantially influenced, however, by strain situa-
tions engendered by short periods of particularly osteogenic
activity (e.g., vigorous and diverse exercise), rather than
by the strain situation experienced during a predominant
activity (e.g., walking) or by the fatigue damage that this
might produce.
Isolating specific components of the physical milieu
that regulate skeletal morphology has been difficult; no
single parameter of the mechanical environment has been
shown to predict bone remodeling reliably in all naturally
observed or experimentally created conditions.222 Perhaps
the limited success in identifying these elusive stimuli has
been due partly to the presumption that structural efficiency
(minimal strain with minimal mass) is an essential goal of
skeletal morphology. That the skeleton has “optimized” its
structure is supported by the similarity in peak strains gener-
ated in the cortex regardless of animal or activity (2000 to
3000 microstrain), indicating a common, peak strain–deter-
mined goal. Contrasting with this perspective is the nonuni-
form, but consistent distribution of normal and shear strains
that exist throughout the stance phase, leaving large areas of
the diaphyseal shaft subjected only to extremely low levels of
strain-energy density.251 Rather than a signal to repair accu-
mulated damage, a new strain milieu needs to be applied for
only a short time to maximize the tissue response.246
The engineering perspective that strain is harmful to
bone, and that remodeling is a repair-driven process needs
to be reconsidered. Instead, there may be some by-product
of strain, such as stress-generated potentials, piezoelectric
currents, or increased perfusion, that enhances the viabil-
ity of the bone cell population.252 Very-low-intensity elec-
tric fields (<10 μV/cm253) and low-magnitude strains (<10
microstrain254), when induced within a specific, hyperphysi-
ologic (10- to 50-Hz) frequency band, influence bone mass
as effectively as stimuli of greater intensity induced at more
“physiologic” frequencies. Strains at this frequency and mag-
nitude are induced as by-products of muscle contractions,
which resonate between 20 and 50 Hz, and they imply that
skeletal diseases such as osteopenia may result not only from
dysfunction of the bone cell population, but also from the
decline of the musculature, as is the case with sarcopenia.255
Perhaps we should be more hesitant to presume skeletal mor-
phology to be a product of dominant strain parameters with
the structural goal of minimizing strain, and instead con-
sider the matrix and cellular advantages of a tissue exposed
to a dynamic functional milieu.
MECHANICAL SIGNALING IN BONE CELLS
Although these data show some relationship of function to
form, they do not suggest the means by which the physical
signal is transduced by the cell and extracellular matrix into
the adaptive process. It has been shown that most eukaryotic
cells, including osteoblasts, stromal cells, and osteocytes,
respond to strain, shear, and pressure.256 Vascular channels
within haversian systems, combined with the lacunae and
canaliculi occupied by cells and the microporosity of the
matrix, may consume 10% of the bone tissue’s volume and
are filled with fluids or cellular components or both. The
deformation of the skeleton caused by functional activity
initiates this fluid to flow, similar to the way in which water
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
flows through a sponge that is stretched or compressed. In  
this way, cells residing in the skeleton are subject to defor-
 
mation, fluid shear, changes in dynamic electric potential,
and transient pressure waves. The forces generated during
exercise stimulate signals within bone cells that lead to  
activation of proliferation and differentiation programs.
Whatever the signal transduction pathway of transforming  
physical information to something the cell population can
perceive and respond to, it is clear that the capacity of bone
tissue to adapt to its functional demands is crucial to the  
skeleton’s structural success. As clinicians attempt to evalu-
ate the cellular mechanisms responsible for the positive  
control of bone mass, the osteogenic potential of physical
stimuli cannot be ignored.
 
SUMMARY  
This chapter has provided an overview of the cells ­responsible
for the regulation of bone mass and the local and systemic
factors that influence the activity of the cells. The cells and  
matrix that compose the tissue of bone reflect a ­sophisticated
interaction of organic and inorganic ­ constituents that  
­contribute to the skeleton’s admirable capacity to serve as
a mineral reservoir and a structural entity. The processes of
 
cell proliferation and differentiation, matrix synthesis and
mineralization, and growth and adaptation all contribute
to this elaborate balance of modeling and remodeling. It  
is hoped that this chapter has diminished the skeleton’s
­reputation as a static entity and has shown its crucial role  
in the dynamic process of mineral homeostasis and its  
­unparalleled capacity to facilitate locomotion. It is neither
solely a ­reservoir of minerals nor uniquely a structure. The
skeleton ­ represents an extremely complex and successful  
­combination of these responsibilities.
 
REFERENCES  
  1. Doty S: Morphological evidence of gap junctions between bone cells.
Calcif Tiss Int 33:509-512, 1981.  
  2. Rodan GA, Martin TJ: Role of osteoblasts in hormonal control of
bone resorption: A hypothesis. Calcif Tissue Int 33:349-351, 1981.  
  3. Owen TA, Aronow M, Shalhoub V, et al: Progressive development
of the rat osteoblast phenotype in vitro: Reciprocal relationships
in expression of genes associated with osteoblast proliferation and  
­differentiation during formation of the bone extracellular matrix.
J Cell Physiol 143:420-430, 1990.
  4. Martin TJ, Allan EH, Fukumoto S: The plasminogen activator and  
inhibitor system in bone remodeling. Growth Regul 3:209-214, 1993.
  5. Yasuda H, Shima N, Nakagawa N, et al: Osteoclast differen-
tiation ­ factor is a ligand for osteoprotegerin/osteoclastogenesis-  
inhibitory ­ factor and is ­ identical to TRANCE/RANKL. Proc
Natl Acad Sci U S A 95:3597-3602, 1998.
  6. Reinholt FP, Hultenby K, Oldberg A, et al: A possible anchor of  
osteoclasts to bone. Proc Natl Acad Sci U S A 87:4473-4475, 1990.
  7. Owen M: Cellular dynamics of bone. In Bourne GH (ed) : The Bio-
chemistry and Physiology of Bone, 2nd ed, Vol III. New York, Aca-
demic Press, 1972, p 271.  
  8. Jaworski ZFG: Lamellar bone: Turnover system and its effector organ.
Calcif Tissue Int 36S:46, 1984.
  9. Jowsey J: Metabolic Diseases of Bone. Philadelphia, WB ­ Saunders,
1977, p 61.  
  10. Parfitt AM: Osteomalacia and related disorders. In Avioli LV, Krane SM,
(eds): Metabolic Bone Disease and Clinically Related Disorders. Phila-
delphia, WB Saunders, 1990, p 329.  
  11. Weinstein RS, Jilka RL, Parfitt AM, et al: Inhibition of
­osteoblastogenesis and promotion of apoptosis of osteoblasts and
osteocytes by glucocorticoids: Potential mechanisms of their delete-  
rious effects on bone. J Clin Invest 102:274-282, 1998.
87
12. Ducy P, Desbois C, Boyce B, et al: Increased bone formation in
­osteocalcin-deficient mice. Nature 382:448-452, 1996.
13. Huang LF, Fukai N, Selby PB, et al: Mouse clavicular development:
Analysis of wild-type and cleidocranial dysplasia mutant mice. Dev
Dynam 210:33-40, 1997.
14. Mundlos S, Otto F, Mundlos C, et al: Mutations involving the
transcription factor CBFA1 cause cleidocranial dysplasia. Cell 89:
773-779, 1997.
15. Otto F, Thornell AP, Crompton AD, et al: CBFA1, a candidate
gene for cleidocranial dysplasia syndrome, is essential for osteoblast
­differentiation and bone development. Cell 89:765-771, 1997.
16. Nakashima K, Zhou X, Kunkel G, et al: The novel zinc finger-
containing transcription factor osterix is required for osteoblast dif-
ferentiation and bone formation. Cell 108:17-29, 2002.
17. Little RD, Carulli JP, Del Mastro RG, et al: A mutation in the LDL
receptor-related protein 5 gene results in the autosomal dominant
high-bone-mass trait. Am J Hum Genet 70:11-19, 2002.
18. Boyden L, Mao M, Belsky J, et al: High bone density due to a mutation
in LDL-receptor-related protein 5. N Engl J Med 346:1513-1521, 2002.
19. Kato M, Patel M, Levasseur S, et al: Cbfa1-independent decrease in
osteoblast proliferation, osteopenia, and persistent embryonic eye
vascularization in mice deficient in Lrp5, a Wnt coreceptor. J Cell
Biol 157:303-314, 2002.
20. Balemans W, Patel N, Ebeling M, et al: Identification of a 52 kb
deletion downstream of the SOST gene in patients with van Buchem
disease. J Med Genet 39:91-97, 2002.
21. Balemans W, Ebeling M, Patel N, et al: Increased bone density
in sclerosteosis is due to the deficiency of a novel secreted protein
(SOST). Hum Mol Genet 10:537-543, 2001.
22. Winkler DG, Sutherland MK, Geoghegan JC, et al: Osteocyte con-
trol of bone formation via sclerostin, a novel BMP antagonist. EMBO
J 22:6267-6276, 2003.
23. Li X, Zhang Y, Kang H, et al: Sclerostin binds to LRP5/6 and ­antagonizes
canonical Wnt signaling. J Biol Chem 280:19883-19887, 2005.
24. Mundlos S, Olsen BR: Heritable diseases of the skeleton. FASEB J
11:227-233, 1997.
25. Shafritz AB, Shore EM, Gannon FH, et al: Overexpression of an
osteogenic morphogen in fibrodysplasia ossificans progressiva. N Engl
J Med 335:555-561, 1996.
26. Shore EM, Xu M, Feldman GJ, et al: A recurrent mutation in the BMP
type I receptor ACVR1 causes inherited and sporadic ­fibrodysplasia
ossificans progressiva. Nat Genet 38:525-527, 2006.
27. Menton DN, Simmons DJ, Chang SL, et al: From bone lining cell to
osteocyte: An SEM study. Anat Rec 209:29-39, 1984.
28. Rubin JE, Rubin CT: The osteoblast, osteoclast and osteocyte. Curr
Opin Orthop 8:34-42, 1997.
29. Curtis TA, Ashrafi SH, Weber DF: Canalicular communication in
the cortices of human long bones. Anat Rec 212:336-344, 1985.
30. VanderMolen M, Rubin C, Donahue H: Decreased hormonal ­respon-
siveness in gap junction deficient osteoblasts. Trans 41st Orthop Res
Soc 20:295, 1995.
31. Skerry T, Bitensky L, Chayen J, et al: Early strain-related changes in
enzyme activity in osteocytes following bone loading in vivo. J Bone
Miner Res 4:783-788, 1989.
32. Inaoka T, Lean JM, Bessho T, et al: Sequential analysis of gene
expression after an osteogenic stimulus: C-FOS expression is induced
in osteocytes. Biochem Biophys Res Commun 217:264-270, 1995.
33. Noble B, Stevens H, Mosley J, et al: Bone loading changes the ­number
and distribution of apoptotic osteocytes in cortical bone. J Bone Miner
Res 12s:36, 1997.
34. Rubin C, Sun Y-Q, Hadjiargyrou M, et al: Increased expression of
MMP-1 in osteocytes precedes bone resorption as stimulated by ­disease:
Evidence for autoregulation of the cell’s mechanical ­ environment?
J Orthop Res 17:354-361, 1999.
35. Albright JA, Skinner C: Bone: Structural organization and remodel-
ing dynamics. In Albright JA, Brand RA (eds): The Scientific Basis
of Orthopaedics, 2nd ed. East Norwalk, Conn, Appleton & Lange,
1987, pp 161-198.
36. Felix R, Hofstetter W, Cecchini MG: Recent developments in the
understanding of the pathophysiology of osteopetrosis. Eur J Endocri-
nol 134:143-156, 1996.
37. Parfitt AM: The coupling of bone resorption to bone formation: A
critical analysis of the concept and of its relevance to the pathogen-
esis of osteoporosis. Metab Bone Dis Relat Res 4:1-6, 1982.
38. Bain SD, Impeduglia TM, Rubin CT: Cement line staining in unde-
calcified thin sections of cortical bone. Stain Tech 65:159-163, 1990.
 88 RUBIN  |  Biology, Physiology, and Morphology of Bone
  39. Parfitt AM: The cellular basis of bone remodeling: The quantum  
concept re-examined in light of recent advances in cell biology of
bone. Calcif Tissue Int 36(Suppl):S37-S45, 1984.
  40. Walker DG: Enzymatic and electron microscopic analysis of isolated  
osteoclasts. Calcif Tissue Res 9:296, 1972.
  41. Baron R: Polarity and membrane transport in osteoclasts. Connect  
Tissue Res 20:109-120, 1989.
  42. Blair HC, Teitelbaum SL, Chiselli R, et al: Osteoclastic bone resorp-
tion by a polarized vacuolar proton pump. Science 245:855-857,  
1989.
  43. Biskobing D, Nanes M, Rubin J: 1,25(OH)2D is required for protein
kinase C upregulation of carbonic anhydrase II in a mono-myelocytic  
cell line. Endocrinology 139:1493-1498, 1994.
  44. Nesbitt SA, Horton MA: Trafficking of matrix collagens through
bone-resorbing osteoclasts. Science 276:266-269, 1997.  
  45. Williams JP, Blair H, Jordan S, et al: Tamoxifen inhibits osteoclas-
tic bone resorption and H+-ATPase activity by an estrogen receptor
independent mechanism. J Bone Miner Res 9:62, 1994.
  46. Jones SJ, Boyde A, Ali NN, et al: A review of bone cell substratum  
interactions. Scanning 7:5-24, 1985.
  47. Silver A, Murrills RJ, Etherington DJ: Microelectrode studies on the  
acid microenvironment beneath adherent macrophages and osteo-
clasts. Exp Cell Res 175:266-276, 1988.
  48. Rodan GA: Bone mass homeostasis and bisphosphonate action. Bone  
20:1-4, 1997.
  49. David P, Nguyen H, Barbier A, et al: The bisphosphonate tiludro-
nate is a potent inhibitor of the osteoclast vacuolar H(+)-ATPase.  
J Bone Miner Res 11:1498-1507, 1996.
  50. Kahn AJ, Simmons DJ: Investigation of cell lineage in bone using a
chimaera of chick and quail embryonic tissue. Nature 258:325, 1975.  
  51. Yasuda H, Shima N, Nakagawa N, et al: Osteoclast differentiation fac-
tor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor  
and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A
95:3597-3602, 1998.  
  52. Lacey DL, Timms E, Tan HL, et al: Osteoprotegerin ligand is a cyto-
kine that regulates osteoclast differentiation and activation. Cell
93:165-176, 1998.  
  53. Felix R, Cecchini M, Fleisch H: MCSF restores in vivo bone resorption
in the op/op osteopetrotic mouse. Endocrinology 127:2592, 1990.
  54. Fan X, Biskobing DM, Fan D, et al: Macrophage colony stimulating fac-
tor downregulates MCSF-receptor expression and entry of progenitors  
into the osteoclast lineage. J Bone Miner Res 12:1387-1395, 1997.
  55. Suda T, Takahashi N, Martin TJ: Modulation of osteoclast differen-  
tiation. Endocr Rev 13:66-80, 1992.
  56. Biskobing DM, Fan D, Fan X, et al: Induction of carbonic anhydrase
II expression in osteoclast progenitors requires physical contact with  
stromal cells. Endocrinology 138:4852-4857, 1997.
57. Kong YY, Yoshida H, Sarosi I, et al: OPGL is a key regulator
of osteoclastogenesis, lymphocyte development and lymph-node
organogenesis. Nature 397:315-323, 1999.
  58. Kong YY, Feige U, Sarosi I, et al: Activated T cells regulate bone loss  
and joint destruction in adjuvant arthritis through osteoprotegerin
ligand. Nature 402:304-309, 1999.  
  59. Simonet WS, Lacey DL, Dunstan CR, et al: Osteoprotegerin: a novel  
secreted protein involved in the regulation of bone density. Cell
89:309-319, 1997.  
  60. Whyte MP, Obrecht SE, Finnegan PM, et al: Osteoprotegerin defi-
ciency and juvenile Paget’s disease. N Engl J Med 347:175-184, 2002.
  61. Jilka RL, Hangoc G, Girasole G, et al: Increased osteoclast devel-  
opment after estrogen loss: Mediation by interleukin-6. Science
257:88-91, 1992.
  62. Roodman G, Kurihara N, Ohsaki Y, et al: Interleukin 6: A potential  
autocrine/paracrine factor in Paget’s disease of bone. J Clin Invest
98:46-52, 1992.
  63. Grigoradis AE, Wang A, Cecchini MG, et al: c-Fos: A key regulator of  
osteoclast-macrophage lineage determination and bone ­remodeling.
Science 266:443-448, 1994.
  64. Tondravi MM, McKercher SR, Anderson K, et al: Osteopetrosis  
in mice lacking haematopoietic transcription factor PU.1. Nature
386:81-84, 1997.
  65. Steingrimsson E, Nii A, Fisher DE, et al: The semidominant Mi(b)  
mutation identifies a role for the HLH domain in DNA binding in addi-
tion to its role in protein dimerization. EMBO J 15:6280-6289, 1996.
  66. Iotsova V, Caamano J, Loy J, et al: Osteopetrosis in mice lacking  
NF-kappaB1 and NF-kappaB2. Nat Med 3:1285-1289, 1997.
67. Takeshita S, Namba N, Zhao JJ, et al: SHIP-deficient mice are
severely osteoporotic due to increased numbers of hyper-resorptive
osteoclasts. Nat Med 8:943-949, 2002.
68. Soriano P, Montgomery C, Geske R, et al: Targeted disruption of the
c-src protooncogene leads to osteopetrosis in mice. Cell 64:693, 1991.
69. Tanaka S, Amling M, Neff L, et al: c-Cbl is downstream of c-Src in
a signalling pathway necessary for bone resorption. Nature 383:528-
531, 1996.
70. Miyauchi A, Alvarez J, Greenfield EM, et al: Recognition of osteopontin
and related peptides by an αvβ3 integrin stimulates immediate cell signals
in osteoclasts. J Biol Chem 266:20369-20374, 1991.
71. McHugh KP, Hodivala-Dilke K, Zheng MH, et al: Mice lacking beta3
integrins are osteosclerotic because of dysfunctional osteoclasts. J Clin
Invest 105:433-440, 2000.
72. Rubin J, Nanes MS: Clinical disorders associated with alterations in
bone resorption. In Bronner F, Farach-Carson MC, Rubin J (eds):
Bone Biology Series: Bone Resorption, Vol II. London, Springer-
­Verlag, 2005, pp108-127.
73. Wolff J: The Law of Remodeling. Maquet P, Furlong R (trans).
Berlin, Springer Verlag, 1986 (original manuscript 1892).
74. Dunbar E, Wysolmerski J, Broadus E: Parathyroid hormone-related
protein: From hypercalcemia of malignancy to developmental regula-
tory molecule. Am J Med Sci 312:287-294, 1996.
75. Vortkamp A, Lee K, Lanske B, et al: Regulation of rate of cartilage
differentiation by Indian Hedgehog and PTH-related protein. Sci-
ence 27:613-663, 1996.
76. Shapiro F, Holtrop ME, Glimcher MJ: Organization and cellular
biology of the perichondrial ossification groove of Ranvier. J Bone
Joint Surg Am 59:703, 1977.
77. Cooper RR, Misol S: Tendon and ligament insertion: A light and
electron microscopic study. J Bone Joint Surg Am 52:1, 1970.
78. Roux W: Gesammelte Abhandlungen-uber Entwicklungsmechanik
der Organismen, Vols I and II. Leipzig, Wilhelm Engelmann, 1895.
79. Carter DR, Orr TE, Fyhrie DP, et al: Influences of mechanical
stress on prenatal and postnatal skeletal development. Clin Orthop
219:237, 1987.
80. Rosenberg LC, Choi HU, Poole AR: Biological processes involved
in endochondral ossification. In Rubin RT, Weiss GB, Putney JB
(eds): Calcium in Biological Systems. New York, Plenum, 1985,
pp 617-624.
81. Eggli PS, Herrmann W, Hunziker EB, et al: Matrix compartments in
the growth plate of the proximal tibia of rats. Anat Rec 211:246, 1985.
82. Floyd WE, Zaleske DJ, Schiller AL, et al: Vascular events associated
with the appearance of the secondary center of ossification in the
murine distal femoral epiphysis. J Bone Joint Surg Am 69:185, 1987.
83. Trelstad RL, Silver FH: Matrix assembly. In Hay ED (ed): Cell Biol-
ogy of Extracellular Matrix. New York, Plenum, 1981, pp 179-215.
84. Urist MF, Mikulski A, Lietze A: Solubilized and insolubilized
bone morphogenetic protein. Proc Natl Acad Sci U S A 76:
1828-1832, 1979.
85. Wozney JM: Bone morphogenetic protein family and osteogenesis.
Mol Reprod Dev 32:160-167, 1992.
86. Elima K: Osteoinductive proteins. Ann Med 24:395-402, 1993.
87. Noda M, Camilliere JJ: In vivo stimulation of bone formation by
­transforming growth factor-beta. Endocrinology 124:2991-2994, 1989.
88. Chenu C, Pfeilschifter J, Mundy GR, et al: Transforming growth fac-
tor-β inhibits formation of osteoclast-like cells in long-term human
marrow cultures. Proc Natl Acad Sci U S A 85:5683-5687, 1988.
89. Pfeilschifter JP, Seyedin S, Mundy GR: Transforming growth factor-β
inhibits bone resorption in fetal rat long bone cultures. J Clin Invest
82:680-685, 1988.
90. Takaishi T, Matsui T, Tsukamoto T, et al: TGF-β induced macro-
phage colony-stimulating factor gene expression in various mesen-
chymal cell lines. Am J Physiol 267:C25-C31, 1994.
91. Stevenson S, Cunningham N, Toth J, et al: The effect of osteogenin (a
bone morphogenetic protein) on the formation of bone in orthotopic
segmental defects in rats. J Bone Joint Surg Am 76:1676-1687, 1994.
92. Hirota S, Takaoka K, Hashimoto J, et al: Expression of mRNA of
murine bone-related proteins in ectopic bone induced by murine
bone morphogenetic protein 4. Cell Tissue Res 277:27-32, 1994.
93. Boden SD, Hair G, Titus L, et al: Glucocorticoid-induced differentia-
tion of fetal rat calvarial osteoblasts is mediated by bone morphoge-
netic protein-6. Endocrinology 138:2820-2828, 1997.
94. Canalis E, McCarthy T, Centrella M: Growth factors and the regula-
tion of bone remodeling. J Clin Invest 81:277-281, 1988.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
  95. Rubin J, Ackert-Bicknell C, Zhu L, et al: Insulin like growth fac-
tor-I (IGF-I) regulates osteoprotegerin (OPG) and RANK Ligand
(RANKL) in vitro and osteoprotegerin in vivo. J Clin Endocrinol
Metab 87:4273-4279, 2002.
  96. Rousseau F, Bonaventure J, Legeai-Mallet L, et al: Mutations in
the gene encoding FGFR3 in achondroplasia. Nature 371:252-254,
1994.
  97. Karaplis AS, Luz A, Glowacki J, et al: Lethal skeletal dysplasia from
targeted disruption of PTHrp gene. Genes Dev 8:277-289, 1994.
  98. Schipani E, Kruse K, Juppner H: A constitutively active mutant
PTH-PTHrp receptor in Jansen-type metaphyseal chondrodysplasia.
Science 268:90-100, 1995.
99. Brown EM, Gamba G, Riccardi D, et al: Cloning and characteriza-
tion of an extracellular Ca2+-sensing receptor from bovine parathy-
roid. Nature 366:575-580, 1993.
100. Diagnosis and management of asymptomatic primary hyperpara-
thyroidism: Consensus Development Conference Statement. Ann
Intern Med 1145:593-597, 1991.
101. McSheehy PMJ, Chambers TJ: Osteoblast-like cells in the presence
of parathyroid hormone release soluble factor that stimulates osteo-
clastic bone resorption. Endocrinology 119:1654-1659, 1986.
102. Donahue HJ, Fryer MJ, Eriksen EF, et al: Differential effects of
parathyroid hormone and its analogues on cytosolic calcium ion
and cAMP levels in cultured rat osteoblast-like cells. J Biol Chem
263:13522-13527, 1988.
103. Rouleau MF, Warshawsky H, Goltzman D: Parathyroid hormone
binding in vivo to renal, hepatic, and skeletal tissues of the rat using
a radioautographic approach. Endocrinology 118:919-931, 1986.
104. Hughes MR, Malloy TJ, Kieback DG, et al: Point mutations in the
human vitamin D receptor gene associated with hypocalcemic rick-
ets. Science 242:1702-1705, 1988.
105. Kitanaka S, Takeyama K, Murayama A, et al: Inactivating ­mutations
in the 25-hydroxyvitamin D3 1-hydroxylase gene in patients with
­pseudovitamin D-deficiency rickets. N Engl J Med 338:653-661,
1998.
106. Pharoah MJ, Heersche JNM: 1,25-Dihydroxyvitamin D3 causes an
increase in the number of osteoclast-like cells in cat bone marrow
cultures. Calcif Tissue Int 37:276-281, 1985.
107. Rubin J, Fan X, Thornton D, et al: Regulation of murine osteoblast
­macrophage colony-stimulating factor secretion by 1,25(OH)2D3.
Calcif Tissue Int 59:291-296, 1996.
108. Horwood NJ, Elliott J, Martin TJ, et al: Osteotropic agents regulate
the expression of osteoclast differentiation factor and osteoprotegerin
in osteoblastic stromal cells. Endocrinology 139:4743-4750, 1998.
109. Stern PH: Vitamin D and bone. Kidney Int 38:S17-S21, 1990.
110. Morrison NA, Qi JI, Tokita A, et al: Prediction of bone density from
vitamin D receptor alleles. Nature 367:284-287, 1994.
111. Hustmyer FG, Peacock M, Hui S, et al: Bone mineral density in relation to
polymorphism at the VDR gene locus. J Clin Invest 94:2130-2134, 1994.
112. Arden NK, Keen RW, Lanchbury JS, et al: Polymorphisms of the
VDR gene do not predict quantitative ultrasound of the calcaneus of
hip axis length. Osteoporosis Int 6:334-337, 1996.
113. Sainz J, Tornout J, Loro L, et al: Vitamin D-receptor gene polymor-
phisms and bone density in prepubertal American girls of Mexican
descent. N Engl J Med 337:77-82, 1997.
114. Austin LA: Heath H III: Calcitonin: Physiology and pathophysiol-
ogy. N Engl J Med 304:269-278, 1981.
115. Arnaud CD, Kolb FO: The calciotropic hormones and metabolic
bone disease. In Greenspan FS, Forsham PH (eds): Basic and Clinical
Endocrinology. Los Altos, Calif, Lange Medical, 1983, pp187-250.
116. Garret JE, Tamir H, Kifor O, et al: Calcitonin-secreting cells of
the thyroid gland express an extracellular calcium-sensing receptor
gene. Endocrinology 136:5202-5211, 1995.
117. Nicholson GC, Moseley JM, Sexton PM, et al: Abundant calcitonin
receptors in isolated rat osteoclasts: Biochemical and autoradiographic
characterization. J Clin Invest 78:355-360, 1986.
118. Chambers TJ, Athanasou NA, Fuller K: Effect of parathyroid
­hormone and calcitonin on the cytoplasmic spreading of isolated
osteoclasts. J Endocrinol 102:281, 1984.
119. Murrills RJ, Dempster DW: The effects of stimulators of
­intracellular cyclic AMP on rat and chick osteoclasts in vitro:
Validation of a ­ simplified light microscope assay of bone resorp-
tion. Bone 11:333-344, 1990.
120. Raisz LG: Local and systemic factors in the pathogenesis of osteopo-
rosis. N Engl J Med 318:818-828, 1988.
89
121. Takahashi S, Goldring S, Katz M, et al: Downregulation of calcitonin
receptor mRNA expression by calcitonin during human osteoclast-
like cell differentiation. J Clin Invest 95:167-171, 1995.
122. Raisz LG, Niemann I: Effect of phosphate, calcium and magnesium
on bone resorption and hormonal responses in tissue culture. Endo-
crinology 85:446-452, 1969.
123. Yamaguchi DT, Hahn TJ, Beeker TG, et al: Relationship of cAMP
and calcium messenger systems in prostaglandin-stimulated UMR-
106 cells. J Biol Chem 263:10745-10753, 1988.
124. Cheng MZ, Zaman G, Rawlinson SC, et al: Mechanical loading and
sex hormone interactions in organ cultures of rat ulna. J Bone Miner
Res 11:502-511, 1996.
125. Eriksen EF, Colvard DS, Berg NJ, et al: Evidence of estrogen receptors
in normal human osteoblast-like cells. Science 241:84-86, 1988.
126. Komm B, Terpening C, Benz D, et al: Estrogen binding, receptor
mRNA, and biologic response in osteoblast-like osteosarcoma cells.
Science 241:81-84, 1988.
127. Colvard DS, Eriksen EF, Keeting PE, et al: Identification of androgen
receptors in normal human osteoblast-like cells. Proc Natl Acad Sci
U S A 86:854-857, 1989.
128. Ernst M, Schmid CH, Froesch ER: Enhanced osteoblast proliferation
and collagen gene expression by estradiol. Proc Natl Acad Sci U S A
85:2307-2310, 1988.
129. Rouleau MF, Warshawsky H, Goltzman D: Parathyroid hormone
binding in vivo to renal, hepatic, and skeletal tissues of the rat using
a radioautographic approach. Endocrinology 118:919-931, 1986.
130. Gray TK, Mohan S, Linkhart TA, et al: Estradiol stimulates in vitro
the secretion of insulin-like growth factors by the clonal osteoblastic cell
line, UMR 106. Biochem Biophys Res Commun 158:407-412, 1989.
131. Oursler MJ, Pederson L, Pyfferoen J, et al: Estrogen modulation
of avian osteoclast lysosomal gene expression. Endocrinology
132:1373-1380, 1993.
132. Shevde NK, Bendixen AC, Dienger KM, et al: Estrogens suppress
RANK ligand-induced osteoclast differentiation via a stromal cell
independent mechanism involving c-Jun repression. Proc Natl
Acad Sci U S A 97:7829-7834, 2000.
133. Khosla S, Atkinson EJ, Dunstan CR, et al: Effect of estrogen versus
testosterone on circulating osteoprotegerin and other cytokine levels
in normal elderly men. J Clin Endocrinol Metab 87:1550-1554, 2002.
134. Smith EP, Boyd J, Frank GR, et al: Estrogen resistance caused by
a mutation in the estrogen-receptor gene in a man. N Engl J Med
16:1056-1061, 1994.
135. Carani C, Qin K, Simoni M, et al: Effect of testosterone and estradiol
in a man with aromatase deficiency. N Engl J Med 337:91-95, 1997.
136. Bilzekian JP, Morishima A, Bell J, et al: Increased bone mass as a
result of estrogen therapy in a man with aromatase deficiency. N Engl
J Med 339:599-603, 1998.
137. Riggs BL, Khosla S, Melton LJ: A unitary model for involutional
osteoporosis. J Bone Miner Res 13:763-773, 1998.
138. Christian JC, Yu P, Slemenda CW, et al: Heritability of bone mass.
Am J Hum Genet 44:429-433, 1989.
139. Pocock NA, Eisman JA, Hopper JL, et al: Genetic determinants of
bone mass in adults. J Clin Invest 80:706-710, 1987.
140. Finkelstein JS, Klibanski A, Neer RM: A longitudinal evaluation of
bone mineral density in adult men with histories of delayed puberty.
J Clin Endocrinol Metab 81:1152-1155, 1996.
141. Han ZH, Palnitkar S, Sudhaker Rao D, et al: Effects of ethnicity and age
or menopause on the remodeling and turnover of iliac bone: Implica-
tions for mechanisms of bone loss. J Bone Miner Res 12:498-508, 1997.
142. Zhao G, Monier-Faugere MC, Langub MC, et al: Targeted overex-
pression of insulin-like growth factor I to osteoblasts of transgenic
mice: Increased trabecular bone volume without increased osteoblast
proliferation. Endocrinology 141:2674-2682, 2000.
143. Adachi JD: Corticosteroid-induced osteoporosis. Am J Med Sci
313:41-49, 1997.
144. Mussolino ME, Looker AC, Madans JH, et al: Risk factors for hip
fracture in white men: The NHANES I epidemiologic follow-up
study. J Bone Miner Res 13:918-924, 1998.
145. Nguyen TV, Sambrook PN, Eisman JA: Bone loss, physical activity
and weight change in elderly women: The Dubbo osteoporosis epide-
miology study. J Bone Miner Res 13:1458-1467, 1998.
146. Zerwekh JE, Ruml LA, Gottschalk F, et al: The effects of 12 weeks
of bed rest on bone histology, biochemical markers of bone turnover,
and calcium homeostasis in 11 normal subjects. J Bone Miner Res
13:1594-1601, 1998.
 90 RUBIN  |  Biology, Physiology, and Morphology of Bone
147. Gong Y, Slee RB, Fukai N, et al: LDL receptor related protein 5
affects bone accrual and eye development. Cell 107:513-523, 2001.
148. Krishnan V, Bryant HU, MacDougald OA: Regulation of bone
mass by Wnt signaling. J Clin Invest 116:1202-1209, 2006.
149. Rubin CT, Hausman MR: The cellular basis of Wolff’s law: Transduc-
tion of physical stimuli to skeletal adaptation. Rheum Dis Clin N Am
14:503-517, 1988.
150. Marcus R: Exercise: Moving in the right direction. J Bone Miner Res
13:1793-1796, 1998.
151. Bradney M, Pearce G, Naghton G, et al: Moderate exercise during
growth in prepubertal boys: Changes in bone mass, size volumetric
density and bone strength—a controlled prospective study. J Bone
Miner Res 13:1814-1821, 1998.
152. Carter D, Wong A: Mechanical stresses and endochondral ossifica-
tion in chondroepiphysis. J Orthop Res 6:148, 1988.
153. Rubin C, Turner AS, Bain S, et al: Anabolism: Low mechanical
signals strengthen long bones. Nature 412:603-604, 2001.
154. Fisher LW, Gehron RP, Tuross N, et al: The Mr 24,000 phosphopro-
tein from developing bone is the NH2-terminal propeptide of the α
chain of type I collagen. J Biol Chem 262:13457-13463, 1987.
155. Noda M, Yoon, Prince CW, et al: Transcriptional regulation of osteo-
pontin production in rat osteosarcoma cells by type β transforming
growth factor. J Biol Chem 263:13916, 1988.
156. Price PA, Otsuka AS, Poser JW, et al: Characterization of gamma-
carboxyglutamic acid containing protein from bone. Proc Natl Acad
Sci U S A 73:1447, 1976.
157. Urist MR, Juo YK, Brownell AG, et al: Purification of bovine bone
morphogenetic protein by hydroxyapatite chromatography. Proc
Natl Acad Sci U S A 81:371, 1984.
158. Termine JD, Belcourt AB, Conn KM, et al: Mineral and ­ collagen-
binding proteins of fetal calf bone. J Biol Chem 256:10403, 1981.
159. Robey PG, Bosky A: The biochemistry of bone. In Marcus R,
Feldman D, Kelsey J: Osteoporosis. San Diego, Calif, Academic
Press, 1996, pp 95-183.
160. Pritchard JJ: General histology of bone. In Bourne GH (ed): The
Biochemistry and Physiology of Bone, 2nd ed, Vol I. New York, Aca-
demic Press, 1972, p15.
161. Ascenzi A, Bonucci E: Relationship between ultrastructure and “pin
test” in osteons. Clin Orthop 121:275, 1976.
162. Eyre DR: Collagen: Molecular diversity in the body’s protein scaffold.
Science 207:1315, 1980.
163. Boskey AL, Posner AS: Bone structure, composition and mineraliza-
tion. Orthop Clin North Am 15:597, 1984.
164. Veis A, Sharkey M, Dickson I: Non-collagenous proteins of bone
and dentin extracellular matrix and their role in organized mineral
deposition. In Wasserman RH (ed): Calcium Binding Proteins and
Calcium Function. New York, Elsevier, 1977, pp 409-418.
165. Glimcher MJ: Studies of the structure, organization and reac-
tivity of bone collagen. In Gibson T (ed): Proceedings of the
­International Symposium on Wound Healing. Nice, France,
­Montreaux ­Foundation of International Cooperative Medical Sci-
ence, 1975, p 253.
166. Herring GM: The chemical structure of tendon cartilage, dentin and
bone matrix. Clin Orthop 60:261, 1968.
167. Skerry TM, Suswillo R, El Haj AJ, et al: Load-induced proteogly-
can orientation in bone tissue in vivo and in vitro. Calcif Tissue Int
46:318-326, 1990.
168. Hauschka PV, Lian JB, Cole DEC, et al: Osteocalcin and matrix
G1a protein: Vitamin K-dependent protein in bone. Physiol Rev
69:990-1047, 1988.
169. Lian JB, Coutts M, Canalis E: Studies of hormonal regulation of
osteocalcin synthesis in cultured fetal rat calvariae. J Biol Chem
260:8706-8710, 1985.
170. Boskey AL, Gadaleta S, Gundberg C, et al: Fourier transform infrared
microspectroscopic analysis of bones of osteocalcin-deficient mice
provides insight into the function of osteocalcin. Bone 23:187-196,
1998.
171. Weiner S, Traub W: Bone structure: From angstroms to microns.
FASEB J 6:879-885, 1992.
172. Posner AS: Crystal chemistry of bone mineral. Physiol Rev 49:760,
1969.
173. Moradian-Oldak J, Weiner S, Addadi L, et al: Electron imaging
and diffraction study of individual crystals of bone, mineralized
tendon and synthetic carbonate apatite. Connect Tissue Res
25:219-228, 1991.
174. Bonar LC, Roufosse AH, Sabine WK, et al: X-ray diffraction studies
of the crystallinity of bone mineral in newly synthesized and density
fractionated bone. Calcif Tissue Int 35:202-209, 1983.
175. Lian JB, Gundberg CM: Osteocalcin: Biochemical considerations
and clinical applications. Clin Orthop 262:267-291, 1988.
176. Rosen CJ, Chesnut CH III, Mallinak NJ: The predictive value of bio-
chemical markers of bone turnover for bone mineral density in early
postmenopausal women treated with hormone replacement or calcium
supplementation. J Clin Endocrinol Metab 82:1904-1910, 1997.
177. Ross PD, Knowlton W: Rapid bone loss is associated with increased
levels of biochemical markers. J Bone Miner Res 13:297-302, 1998.
178. Greenspan SL, Parker RA, Ferguson L, et al: Early changes in bio-
chemical markers of bone turnover predict the long-term response to
alendronate therapy in representative elderly women: A randomized
clinical trial. J Bone Miner Res 13:1431-1438, 1998.
179. Seemon E: From density to structure: Growing up and growing old on
the surfaces of bone. J Bone Miner Res 12:509-521, 1997.
180. Black DM, Bilezikian JP, Ensrud KE, et al: One year of alendronate
after one year of parathyroid hormone (1-84) for osteoporosis. N Engl
J Med 353:555-565, 2005.
181. Howard GM, Nguyen TV, Harris M, et al: Genetic and environmen-
tal contributions to the association between quantitative ultrasound
and bone mineral density measurements: A twin study. J Bone Miner
Res 13:1318-1327, 1998.
182. Genant HK, Jiang Y: Advanced imaging assessment of bone quality.
Ann N Y Acad Sci 1068:410-428, 2006.
183. Christoffersen J, Landis WJ: A contribution with review to the
description of bone and other calcified tissues in vivo. Anat Rec
230:435-450, 1991.
184. Jowsey J: Microradiography: A morphologic approach to quantitating
bone turnover. Excerpta Medica International Congress Series No.
270. Amsterdam, Excerpta Medica Foundation, 1972, p 114.
185. Glimcher MJ, Lian JB (eds): The Chemistry and Biology of ­Mineralized
Tissues. Proceedings of the Third International ­ Conference on the
Chemistry and Biology of Mineralized Tissues. New York, Gordon &
Breach Science, 1988.
186. Wuthier RE, Jajeska RJ, Collins GM: Biosynthesis of matrix vesicles in
epiphyseal cartilage, I: In vivo incorporation of 32P-orthophosphate into
phospholipid of chondrocyte, membrane and matrix vesicle fractions.
Calcif Tissue Res 23:135-139, 1977.
187. Kerr JFR, Wyllie AH, Currie AR: Apoptosis: A basic biological phe-
nomenon with wide ranging implications in tissue kinetics. Br J Can-
cer 26:239, 1972.
188. Russell RGG, Caswell AM, Hearn PR, et al: Calcium in mineralized
tissues and pathological calcification. Br Med Bull 42:435, 1986.
189. Anderson HC: Electron microscopic studies of induced cartilage
development and calcification. J Cell Biol 35:81, 1967.
190. Anderson HC: Matrix vesicle calcification: Review and update.
In Peck WA (ed): Bone and Mineral Research, Vol 3. New York,
Elsevier, 1985, pp 109-150.
191. Wuthier RE: Lipid composition of isolated cartilage cells, membranes
and matrix vesicles. Biochim Biophys Acta 409:128, 1975.
192. Endo A, Glimcher MJ: The potential role of phosphoproteins in
the in vitro calcification of bone collagen. In Goldberg VM (ed):
Transactions of the 32nd Meeting of Orthopaedic Research Society.
Chicago, Adept Printing, 1986, p 221.
193. Hartgerink JD, Beniash E, Stupp SI: Self-assembly and mineralization
of peptide-amphiphile nanofibers. Science 294:1684-1688, 2001.
194. Puzas JE: Phosphotyrosine phosphatase activity in bone cells: An old
enzyme with a new function. Adv Protein Phosphatases 3:237-256, 1986.
195. Anderson HC: Biology of disease: Mechanism of mineral formation
in bone. Lab Invest 60:320, 1989.
196. Wuthier RE: The role of phospholipids in biological calcification:
Distribution of phospholipase activity in calcifying epiphyseal carti-
lage. Clin Orthop 90:191, 1973.
197. Peress NS, Anderson HC, Sajdera SW: The lipids of matrix vesicles
from bovine fetal epiphyseal cartilage. Calcif Tissue Res 14:275, 1974.
198. Termine JD, Belcourt AB, Conn KM, et al: Mineral and collagen-
binding proteins of fetal calf bone. J Biol Chem 256:10403, 1981.
199. Hynes RO: The molecular biology of fibronectin. Annu Rev Cell
Biol 1:67, 1985.
200. Glimcher MJ, Krane SM: The organization and structure of
bone and the mechanism of calcification. In Ramachandran GN,
Gould BS (eds): Treatise on Collagen, Vol 11B. Biology of Col-
lagen. New York, Academic Press, 1968, p 68.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
201. Baylink D, Wengedal J, Thompson E: Loss of protein polysaccharides
at sites where bone mineralization is initiated. J Histochem Cyto-
chem 20:279, 1972.
202. Glimcher MJ: Composition, structure and organization of bone and
other mineralized tissues and the mechanism of calcification. In Hand-
book of Physiology: Endocrinology, Vol VII. Baltimore, ­ Williams &
Wilkins, 1976, p 25.
203. Parfitt AL: Bone histomorphometry: Proposed system for standardization
of nomenclature, symbols and units. Calcif Tissue Int 42:284-286, 1988.
204. Hayes WC, Gerhart TN: Biomechanics of bone: Applications for
assessment of bone strength. In Peck WA (ed): Bone and Mineral
Research, 3rd ed. New York, Elsevier, 1985, p 259.
205. Lanyon LE: Analysis of surface bone strain in the calcaneus of sheep
during normal locomotion: Strain analysis of the calcaneus. J Bio-
mech 6:41-49, 1973.
206. Ascenzi A: The micromechanics versus the macromechanics of cortical
bone: A comprehensive presentation. J Biomech Eng 110:357-363, 1988.
207. Martin RB, Burr DB, Sharkey N: Skeletal Tissue Mechanics. New
York, Springer-Verlag, 1998.
208. Jones BH, Harris JM, Vinh TN, et al: Exercise-induced stress frac-
tures and stress reactions of bone: Epidemiology, etiology, and clas-
sification. Exerc Sport Sci Rev 17:379-422, 1989.
209. Rubin CT, Gross T, McLeod K, et al: Morphologic stages in lamellar
bone formation stimulated by a potent mechanical stimulus. J Bone
Miner Res 10:488-495, 1995.
210. Albright JA, Skinner HCW: Bone: Structural organization and remod-
eling dynamics. In Albright JA, Brand RA (eds): The ­Scientific Basis of
Orthopaedics, 2nd ed. East Norwalk, Conn, Appleton & Lange, 1987,
pp 161-198.
211. Frost HM: Secondary osteon population densities: An algorithm
for estimating the missing osteons. Yearbook Phys Anthropol 30:
221-238, 1987.
212. Ascenzi A, Benvenuti A: Orientation of collagen fibers at the bound-
ary between two successive osteonic lamellae and its mechanical
interpretation. J Biomech 19:455-463, 1986.
213. Schaffler MB, Burr DB, Fredrickson RG: Morphology of the osteonal
cement line in human bone. Anat Rec 217:223-228, 1987.
214. Garn SM: The course of bone gain and the phases of bone loss.
Orthop Clin North Am 3:503, 1972.
215. Ruff CV, Hayes WC: Subperiosteal expansion and cortical remodel-
ing of the human femur and tibia with aging. Science 217:945, 1982.
216. Enlow DH: Principles of Bone Remodeling. Springfield, Ill, Charles
C Thomas, 1963.
217. Koch JC: The laws of bone architecture. Am J Anat 21:177, 1917.
218. Lanyon LE, Rubin CT, Baust G: Modulation of bone loss during cal-
cium insufficiency by controlled dynamic loading. Calcif Tissue Int
38:209-216, 1986.
219. Bain SD, Rubin CT: Metabolic modulation of disuse osteopenia:
Endocrine-dependent site specificity of bone remodeling. J Bone
Miner Res 5:1069-1075, 1990.
220. Rubin CT, Bain S: McLeod KJ: Suppression of the osteogenic
response in the aging skeleton. Calcif Tissue Int 50:306-313, 1992.
221. Rubin CT: The benefits and consequences of structural adaptation in
bone. In Fitzgerald R (ed): Non-Cemented Total Hip Arthroplasty.
New York, Raven Press, 1988, pp 41-48.
222. Brown TD, Pedersen DR, Gray ML, et al: Toward an identification of
mechanical parameters initiating periosteal remodeling: A combined
experimental and analytic approach. J Biomech 23:893-905, 1990.
223. Radin EL, Martin RB, Burr DB, et al: Effects of mechanical loading
on the tissues of the rabbit knee. J Orthop Res 2:221-234, 1984.
224. O’Connor JA, Lanyon LE, MacFie H: The influence of strain rate on
adaptive bone remodeling. J Biomech 15:767-781, 1982.
225. Rubin C, Gross T, Donahue H, et al: Physical and environmental
influences on bone formation. In Brighton CT, Friedlaender GE,
Lane JM (eds): Bone Formation and Repair. Rosemont, Ill, American
Academy of Orthopaedic Surgeons, 1994, pp 61-78.
226. Bassett CA: Fundamental and practical aspects of therapeutic uses
of pulsed electromagnetic fields (PEMFs). Crit Rev Biomed Eng
17:451-529, 1989.
227. Heckman J, Ryaby J, McCabe J, et al: Acceleration of tibial fracture
healing by noninvasive low intensity ultrasound. J Bone Joint Surg
Am 76:165-374, 1994.
228. Rubin C, Turner AS, Muller R, et al: Quantity and quality of trabecu-
lar bone in the femur are enhanced by a strongly anabolic, noninva-
sive mechanical intervention. J Bone Miner Res 17:349-357, 2002.
91
229. Shim SS: Physiology of blood circulation of bone. J Bone Joint Surg
Am 50:812, 1968.
230. Sledge CB: Epiphyseal injuries. In Cave EF, Burke JF, Boyd RJ,
(eds): Trauma Management. Chicago, Year Book Medical, 1974,
pp 152-161.
231. Cowin SC: Bone Mechanics. Boca Raton, CRC Press, 2001.
232. Ascenzi A, Bell GH: Bone as a mechanical engineering problem. In
Bourne GH (ed): The Biochemistry and Physiology of Bone, Vol 1:
Structure. New York, Academic Press, 1972, p 311.
233. Reilly DT, Burstein AH: The elastic and ultimate properties of com-
pact bone tissue. J Biomech 8:393-405, 1975.
234. Carter DR, Harris WH, Caler WE: The mechanical and biological
response of cortical bone to in vivo strain histories. In Cowin SC,
(ed): Mechanical Properties of Bone, Vol 45. New York, American
Society of Mechanical Engineers, 1981, pp 81-92.
235. Curry JD: Mechanical properties of bone with greatly differing func-
tions. J Biomech 12:313, 1979.
236. Lanyon LE, Rubin CT: Functional adaptation in skeletal structures.
In Hildebrand M, Bramble DM, Leim KF, Wake DB (eds): ­Functional
Vertebrate Morphology. Cambridge, Harvard University Press, 1985,
pp 1-25.
237. Woo SLY: The relationships of changes in stress levels on long bone
remodeling. In Cowen S (ed): Mechanical Properties of Bone, AMD,
Vol 45. New York, American Society of Mechanical ­Engineers, 1981,
p 107.
238. Rubin CT, Lanyon LE: Limb mechanics as a function of speed and
gait: A study of functional strains in the radius and tibia of horse and
dog. J Exp Biol 101:187-211, 1982.
239. Gross T, McLeod K, Rubin C: Characterizing bone strain distri-
butions in vivo using three triple rosette strain gauges. J Biomech
25:1081-1087, 1992.
240. Rubin CT: Skeletal strain and the functional strain significance of
bone architecture. Calcif Tissue Int 36:S11-S18, 1984.
241. Lanyon LE: The influence of function on the development of bone
curvature: An experimental study on the rat tibia. J Zool Lond
192:457-466, 1980.
242. Rubin CT, Lanyon LE: Dynamic strain similarity in vertebrates: An
alternative to allometric limb bone scaling. J Theoret Biol 107:321-327,
1984.
243. Rubin CT, Lanyon LE: Osteoregulatory nature of mechanical stimuli:
Function as a determinant for adaptive remodeling in bone. J Orthop
Res 5:300-310, 1987.
244. Jones HH, Pries JD, Hayes WC, et al: Humeral hypertrophy in
response to exercise. J Bone Joint Surg Am 59:204-208, 1977.
245. Donaldson CL, Hulley SB, Vogel JM, et al: Effect of prolonged bed
rest on bone mineral. Metabolism 19:1071, 1970.
246. Rubin CT, Lanyon LE: Regulation of bone mass by mechanical
­loading. Calcif Tissue Int 37:411, 1985.
247. Lanyon LE, Goodship AE, Pye C, et al: Mechanically adaptive bone
remodeling: A quantitative study on function adaptation in the radius
following ulna osteotomy in sheep. J Biomech 15:141-154, 1982.
248. Lanyon LE, Rubin CT: Static versus dynamic loads as an influence
on bone remodeling. J Biomech 17:897, 1984.
249. Rubin CT, Lanyon LE: Regulation of bone formation by applied
dynamic loads. J Bone Joint Surg Am 66:397-402, 1984.
250. Sun Y, McLeod K, Rubin C: Upregulation of collagen type 1 mRNA
following mechanical loading as measured by in situ RT PCR. Trans
41st Orthop Res Soc 20:290, 1995.
251. Rubin CT, McLeod KJ: Biologic modulation of mechanical ­influence
in bone remodeling. In Mow VC, Ratcliffe A, Woo SLY (eds):
­Biomechanics of Diarthrodial Joints, Vol II. New York, Springer-
­Verlag, 1990, pp 97-118.
252. You L, Cowin SC, Schaffler MB, et al: A model for strain amplifi-
cation in the actin cytoskeleton of osteocytes due to fluid drag on
pericellular matrix. J Biomech 34:1375-1386, 2001.
253. McLeod KJ, Rubin CT: The effect of low frequency electric fields on
osteogenesis. J Bone Joint Surg Am 74:920-929, 1992.
254. Rubin CT, Sommerfeldt DW, Judex S, et al: Inhibition of osteo-
penia by low magnitude, high-frequency mechanical stimuli. Drug
Discov Today 6:848-858, 2001.
255. Fritton SP, McLeod KJ, Rubin CT: Quantifying the strain history
of bone: Spatial uniformity and self-similarity of low-magnitude
strains. J Biomech 33:317-325, 2000.
256. Rubin J, Rubin C, Jacobs C: Molecular pathways of mechanical
signaling in bone. Gene 367:1-16, 2006.
 5 Muscle: Anatomy,
KEY POINTS
Muscles have a complex developmental process
and structure.
Various types of muscle fibers are specialized in terms of
metabolic requirements, fatigue susceptibility, speed, and
power.
The structure, function, and protein isoform expression can
change rapidly and are sensitive to the type of innervation
and level of activity (i.e., plasticity).
The smallest functional unit of muscle, the sarcomere, is com-
posed of an almost crystalline array of filamentous proteins
that transduce metabolic energy into motion and work.
Muscles are connected to the skeleton through collagenous
tendons and the epimysium.
Skeletal muscle contraction is controlled by the central
nervous system through motoneurons, the neuromuscular
junction, and the intricate internal membrane system of the
muscle fiber.
Force is transmitted to the exterior through two sets of
protein cell adhesion complexes: integrins and dystroglycans.
Mature muscle fibers can regenerate after insult using
components of the myogenesis process.
Approximately 660 skeletal muscles support and move
the body under the control of the central nervous system.
They constitute up to 40% of the adult human body mass.
Most skeletal muscles are fastened by collagenous tendons
across joints in the skeleton. The transduction of chemi-
cal energy into mechanical work by the muscle cells leads
to muscle shortening and consequent movement. A high
degree of specialization in this tissue is evident from the
intricate architecture and kinetics of the intracellular mem-
brane systems, the contractile proteins, and the molecular
components that transmit force extracellularly to the base-
ment membrane and tendons. Muscle cells normally exhibit
wide variations in activity level and are able to adapt in
size, isoenzyme composition, membrane organization, and
energetics. In pathologic states, they often become decon-
ditioned. These examples of plasticity can be surprisingly
swift and extensive. This chapter outlines the structure
and function of muscle and its relationship to the associ-
ated connective tissue. It also introduces the basis for the
highly adaptive response to altered functional demands and
diseases. Two excellent Web sites can be accessed for further
­information.1,2
Physiology, and
Biochemistry
Jody A. Dantzig  • 
Eugenia C. Pacheco-Pinedo  • 
Yale E. Goldman
MUSCLE DEVELOPMENT
During embryogenesis, connective tissue, bone, and skeletal
muscle are derived from mesodermal cells of the somites
(Fig. 5-1). Developmental studies have shown that highly
regulated sequential expression of transcription factors such
as Sonic Hedgehog, bone morphogenetic proteins, and oth-
ers3-5 are secreted from nearby structures, including the neu-
ral tube, notochord, dorsal ectoderm, and lateral mesoderm.3
These patterning factors participate in the commitment and
delamination of mesodermal muscle precursors, which later
migrate to the limb buds under the influence of other mor-
phogenetic transcription factors.6-8 Once in the limb bud,
the cells proliferate and differentiate under the stimulation
of muscle regulatory factors such as myogenesis determining
factor (MyoD), myogenic factor 5 (Myf-5), myogenic regula-
tory factor 4 (Mrf-4), and myogenin6,8; Myf-5 strongly pro-
motes myoblast proliferation, whereas MyoD predominantly
induces cell cycle arrest and differentiation (see Fig. 5-1).
The migrating mesodermal precursors withdraw from
the cell cycle, differentiate into spindle-shaped myoblasts
(see Fig. 5-1), and begin to synthesize embryonic isoforms
of muscle-specific proteins9 (Fig. 5-2A). Myoblasts align
in columns and fuse to produce multinucleated primary myo-
tubes (see Figs. 5-1 and 5-2B and C). The contrac­tile organ-
elle, the myofibril, comprises long columns of sarcomeres
and self-assembles on cytoskeletal scaffolding (see Fig. 5-2D
and E).10-12 Myofibrils first appear near the periphery of the
myotubes (see Fig. 5-2D) and fill in toward the center of the
newly formed skeletal muscle fibers. Myofibrillogenesis con-
tinues until the cytoplasm is packed with parallel, laterally
aligned sarcomeres (see Fig. 5-2E). Myoblasts continue to
proliferate, and later generations elongate, fuse, and differ-
entiate within the basal laminae of the primary myotubes,
forming independent secondary myotubes.11,12 The nuclei
migrate from the center to the periphery, where they remain
in mature, multinucleated muscle fibers (see Fig. 5-2D and
E). Central nuclei in adult muscle biopsies are thus diagnos-
tic of abnormal muscle cell turnover (see Chapter 78). Both
primary and secondary myotubes develop into phenotypi-
cally distinct muscle fiber types by sequentially expressing
a series of embryonic, neonatal, and mature isoforms of the
contractile proteins (Table 5-1).5,12 Some of the develop-
mental and mature isoforms of the contractile proteins that
appear in fast, slow, and cardiac muscle are encoded by mul-
tigene families, whereas others are differentially expressed
by alternative splicing of messenger RNA.
An extracellular matrix of type IV collagen, proteogly-
cans, fibronectin, and laminin is secreted by the myotubes
to form the basal lamina, which fully ensheathes the fiber
membrane (sarcolemma) except at the neuromuscular
93
 94 DANTZIG  |  Muscle: Anatomy, Physiology, and Biochemistry

Noggin Epaxial Pitx2

Shh dermomyotome Pax-3
Wnts Neural Hypaxial C-Met Pax 3
BMP tube dermomyotome C-Met / HGF Mesodermal
Notch Lbx1 progenitor cells

Delamination Pax-3
C-Met
Presomitic and Sclerotome Migration Msx-1
somitic stages Notochord Mox-2
Mesodermal
precursor cells Six
Somite
Myf-5
Muscle damage Myo D
Numb
IGF-1
Pitx-2 Proliferation
Satellite cell activation
Pax-7

Notch
Replenishment Myoblast precursors
in the limb bud

Satellite Innervation Differentiation Determination

cells and maturation and fusion

Sarcolemma
Basal Lamina Myf-5
Multinucleated MyoD
Myofiber Mrf4 Myogenin Myoblasts Cyclin D1
early myotube
MLP Mef 2 Id
Pax7 Six-1 Rb
MyoD
Myf-6(Mrf4)
Slug(snail)
Figure 5-1  Myogenesis and cell regeneration of skeletal muscle. The long red arrow indicates the initiation of muscle regeneration due to disease or
injury. The somite is composed of a dermomyotome and a sclerotome. BMP, bone morphogenetic protein; C-Met, hepatocyte growth factor receptor;
HGF, hepatocyte growth factor; Id, originally identified as dominant negative antagonists of the basic helix-loop-helix transcription factor family; IGF-1, 
insulin-like growth factor 1; Lbx1, ladybird homeobox homolog 1; Mef-2, myocyte enhancing factor 2; MLP, muscle lim protein; Mox-2, membrane
glycoprotein; Mrf-4, myogenic regulatory factor 4; Msx-1, homeobox msh-like 1; Myf-5, myogenic factor 5; Myf-6, myogenic factor 6; MyoD, myogenesis
determining factor; Pax-3, paired box gene 3; Pax-7, paired box gene 7; Pitx2, paired-like homeo­domain transcription factor 2; Rb, product of the reti-
noblastoma tumor suppressor gene; Shh, Sonic Hedgehog; Six-1, homolog of Drosophila sine oculis homeobox 1; Slug(Snail), muscle LIM protein; Wnt,
wingless-type mouse mammary tumor virus integration site family.

j­unction.13 Cell matrix interactions also feed back to the
gene expression apparatus, as shown by experiments in
which the lineage of cultured mesenchymal stem cells was
modulated by the mechanical stiffness of the substrate.14
Under normal conditions, the number of muscle fibers
within a skeletal muscle is virtually constant throughout
life. Myogenic stem cells (satellite cells) remain situated
in mature muscle fibers between the sarcolemma and basal
lamina (see Fig. 5-1), providing a reservoir for muscle repair
and growth.15 When a muscle fiber is damaged or necrosed,
mitogenic factors and peptide regulators released from the
damaged cells5,16 trigger satellite cells to proliferate and
migrate into the affected area, guided by the basal lamina.
Some satellite cells differentiate into myoblasts (see Fig. 5-1,
red arrow, and following steps), fuse, and form new muscle
fibers. Others replenish the satellite cell niche. The supply
of these stem cells is limited, however, which diminishes
the potential for repair in severe degenerative conditions.
Recent studies suggest that other stem cell populations,
such as muscle side populations17 and a small percentage of
bone marrow cells,18-21 may also participate in muscle repair
and satellite cell replenishment.
Many of the genes involved in muscle myogenesis are
also induced in the muscle repair process.5,6 For this reason,
knowledge of the mechanism of myogenesis may assist us
in understanding regeneration and lead to new therapeutic
strategies for treating muscle injuries and diseases.
STRUCTURE
MUSCLE TISSUE
Parallel, aligned bundles of skeletal muscle fibers make up
approximately 85% of muscle tissue. Nerves, blood sup-
ply, and connective tissue structures that provide support,
elasticity, and force transmission to the skeleton (discussed
later) constitute the remaining volume. Muscle fibers range
in length from a few millimeters to many centimeters and
range in diameter from 10 to 150 μm. This elongated shape
is determined by the organization of the contractile proteins
that occupy the majority of the sarcoplasm. Each muscle
has a limited range of shortening that is amplified into large
motions by lever systems of the skeleton, usually operating
at a mechanical disadvantage. Variations in the geometric
arrangements of the fibers—parallel, fan shaped, fusiform
(spindle-like), or pennate (feather-like)—determine some
of the mechanical properties. For example, the slant of the
fibers in a pennate muscle increases the magnitude of force
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE 95

Actin Muscle myosin II filaments

Non-muscle α-actinin Titin
Muscle α-actin Nuclei

Non-muscle myosin IIB

Stress fibers Premyofibrils Nascent myofibrils

A B C

Myoblasts Fusing myoblasts Primary myotube

Mature myofibrils
D

Secondary myotubes
E
Mature myofibrils

Myotubes
Figure 5-2  Developmental progression of myoblasts during fusion into myotubes. A, Unicellular myoblasts. B, Initial fusion of myoblasts. 
C, Multinucleated myotubes. D, Myoblast fusing to multinucleated myotube; interface between myotube and mature myofibril. E, Mature myofibril.

generation at the expense of speed and range of movement,
compared with a similarly sized muscle with fibers aligned
parallel to the tendons.22 Muscles designed for strength
(e.g., gastrocnemius) are typically pennate, whereas those
designed for speed (e.g., biceps) tend to have parallel fibers.
Muscles are commonly arranged around joints as antagonis-
tic pairs, facilitating bidirectional motion. When one muscle
(the agonist) contracts, another (its antagonist) is relaxed
and passively extended. Their roles reverse to actively gen-
erate the opposite motion, unless it occurs passively by the
force of gravity.
An extensive network of connective tissue, forming
the endomysium, surrounds each muscle fiber. Fine nerve
branches and the small capillaries, necessary for the exchange
of nutrients and metabolic waste products, penetrate this
layer. The endomysium is continuous with the perimysium,
a connective tissue network that ensheathes small parallel
bundles of muscle fibers known as fasciculi, intrafusal fibers,
larger nerves, and blood vessels. The epimysium encom-
passes the whole muscle. All three layers of connective tis-
sue contain collagen, mostly types I, III, IV, and V; types
IV and V predominate in the basement membranes sur-
rounding each skeletal muscle fiber. The α12α2-chain com-
position of the collagen IV isoform is the most prevalent
and provides the mechanical stability and flexibility of the
basal lamina.23,24 The perimysium and endomysium merge
at the junction between the muscle fibers and the tendons,
aponeuroses, and fasciae. These layers give the attachment
sites great tensile strength and distribute axial force into
shear forces over a larger surface area.
FIBER TYPES
Muscles adapt to their specific functions. In any given
muscle, part of this adaptation arises from its composition
and the organization of fiber types. Fibers can be classified
according to their size, twitch duration, speed of contrac-
tion, balance between aerobic and glycolytic metabolism,
and resistance to fatigue (Table 5-2). In addition, isoen-
zymes of the signaling, regulatory, and contractile proteins
and the surface area of the sarcoplasmic reticulum (SR)
membranes are key features that distinguish the functional
properties of the different fiber types. For instance, the dura-
tion of the twitch is influenced by the rates of release and
reuptake of calcium ions (Ca2+) by the SR, and the velocity
of shortening is determined by myosin isoenzyme composi-
tion. Type IIB (fast) fibers are less “red” than the other fiber
types because they contain less of the iron-heme–containing
proteins, myoglobin, and mitochondrial cytochromes. Clas-
sification schemes are not, however, absolute, because some
groups of fibers have composite or intermediate functional,
ultrastructural, and histochemical characteristics.
During development, fiber-type specificity may be partly
determined before innervation.25 Although the biological
events and signals responsible for designating functional
specialization in muscle fibers are not fully understood,
 96 DANTZIG  |  Muscle: Anatomy, Physiology, and Biochemistry

Table 5-1  Signaling and Contractile Proteins of Skeletal Muscle

Protein Molecular Weight (kD) Subunits (kD) Location Function
Acetylcholine receptor 250 5 × 50 Postsynaptic membrane of   Neuromuscular signal trans-
neuromuscular junction mission
Annexins 38 — F-actin binding protein Membrane repair
Dihydropyridine receptor 380 1 × 160 T tubule membrane Voltage sensor
1 × 130
1 × 60
1 × 30
Dysferlin 230 — Periphery of myofibers Membrane repair
Ryanodine receptor 1800 4 × 450 Terminal cisternae of SR SR Ca2+ release channel
Ca -ATPase
2+
110 — Longitudinal SR Uptake of Ca2+ into SR
Calsequestrin 63 — SR terminal cisternae lumen Binding and storage of Ca2+
Troponin 78 1 × 18 Thin filament Regulation of contraction
1 × 21
1 × 31
Tropomyosin 70 2 × 35 Thin filament Regulation of contraction
Myosin 500 2 × 220 Thick filament Chemomechanical energy
2 × 15 transduction
2 × 20
Actin 42 — Thin filament Chemomechanical energy
transduction
MM creatine   40 — M line ATP buffer, structural protein
phosphokinase
α-Actinin 190 2 × 95 Z line Structural protein
Titin 3000 — From Z line to M line Structural protein
Nebulin 600 — Thin filaments, in the I band Structural protein
Dystrophin 400 — Subsarcolemma Structural integrity of sarco-
lemma
ATP, adenosine triphosphate; SR, sarcoplasmic reticulum.

classic cross-innervation experiments demonstrated that
innervation can dynamically specify and modify the type of
muscle fiber.26 After cross-innervation, the functional and
histological properties listed in Table 5-2 shift toward the
target fiber type over a few weeks’ time, indicating the ability
of muscles to adapt and remodel in accordance with the pat-
tern of neuronal activity.
EVENTS DURING MUSCLE CONTRACTION
NEURAL CONTROL
Normally, a skeletal muscle fiber is activated briefly and
then relaxes. This twitch, lasting for 5 to 40 msec, is initi-
ated by an action potential propagated from the central
nervous system along an alpha motoneuron, synaptic
transmission across the neuromuscular junction, and an
action potential in the muscle sarcolemma. Muscle fibers
are typically innervated at one or two sites along their
length by branches of an alpha motoneuron axon arising
from the ventral horn of the spinal column. A motoneu-
ron and the approximately 5 to 1600 homogeneous muscle
fibers it innervates constitute a motor unit. Although the
spatial domains of different units are intermingled, when
an alpha motoneuron is excited, all fibers in the motor unit
are triggered to contract together. Functional properties,
such as speed and susceptibility to fatigue, vary with the
dynamic requirements set by the neuronal firing pattern
and the mechanical load, but they are homogeneous within
a motor unit. The level of muscle activity is controlled by
varying the firing rate of twitches and the number of active
motor units. As activity increases, more and larger units
are recruited.
A second class of fibers, the intrafusal fibers, are inner-
vated by gamma motoneurons and control the sensitivity of
the Golgi tendon organs and spindle receptors that provide
local feedback to the spinal cord regarding muscle length
and force. Afferent feedback pathways modulate activity to
control the desired movement. The same feedback system
generates the monosynaptic stretch reflex.
NEUROMUSCULAR TRANSMISSION
At the neuromuscular junction, the axon tapers and loses
its myelin sheath. The postsynaptic sarcolemmal mem-
brane (the motor end plate) is indented into folds that
increase its surface area (Fig. 5-3). Mitochondria and nuclei
are ­ concentrated in this region. The junctional cleft is a
50-nm-wide space between the presynaptic axonal mem-
brane and the sarcolemma.
When the nerve action potential reaches the presynaptic
terminal, local Ca2+ channels are gated open for the influx
of Ca2+. This triggers the fusion of acetylcholine-loaded
membrane vesicles with the neuronal presynaptic mem-
brane.27 Exocytosed acetylcholine rapidly diffuses across the
junctional cleft and binds to nicotinic acetylcholine-gated
ion channels in the crests of the postsynaptic membrane
folds. Ligand gating of the acetylcholine receptors increases
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE 97

Table 5-2  Classification of Muscle Fiber Types

General Features I IIA IIB IIC
Size Moderate Small Large Small
Mitochondria Many Intermediate Few Intermediate
Capillary blood supply Extensive Sparse Sparse Sparse
SR membrane Sparse Extensive Extensive Extensive
Z line Wide Wide Narrow Narrow
Protein isoforms
Myosin heavy chain (MHC) MHCI MHCIIA MHCIIB MHCI, MHCIIA
Myosin essential light chain   ELC1s ELC1f, ELC3f ELC1f, ELC3f ELC1f, ELC3f, ELC1s
(ELC)
Myosin regulatory light   RLC2s RLC2f RLC2f RLC2f, RLC2s
chain (RLC)
Regulatory proteins Slow Fast Fast Fast
Mechanical properties
Contraction time Slow/sustained Fast twitch Fast twitch Moderate twitch
SR calcium ATPase rate Low High High High
Actomyosin ATPase rate Low High High Moderate
Shortening velocity Slow Fast Fast Moderate
Resistance to fatigue High Moderate Low Moderate
Metabolic profile
Oxidative capacity High Intermediate Low High
Glycolytic capacity Moderate High High High
NADH-TR/SDH/MDH High Moderate Low Moderate
LDH and phosphorylase Low Medium High Moderate
Glycogen Low High High Variable
Myoglobin High Medium Low High
f, fast; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; NADH-TR, nicotinamide adenine dinucleotide tetrazolium reductase; s, slow; SDH, succinate
dehydrogenase; SR, sarcoplasmic reticulum.

their cation permeability, locally depolarizing the muscle
cell. This change of membrane potential initiates a regen-
erative action potential, mediated by voltage-gated sodium
(Na+) and potassium (K+) channels. The action potential
propagates at velocities up to 5 m/sec from the motor end
plate throughout the sarcolemma. Thus, the entire fiber and
motor unit contract at the same time.
EXCITATION-CONTRACTION COUPLING
Invaginations of the sarcolemma at regular intervals consti-
tute the transverse tubule (T tubule) network, which per-
vades the fiber and surrounds the contractile apparatus with
connected longitudinal and lateral segments (Fig. 5-4). The
lumen of this network is open to the extracellular space,
and it contains the high-Na+ and low-K+ concentrations
of interstitial fluid.28 Action potentials at the surface mem-
brane invade the entire T tubular system. A specialized type
of endoplasmic reticulum forms the entirely intracellular
SR. Prevalent structures termed triads contain a T tubule
flanked by two terminal cisternae of the SR to form junc-
tional complexes (see Fig 5-4). Terminal cisternae contain
the oligomers of the Ca2+-binding protein calsequestrin,
which provide the fiber with an internal reservoir of Ca2+.
Dihydropyridine receptors (DHPRs) are Ca2+ channels
localized in the T tubule membranes facing the cytoplasmic
domain of SR Ca2+-release channels (the ryanodine recep-
tors [RyRs], or foot proteins) in the terminal cisternae mem-
branes.29 These membrane proteins are further characterized
in Table 5-1.
When an action potential depolarizes the T tubular
membrane, the DHPRs, which are primarily voltage ­sensors
in skeletal muscle (but not in myocardium), transfer a sig-
nal from the T tubules to the RyRs by direct interprotein
coupling. Ca2+ is then released cooperatively through the
RyRs from the SR into the myoplasm, where it activates the
contractile machinery.30 This sequence of events is termed
excitation-contraction coupling.
Mutations in the α-subunit of the DHPR in dysgenic
mice lead to paralysis because, in these mutants, depolar-
ization of the skeletal muscle membrane does not initiate
the release of Ca2+ from the SR. Excitation-contraction
coupling can be restored in cultured cells from these mice
by transfection with the complementary DNA encoding
for the DHPR,31 and transfections using chimeric con-
structs32 have pinpointed the domain within the DHPR that
specifies skeletal- or cardiac-type excitation-­contraction
­coupling.33 ­ Isoforms of the RyRs also help determine the
characteristics of the coupling between T tubules and the
SR.34 Channelopathies in human skeletal and heart muscle
have been linked to DHPR mutations.35,36 Human malignant
hyperthermia occurs in individuals with mutant RyRs that
become trapped in the open state after exposure to halo-
thane anesthetic agents.37
CONTRACTILE APPARATUS
The specific locations and functions of the contrac-
tile proteins are listed in Table 5-1. Myofibrils are long,
1-μm-diameter cylindrical organelles that contain the con-
tractile protein arrays responsible for work production, force
generation, and shortening (Fig. 5-5). Each myofibril is a
column of sarcomeres, the basic contractile units, which are
approximately 2.5 μm long and delimited by Z lines (see
 98 DANTZIG  |  Muscle: Anatomy, Physiology, and Biochemistry

Fig. 5-5D and E) containing the densely packed structural contraction,39 resulting in the cross-striated histological
protein α-actinin. The contractile and structural proteins appearance of skeletal and cardiac muscles. This highly
within each sarcomere form a highly ordered, nearly crystal- periodic organization has facilitated biophysical studies of
line lattice of interdigitating thick and thin myofilaments muscle by sophisticated structural38 and spectroscopic tech-
(see Fig. 5-5E, I, and J).38 Myofilaments are remarkably uni- niques.40,41
form in both length and lateral registration, even ­ during Thick filaments (1.6 μm long) containing the motor pro-
tein myosin are located in the center of the sarcomere in
the optically anisotropic A band (see Fig. 5-5D). The thick
filaments are organized into a hexagonal lattice stabilized by
M protein42 and muscle-specific creatine phosphokinase43 in
Muscle fiber the M line (see Fig. 5-5D and E). Myosin (see Fig. 5-5K) is a
highly asymmetric 470-kD protein containing two 120-kD
globular NH2-terminal heads, termed cross-bridges or sub-
Nerve
fragment-1 (S-1; see Fig. 5-5L), and an α-helical coiled-coil
Motor rod. Two light chains—essential and regulatory, ranging
end plate from 15 to 22 kD—are associated with the heavy chain in
each S-1 (see Fig. 5-5L). The rod portions of approximately
300 myosin molecules polymerize in a three-stranded helix
to form the backbone of each thick filament (see Fig. 5-5K).
The cross-bridges, protruding from these backbones, con-
10µm
A tain adenosine triphosphatase (ATPase) and actin-binding
sites responsible for the conversion of chemical energy into
mechanical work. Besides their role in muscle contraction,
at least 20 classes of nonmuscle myosins accomplish diverse
Myofibrils
Synaptic tasks in cell motility such as chemotaxis, cytokinesis, pino-
Cleft cytosis, targeted vesicle transport, and signal transduction.44
Postsynaptic Thus, myosin is the target for mutations leading to a number
Membrane
Synaptic of inherited muscular and neurologic diseases.45,46
Vesicles Thin filaments (see Fig. 5-5I) are double-stranded heli-
cal polymers of actin that extend 1.1 μm from each side
of the Z line and occupy the optically isotropic I band
(see Fig. 5-5D and E). A regulatory complex containing
one tropomyosin molecule and three troponin subunits
Presynaptic (TnC, TnT, and TnI) is associated with each succes-
B Terminal 1µm sive group of seven actin monomers along the thin fila-
ment (see Fig. 5-5J).38 In the region where the thick and
Figure 5-3  Neuromuscular junction. A, Scanning electron micrograph thin filaments overlap, the thin filaments are positioned
of an alpha motoneuron innervating several muscle fibers in its motor within the hexagonal lattice equidistant from three thick
unit. B, Transmission electron micrograph. (A, From Bloom W, Fawcett DW:
A Textbook of Histology, 10th ed. Philadelphia, WB Saunders, 1975.
filaments (see Fig. 5-5F). Both sets of filaments are polar-
B, Courtesy of Dr. Clara Franzini-Armstrong, University of Pennsylvania, ized. In an active muscle, an interaction between the
Philadelphia.) two filaments causes a concerted translation of the thin

Myofibril
Ca2+-release
channels

Transverse (T)
tubules formed
from invaginations
of plasma
membrane
Sarcoplasmic
reticulum
0.5µm

Figure 5-4  Membrane systems that relay the excitation signal from the sarcolemma to the cell interior. In the electron micrograph, two T tubules
are cut in cross section. The electron densities spanning the gap between the T tubules and the sarcoplasmic reticulum membranes are the ryanodine
receptors, channels that release calcium into the myoplasm. (From Alberts B, Bray D, Lewish J, et al: Molecular Biology of the Cell, 2nd ed. New York, Garland,
1989. Micrograph courtesy of Dr. Clara Franzini-Armstrong, University of Pennsylvania, Philadelphia.)
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE 99

A
Muscle

Muscle fasciculus
C

Muscle fiber
H Z A I
band line band band
D

M line Z Sarcomere Z Myofibril

I
H E Troponin Tnl Tnc TnT
complex

Actin
Tropomyosin
Sarcomere Actin thin filament

J
F G H Single myosin
molecule Subfragment 1

Tail
Myosin filament
L K
ATP pocket RLC
S2
Actin ELC
m ain
S1
binding do Light meromyosin
h ain
site tc
h
Motor domain Lig
Myosin molecule
Myosin subfragment 1

Figure 5-5  Components of the contractile apparatus. Components are shown at successively increasing magnifications from the whole muscle 
(A) to the molecular level (I-L). The myofibril (D) shows the banding pattern created by the lateral alignment of the myofilaments (I, J) in the sarcomeres
(D, E). Parts F to H show the cross-sectional structure of the filament lattice at various points within the sarcomere. Myosin is shown at the single two-
headed molecule level (K), and the crystal structure of the globular motor domain is shown (L, S-1) with the essential and regulatory light chains. (From
Juanqueira LC, Carneiro J, Long JA: Basic Histology, 5th ed. Norwalk, Conn, Appleton-Lange, 1986. Modified from Bloom W, Fawcett DW: A Textbook of Histol-
ogy, 10th ed. Philadelphia, WB Saunders, 1975; and Rayment I, Rypniewski WR, Schmidt-Base K, et al: Three-dimensional structure of myosin subfragment-1: A
molecular motor. Science 261:50, 1993.)

fi­ laments toward the M line, which shortens the sarco-
mere and thus the whole muscle. Actin is ubiquitous in
the cytoskeleton of eukaryotic cells and, like myosin, ful-
fills many roles in determining cell shape and motion.47,48
Control of the actin cytoskeleton and diseases due to
mutations in actin-binding proteins are being intensively
investigated.49
Two of the largest identified proteins, titin and nebulin,
function in the assembly and maintenance of the sarcomeric
structure. Individual titin molecules (∼3000 kD) are associ-
ated with the thick filament and extend from the M line to
the Z line.50 Titin contains repeating fibronectin-like immu-
noglobulin and unusual proline-rich domains that confer
molecular elasticity on the resting sarcomere.51 Nebulin
 100 DANTZIG  |  Muscle: Anatomy, Physiology, and Biochemistry
ADP
Pi
(b)
ADP
Pi
Figure 5-6  The actomyosin cross-bridge cycle. Myo-
sin molecules normally have two globular head regions
(cross-bridges), but for clarity, only one is shown. The
⊗ within the globular domain of myosin represents a
hinge point with maximal flexibility. Each head binds Atached
with two actin monomers. The sequence of reactions (a)
is attachment (a); the force-generating transition (b);
orthophosphate (Pi) release (c); force generation and
filament sliding (d); adenosine diphosphate (ADP) re-
lease (e); adenosine triphosphate (ATP) binding and
Actin
detachment (f); and ATP hydrolysis (g). The shadowed
thin filament
heads near the detached and force-generating myosin
heads indicate high mobility of the cross-bridges in
these states.
(∼800 kD) is associated with the Z line and thin filaments.50
Protein connections from the contractile apparatus through
the sarcolemma to the extracellular matrix are described
later in this chapter. The cytoskeleton of muscle fibers also
contains cytoplasmic actin, microtubules, and intermediate
filaments.52
FORCE GENERATION AND SHORTENING
At rest, the thin-filament regulatory proteins troponin and
tropomyosin inhibit contraction (see Fig. 5-5I). During a
twitch, Ca2+ released from the SR binds to TnC, relieving
this inhibition and thus allowing cross-bridges to attach
to actin. A contraction results from a cyclic interaction
between actin and myosin (the cross-bridge cycle) that pro-
duces a relative sliding force between the thin and thick
filaments.53 The energy source is the hydrolysis of adenosine
triphosphate (ATP) to adenosine diphosphate (ADP) and
orthophosphate (Pi).
A simplified model of the chemomechanical events in
the cross-bridge cycle is illustrated in Figure 5-6. Motor
proteins, including myosin, can now be studied by single-
molecule biophysical techniques, which provide unprec-
edented details of their dynamics.54 When Ca2+ is present,
a complex of myosin, ADP, and Pi attaches to the thin fila-
ment (step a in Fig. 5-6), and a structural change within
the myosin S-1 initiates force production and the release
of Pi (steps b and c).55,56 The conformational change in
the cross-bridge that leads to force generation is a tilting
motion of the light-chain region.41,57,58 The filament sliding
that leads to shortening of the sarcomere occurs during a
strain-dependent transition between two ADP states (step
d). After ADP is released (step e), ATP binds to the active
M-line
Pi
ADP
ADP
(c) (d)
Z-line
Force generation and
filament sliding
ADP
(e)
Rigor
(f)
ADP
ATP ATP
Pi
(g)
Myosin
thick filament
Hydrolysis
Primed Detached
site and dissociates myosin from actin (step f). Myosin
then hydrolyzes ATP (step g) to form the ternary myosin-
ADP-Pi complex, which can reattach to actin for the next
cycle.
If the mechanical load on the muscle is high, the con-
tractile apparatus produces a force without changing length
(an isometric contraction). If the load is moderate, the thin
filaments slide actively toward the center of the sarcomere,
resulting in shortening of the whole muscle. The width
of the muscle increases during shortening, so the volume
stays constant. Work production (concomitant force and
sliding) is associated with an increase in the ATPase rate.
The thermodynamic efficiency (mechanical power divided
by energy liberated by ATPase activity) approaches 50%—
a remarkable figure, considering that manufactured com-
bustion engines seldom achieve ­ efficiencies greater than
20%.
RELAXATION
The twitch is terminated by reversal of all the steps in acti-
vation. Ca2+ released from the SR is taken up again by Ca2+-
ATPase pumps located in longitudinal membranes of the
SR. The myoplasmic Ca2+ concentration then decreases,
and Ca2+ dissociates from TnC, deactivating the thin fila-
ment. When the number of attached cross-bridges declines
below a certain threshold, tropomyosin inhibits further
cross-bridge attachment, and tension declines to the rest-
ing level. Ca2+ diffuses within the longitudinal SR to the
calsequestrin sites in terminal cisternae, ready to be released
in the next twitch. Myosin continues to hydrolyze ATP at a
low rate in relaxed muscle, accounting for a sizable propor-
tion of basal metabolism.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE 101

the membrane. Its absence or truncation causes Duchenne’s

TRANSMISSION OF FORCE
TO THE EXTERIOR
CELL MATRIX ADHESIONS
The muscle cell is closely connected to the basal lamina
along its entire surface. Several transmembrane macromo-
lecular complexes link the myofibrils and actin cytoskel-
eton to laminins and collagen in the extracellular matrix.
Attachment complexes for muscle, analogous to the focal
adhesions of motile and epithelial cells and to the adhesion
plaques and intercalated disks in cardiac muscle, contain fil-
amentous actin, vinculin, talin, and integrin (primarily the
α7β1 isoform), which is the transmembrane link to laminin
(Fig. 5-7). In muscle, the main laminin isoforms are lam-
inin-2 (α2β1γ1) and laminin-4 (α2β2γ1), which are collec-
tively termed merosin. In addition to providing mechanical
coupling between the cytoskeleton and the extracellular
matrix, the laminin-integrin system may provide a signaling
pathway to regulate localized protein expression.59 Defects
in the expression of many of the cytoskeletal proteins lead
to various forms of muscular dystrophy (Table 5-3).60
A specialized linkage between the cytoskeleton and
basal lamina in muscle, complementary to the integrin focal
adhesion system, is the dystrophin-glycoprotein complex
(see Fig. 5-7). Dystrophin, a 427-kD peripheral cytoskeletal
protein, has been postulated to function as a mechanical
link between the cytoskeleton and the cell membrane or as
a shock absorber, or to contribute mechanical strength to
and Becker’s muscular dystrophies.61 The N-terminus of dys-
trophin binds to actin via a region with sequence homology
to the actin-binding domain of α-actinin. This end of the
dystrophin molecule may be linked to the basal lamina via
the same proteins described previously for the focal adhe-
sion-like complexes (see Fig. 5-7). The C-terminus binds to
a transmembrane dystroglycan-sarcoglycan complex, which
in turn binds to the laminins. Muscular dystrophies of var-
ied severity are associated with a loss of these components
(see Table 5-3).62 Dystroglycans are also required for early
embryonic development of muscle, possibly organizing lam-
inin localization and assembly.63,64 Utrophin, a smaller dys-
trophin-related protein (395 kD), may also link the actin
cytoskeleton to the dystroglycans, especially near the neu-
romuscular junction and in nonmuscle cells. Overexpres-
sion of this protein or truncated dystrophin constructs are
promising avenues for gene therapy in Duchenne’s muscular
dystrophy.65 The unusual intricacy of cell matrix connection
systems in muscle may relate to the high forces generated
during contraction.
MYOTENDINOUS JUNCTION
The force of muscle contraction is transmitted to the skel-
eton via the tendons, which are composed of type I and
III collagens, blood vessels, lymphatic ducts, and fibroblasts.
At the ends of the muscle fibers, myofibrils are separated
by invaginations of sarcolemma filled with long bundles of

Basal lamina Collagen

Laminin
Laminin
Dysferlin Integrins
α Dystroglycans
Sarcospan

α β
A1 A2 β ζ ε δ γ β α Sarcolemma
Annexins

nNOS Vinculin
Sarcoglycans Caveolin 3
α1
Talin
α1 Syntrophins Dystrophin N

α-dystrobrevin C

Cytoplasm

Actin

Syncoilin

Figure 5-7  Connections between the muscle cytoskeleton and extracellular matrix. Actin is linked through integrins to the matrix, as in many cell
types. Dystrophin forms an extra link through the dystroglycan-sarcoglycan complex of glycosylated proteins. The helical section of dystrophin is ho-
mologous to spectrin and may form homodimers or oligomers. Dystrophin links two intricate systems connecting the sarcolemma to the basal lamina.
The COOH-terminus of dystrophin (N) is associated with the sarcoglycans, dystroglycans, dystrobrevin, syncoilin, neuronal nitric oxide synthase (nNOS),
and syntrophins. The NH3-terminus links actin, vinculin, and the integrins with laminin and the basal lamina. These two adhesion systems provide a sup-
portive substructure to maintain the integrity of the sarcolemma. The annexins and dysferlin have a role in muscle regeneration.
 102 DANTZIG  |  Muscle: Anatomy, Physiology, and Biochemistry

Table 5-3  Classification of Muscular Dystrophies

Disease Genetic Locus Inheritance Protein Outcome
Duchenne’s, Becker’s XR Dystrophin Lethal
Emery-Dreifuss XR Emerin, laminins A and C 40% lethality
Limb-girdle muscular dystrophies Less severe forms can
(LGMDs) emerge during first 3
LGMD 1A 5q31 AD Myotilin decades of life, leading to
LGMD 1B 1q11-q21 AD Laminin A or C loss of ambulation after
LGMD 1C 3p35 AD Caveolin age 30 yr; most severe
LGMD 1D 6q23 AD — forms start at age 3-5 yr
LGMD 1E 7q AD — and progress rapidly
LGMD 1F 7q32 AD —
LGMD 1G 4p21 AD —
LGMD 2A 15q15.1-q21.1 AR Calpain 3
LGMD 2B 2p13 AR Dysferlin
LGMD 2C 13q12 AR γ-Sarcoglycan
LGMD 2D 17q12-q21.33 AR α-Sarcoglycan
LGMD 2E 4q12 AR β-Sarcoglycan
LGMD 2F 5q33-q34 AR δ-Sarcoglycan
LGMD 2G 17q11-q12 AR Telethonin
LGMD 2H 9q31-q34.1 AR E3 ubiquitin ligase (TRIM32)
LGMD 2I 19q13.3 AR Fukutin-related protein
LGMD 2J 2q24.3 AR Titin
LGMD 2K 9q34 AR Protein  
O-mannoslytransferase
CMDs with CNS involvement
Fukuyama CMD 9q31 AR Fukutin LE, 11-16 yr
Walker-Warburg CMD 1p32 AR O-Mannosyltransferas LE, <3 yr
Muscle-eye-brain CMD 1p32-34 AR O-MNAGAT LE, 10-30 yr
CMDs without CNS involvement
Merosin-deficient classic type 6q2 AR Merosin (laminin A2) Many patients never walk;
others have LGMD pat-
tern
Merosin-positive classic type 4p16.3 AR Selenoprotein N1, collagen Course stabilizes in late
VI α2 childhood; many patients
continue to walk into
adulthood
Integrin-deficient CMD 12q13 AR Integrin α7 Presents early in infancy
with hypotonia and
delayed milestones
Other dystrophies
Facioscapulohumeral 4q35 AD — 20% wheelchair bound
Oculopharyngeal 14q11.2-q13 AD/AR Polyadenylate binding   Onset ∼48 yr; 100% symp-
protein nuclear 1 tomatic by age 70 yr
Myotonic dystrophy 19q13.3 AD DMPK, CCHC-type zinc   50% show signs by age  
finger, CNBP 20 yr; variable severity
AD, autosomal dominant; AR, autosomal recessive; CCHC, cysteine and histidine amino acid sequence; CMD, congenital muscular dystrophy; CNBP, cellular
nucleic acid binding protein; CNS, central nervous system; DMPK, dystrophia myotonica protein kinase; LE, life expectancy; O-MNAGAT, O-mannose β-1,2-N
acetylglucosaminyl transferase; XR, X chromosome related.

collagen arising from the tendon. These membrane folds
increase the surface area for bearing the mechanical load
by approximately 30-fold. Instead of terminating in the Z
disks, actin filaments insert into a subsarcolemmal matrix
containing α-actinin, vinculin, talin, and integrin. The
force is transmitted through laminin to the collagen of the
tendon.
ENERGETICS
Metabolic pathways in muscle cells are specialized for the
variable, and at times extreme, rates of ATP splitting by the
contractile apparatus and membrane ionic pumps. Because
the muscle tissue compartment is large, and because certain
protein isoforms are specific to muscle tissue, circulating
levels of some metabolic enzymes (e.g., lactate dehydroge-
nase, creatine phosphokinase) are useful in the diagnosis of
sarcolemmal disruption. Among the dozens of enzymes pres-
ent, only the most important ones related to normal muscle
function are mentioned here.
BUFFERING OF ADENOSINE TRIPHOSPHATE
CONCENTRATION
The ATP content (∼5 mM) is sufficient for only a few seconds
of contraction, so rapid and effective buffering of ATP during
contraction is essential for the maintenance of activity. ADP
formed by the hydrolysis of ATP is rephosphorylated by trans-
fer of a phosphate group from creatine phosphate (20 mM in
a resting cell), by creatine phosphokinase located within the
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
M line of the sarcomere, in the myoplasm, and between the
inner and outer membranes of the mitochondria. Adenyl-
ate kinase, known in muscle as myokinase, catalyzes the
transfer of a phosphate group between two ADP molecules,
forming ATP and adenosine monophosphate (AMP). The
by-products of the rapid enzymatic reactions that maintain
ATP concentration are, therefore, creatine, Pi, and AMP.
Some of the AMP is converted to inosine monophosphate
by adenylate deaminase. A creatine phosphate shuttle has
been proposed to enhance energy flux.66 According to this
hypothesis, creatine phosphate is split within the contractile
apparatus, and creatine is predominantly rephosphorylated
in the mitochondria.
GLYCOLYSIS
Muscles use glucose as fuel when possible; otherwise,
they use fatty acids and ketone bodies (acetoacetate and
3-hydroxybutyrate). The muscle compartment contains
most of the body storage of glycogen, which is converted
to glucose 6-phosphate for local use. Muscle fibers lack
glucose-6-phosphatase and thus do not export glucose.
During intense activity, especially in anaerobic condi-
tions, the rate of glycolysis and the production of pyru-
vate exceed the rate of pyruvate consumption by the citric
acid cycle. The excess pyruvate is reduced to lactate by
lactate dehydrogenase, which has tissue-specific isoforms.
The lactate dehydrogenase reaction also produces nicotin-
amide adenine dinucleotide (NAD+), which is necessary
for glycolysis, but otherwise, lactate is not useful within
the muscle. Lactate is freely permeable through the sar-
colemma, and a local increase in the extracellular lactate
concentration or acidification produces exertional pain
(“the burn”). Lactate is transported through the blood
to the liver, where it is converted back to pyruvate and
then glucose, which is released into the blood for use by
other tissues, such as muscle and brain. This sequence of
steps, termed the Cori cycle, transfers some of the high
metabolic load to the liver and “buys time” until oxidative
metabolism is available.
OXIDATIVE PHOSPHORYLATION
In aerobic conditions, pyruvate enters the mitochondria,
where it is oxidized to carbon dioxide and water, gener-
ating reduced NAD (NADH). A hydrogen ion (H+) gra-
dient across the mitochondrial membrane is produced by
the electron transport chain, and, finally, ADP is phos-
phorylated to ATP by the mitochondrial ATP synthase.
This remarkable rotary motor generator has been studied
extensively using single-molecule biophysical techniques.67
By combining glycolysis and oxidative phosphorylation, up
to 38 ATP molecules can be generated by the oxidation
of each molecule of glucose. This process is energetically
much more favorable than the production of lactate, but
it can occur only when molecular oxygen is available.
Myoglobin is an iron-heme complex protein that facili-
tates oxygen transport within the muscle cells. The tissue
hydrostatic pressure in a contracting muscle often exceeds
arterial perfusion pressure, so the strongest contractions
are anaerobic. The content of oxidative enzymes, myoglo-
bin, and mitochondria determines the predominant type of
103
energy metabolism and varies in different muscle types, as
discussed previously (see Table 5-2).
FATIGUE AND RECOVERY
During intense or prolonged activity, muscle fatigue is
caused by alterations of metabolite levels that suppress force
­generation at the contractile apparatus,68 in excitation-con-
traction coupling, or both.69 Markedly increased ­myoplasmic
Pi and H+ concentrations and decreased creatine phosphate
levels have been detected by magnetic resonance spectros-
copy.70 When the creatine phosphate level declines, main-
tained activity depends on glycogenolysis until glycogen
stores are depleted. During prolonged intense activity, the
respiratory and circulatory systems are unable to meet the
oxygen demands of tissues. Force production declines well
before the ATP concentration is compromised.
The chemomechanical link between Pi release and force
generation (see Fig. 5-6) implies that the increase of myoplas-
mic Pi in fatigued muscle reduces the magnitude of force sim-
ply by mass action.56 Decreased muscle pH, partly from lactate
accumulation, and insufficient acetylcholine at the neuro-
muscular junction, leading to failure of synaptic transmission,
also contribute to decreased work production. Because the
respiratory and circulatory systems do not supply sufficient
oxygen to support the metabolism during intense activity,
an oxygen debt is incurred. Blood flow and oxygen uptake
continue at an enhanced level after the period of exercise to
reclaim this energy. Rephosphorylation of creatine can take
place within a few minutes, but glycogen resynthesis requires
several hours. Recovery processes also involve the restoration
of ionic gradients across the membrane-bound compartments
and require the consumption of additional energy.
PLASTICITY
The strength and endurance of a muscle are altered dramati-
cally within weeks after changes in the demand for its use,
its mobility, or its hormonal or metabolic environment. The
effects of this adaptive response should be considered in any
clinical situation that causes a substantial shift in these fac-
tors and in terms of the patient’s long-term quality of life.
ADAPTATION TO EXERCISE
Exercise leads to adaptations in the muscle fibers, includ-
ing alterations in specific contractile, regulatory, structural,
and metabolic proteins, as well as optimization of motor
unit recruitment. The frequency, intensity, and duration
of a training stimulus and the external load influence the
adaptive response.71 Trophic factors liberated from the
nerve play a minor role, if any. Strength training causes
cross-­sectional hypertrophy of fast type IIB fibers (see Table
5-2), the expression of fast myosin isoforms, an increase in
amino acid uptake, and decreased synthesis of mitochondrial
proteins. Endurance training enhances the oxidative capac-
ity and volume density of mitochondria in oxidative type I
and IIA fibers, redistributes blood flow to these motor units,
and increases the synthesis of contractile proteins. Long-term
hyperplasia does not usually occur after training in humans.
Although hypertrophy of preexisting fiber types is observed,
training also induces changes in myosin isoform distribution,
 104 DANTZIG  |  Muscle: Anatomy, Physiology, and Biochemistry
especially in fibers already containing more than one isoform.
There is little evidence that voluntary training regimens can
switch fibers between the major categories.
When physical activity is reduced, such as during limb
immobilization, the cross section of the fibers decreases, and
endurance is reduced as a result of a decreased metabolic
reserve. Acute exercise hypertrophy and disuse atrophy are
both reversible, but after extensive alterations, the restora-
tion may be incomplete.72
ENDOCRINE CONTROL
Endocrine factors also participate in adaptation. For example,
normal circulating levels of thyroid hormones are required
during muscle development and differentiation.73 Experi-
mental alterations of thyroid hormone concentrations pro-
mote changes in the relative levels of myosin and regulatory
protein isoforms, as well as changes in the activity of some
metabolic enzymes.74 An intact nerve supply is required for
these thyroid effects. Myogenesis is partly controlled by
the release of growth hormone from the pituitary via the
production of insulin-like growth factor 1 (IGF-1) in the
liver. IGF-1 activates satellite cells for muscle regeneration
and stimulates the hypertrophy of mature muscle cells.75,76
Abuse of growth factors, drugs, steroids, and metabolites by
athletes (primarily young men) to increase muscle mass and
strength is a significant medical and societal problem.77,78
The use of androgenic-anabolic steroids (e.g., synthetic
testosterone) to increase muscle mass and improve body
image induces an increase in lean muscle mass as a result
of skeletal muscle hypertrophy. Along with the increase in
cross-sectional area of the muscle comes strength enhance-
ment, with or without concurrent exercise training. Side
effects of these performance-enhancing drugs include acne,
erratic aggression, hirsutism, and male pattern baldness.
These compounds also decrease high-density lipoprotein
levels, increase erythropoiesis, increase bone density, and
elevate liver enzymes. Most of the physiological changes are
reversible. Acute cessation can lead to severe depression in
many individuals.
As in other tissues, insulin regulates the entry of glucose
through the cell membrane. In muscle, it also promotes the
uptake of branched-chain amino acids (valine, leucine, and
isoleucine), which are necessary for rapid protein synthesis,
and it inhibits protein degradation. Muscle weakness and
fatigue may also be symptoms of changes in serum insulin
and glucose levels.
AGING
Normal individuals exhibit an approximately 30% decrease
in total muscle mass between the ages of 30 and 80 years.
The cross-sectional area of the fibers decreases, leading to a
­significant loss of strength. Gradual decreases in muscle activ-
ity, as well as neuronal changes, contribute to this muscle
atrophy, which can exacerbate age-related joint degeneration
and make joints less stable and more prone to injury.
A decline in estrogen and testosterone levels in aging
individuals has been hypothesized to affect muscle strength.
Aging also produces a marked decrease in the level of IGF-1
and the subsequent ability to repair or build new muscle.
Recombinant IGF-1 delivered via virus-mediated gene
transfer has been shown to increase the muscle mass and
strength of the aged mouse.79 Further manipulations of
specific growth factors may ameliorate muscle loss due to
muscular dystrophies, other chronic illnesses, and aging.80,81
Muscle mass, strength, and flexibility are major factors in
the maintenance of an independent and productive lifestyle
among the elderly.
SUMMARY
The complex functional capacity of muscle to produce finely
tuned and coordinated movements is ultimately expressed
as the transduction of chemical to mechanical energy by
actomyosin. A twitch is initiated by an action potential
propagated from the central nervous system along an alpha
motoneuron, neuromuscular chemical transmission, direct
protein-protein communication at the T tubule–SR junc-
tion, Ca2+ diffusion in the myoplasm, and Ca2+ binding to
thin-filament regulatory proteins. Because the central ner-
vous system controls activity through the recruitment of
motor units, the gradation and coordination of movement
depend on the pattern of connections between the alpha
motoneurons and the muscle fibers and on the variation of
properties among motor units. The development, mainte-
nance, and aging of the muscular system involve a complex
series of genetic programs and cellular interactions that are
beginning to be understood at the molecular level. Adapta-
tion of motor unit properties is evident not only in training
regimens but also in reduced activity caused by pain or joint
immobilization and in compromised metabolic, hormonal,
or nutritional conditions. Hence, the plasticity of muscle
affects the clinical course of many diseases. In addition to its
importance in pathophysiology, muscle serves as an excel-
lent substrate for understanding the molecular basis of cell
development, protein structure-function relationships, cell
signaling, and energy transduction processes.
REFERENCES
  1. Myosin Home Page, hosted by the Myosin Group at the MRC Labora-
tory of Molecular Biology and the Cambridge Institute for Medical
Research. http://www.mrc-lmb.cam.ac.uk/myosin/myosin.html.
  2. Illingworth JA: Muscle structure and function. School of Biochem-
istry and Molecular Biology, University of Leeds. http://www.bmb.
leeds.ac.uk/illingworth/muscle/.
   3. Blagden CS, Hughes SM: Extrinsic influences on limb muscle organi-
zation. Cell Tissue Res 296:141, 1999.
   4. Diehl AG, Zareparsi S, Qian M, et al: Extraocular muscle morpho-
genesis and gene expression are regulated by Pitx2 gene dose. Invest
Ophthalmol Vis Sci 47:1785, 2006.
  5. Buckingham M, Bajard L, Chang T, et al: The formation of skeletal
muscle: From somite to limb. J Anat 202:59, 2003.
  6. Muntoni F, Brown S, Sewry C, et al: Muscle development genes:
Their relevance in neuromuscular disorders. Neuromuscul Disord
12:438, 2002.
  7. Parker MH, Seale P, Rudnicki MA: Looking back to the embryo:
Defining transcriptional net works in adult myogenesis. Nat Rev
Genet 4:495, 2003.
  8. Kitzmann M, Fernandez A: Crosstalk between cell cycle regulators
and myogenic factor MyoD in skeletal myoblasts. Cell Mol Life Sci
58:571, 2001.
  9. Buckley PA, Konigsberg IR: Myogenic fusion and the duration of the
post-mitotic gap (G1). Dev Biol 37:193, 1974.
10. Holtzer H, Hijikata T, Lin ZX, et al: Independent assembly of 1.6 μm
long bipolar MHC filaments and I-Z-I bodies. Cell Struct Funct 22:83,
1997.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
11. Sanger JW, Chowrashi P, Shaner NC, et al: Myofibrillogenesis in skel-
etal muscle cells. Clin Orthop 403S:S153, 2002.
12. Sanger J, Kang S, Siebrands CC, et al: How to build a myofibril.
J Muscle Res Cell Motil 26:343, 2005.
13. Kühl U, Öcalan M, Timpl R, et al: Role of muscle fibroblasts in the
deposition of type-IV collagen in the basal lamina of myotubes. Dif-
ferentiation 28:164, 1984.
14. Engler AJ, Sen S, Sweeney HL, Discher DE: Matrix elasticity directs
stem cell lineage specification. Cell 126:677, 2006.
15. Bischoff R: Interaction between satellite cells and skeletal muscle
fibers. Development 109:943, 1990.
16. Florini JR: Hormonal control of muscle growth. Muscle Nerve 10:577,
1987.
17. Muskiewicz KR, Frank NY, Flint AF, Gussoni E: Myogenic potential
of muscle side and main population cells after intravenous injection
into sub-lethally irradiated mdx mice. J Histochem Cytochem 53:861,
2005.
18. Bittner RE, Schofer C, Weipoltshammer K, et al: Recruitment of
bone-marrow-derived cells by skeletal and cardiac muscle in adult dys-
trophic mdx mice. Anat Embryol (Berl) 199:391, 1999.
19. Shi D, Reinecke H, Murry CE, et al: Myogenic fusion of human bone
marrow stromal cells, but not hematopoietic cells. Blood 104:290,
2004.
20. Ojima K, Uezumi A, Miyoshi H, et al: Mac-1 low early myeloid cells
in the bone marrow-derived SP fraction migrate into injured skeletal
muscle and participate in muscle regeneration. Biochem Biophys Res
Commun 321:1050, 2004.
21. Dezawa M, Ishikawa H, Itokazu Y, et al: Bone marrow stromal cells
generate muscle cells and repair muscle degeneration. Science
309:314, 2005.
22. Gowitzke BA, Milner M: Scientific Bases of Human Movement, 3rd
ed. Baltimore, Williams & Wilkins, 1988, pp 144–145.
23. Hudson BG, Reeders ST, Tryggvason K: Type IV collagen: Structure,
gene organization, and role in human diseases. Molecular basis of
Goodpasture and Alport syndromes and diffuse leiomyomatosis. J Biol
Chem 268:26033, 1993.
24. Kühn K: Basement membrane (type IV) collagen. Matrix Biol 14:439,
1994.
25. Miller JB, Stockdale FE: What muscle cells know that nerves don’t tell
them. Trends Neurosci 10:325, 1987.
26. Buller AJ, Eccles JC, Eccles RM: Differentiation of fast and slow mus-
cles in the cat hind limb. J Physiol 150:399, 1960.
27. Südhof TC: The synaptic vesicle cycle: A cascade of protein-protein
interactions. Nature 375:645, 1995.
28. Somlyo AV, Gonzalez-Serratos H, Shuman H, et al: Calcium release
and ionic changes in the sarcoplasmic reticulum of tetanized muscle:
An electron-probe study. J Cell Biol 90:577, 1981.
29. Franzini-Armstrong C, Protasi F: Ryanodine receptors of striated mus-
cles: A complex channel capable of multiple interactions. Physiol Rev
77:699, 1997.
30. Rios E, Brum G: Involvement of dihydropyridine receptors in
excitation-contraction coupling in skeletal muscle. Nature 325:717,
1987.
31. Tanabe T, Beam KG, Powell JA, Numa S: Restoration of excitation-
contraction coupling and slow calcium current in dysgenic muscle
by dihydropyridine receptor complementary DNA. Nature 336:134,
1988.
32. Tanabe T, Beam KG, Adams BA, et al: Regions of the skeletal muscle
dihydropyridine receptor critical for excitation-contraction coupling.
Nature 346:567, 1990.
33. Nakai J, Ogura T, Protasi F, et al: Functional nonequality of the car-
diac and skeletal ryanodine receptors. Proc Natl Acad Sci U S A
94:1019, 1997.
34. Murayama T, Ogawa Y: Roles of two ryanodine receptor isoforms
coexisting in skeletal muscle. Trends Cardiovasc Med 12:305, 2002.
35. Barchi RL: Ion channel mutations and diseases of skeletal muscle.
Neurobiol Dis 4:254, 1997.
 36. Ptácek LJ: Channelopathies: Ion channel disorders of muscle as a par-
adigm for paroxysmal disorders of the nervous system. Neuromuscul
Disord 7:250, 1997.
 37. Gillard EF, Otsu K, Fujii J, et al: A substitution of cysteine for arginine
614 in the ryanodine receptor is potentially causative of human malig-
nant hyperthermia. Genomics 11:751, 1991.
 38. Squire J: The Structural Basis of Muscular Contraction. New York,
Plenum Press, 1981.
105
 39. Sosa H, Popp D, Ouyang G, Huxley HE: Ultrastructure of skeletal
muscle fibers studied by a plunge quick freezing method: Myofilament
lengths. Biophys J 67:283, 1994.
 40. Thomas DD: Spectroscopic probes of muscle cross-bridge rotation.
Ann Rev Physiol 49:691, 1987.
 41. Irving M, Allen TS, Sabido-David C, et al: Tilting of the light-chain
region of myosin during step length changes and active force genera-
tion in skeletal muscle. Nature 375:688, 1995.
 42. Chowrashi PK, Pepe FA: M-band proteins: Evidence for more than
one component. In Pepe FA, Sanger JW, Nachmias VT (eds): Motil-
ity in Cell Function. New York, Academic Press, 1979.
43. Walliman T, Pelloni G, Turner DC, Eppenberger HM: Removal of the
M-line by treatment with Fab′ fragments of antibodies against MM-
creatine kinase. In Pepe FA, Sanger JW, Nachmias VT (eds): Motility
in Cell Function. New York, Academic Press, 1979.
 44. Mermall V, Post PL, Mooseker MS: Unconventional myosins in
cell movement, membrane traffic, and signal transduction. Science
279:527, 1998.
 45. Hasson T: Unconventional myosins, the basis for deafness in mouse
and man. Am J Hum Genet 61:801, 1997.
46. Redowicz MJ: Myosins and deafness. J Muscle Res Cell Motil
20:241, 1999.
 47. Small JV, Rottner K, Kaverina I, Anderson KI: Assembling an actin
cytoskeleton for cell attachment and movement. Biochim Biophys
Acta 1404:271, 1998.
 48. Sheterline P, Clayton J, Sparrow JC (eds): Actin, 4th ed. New York,
Oxford University Press, 1998.
49. Ramaekers FC, Bosman FT: The cytoskeleton and disease. J Pathol
204:351, 2004.
 50. Wang K: Sarcomere-associated cytoskeletal lattices in striated muscle.
In Shay JW (ed): Cell and Muscle Motility, vol 6. New York, Plenum
Press, 1985.
 51. Labeit S, Kolmerer B: Titins: Giant proteins in charge of muscle ultra-
structure and elasticity. Science 270:293, 1995.
 52. Toyama Y, Forry-Schaudies S, Hoffman B, Holtzer H: Effects of Taxol
and Colcemid on myofibrillogenesis. Proc Natl Acad Sci U S A
79:6556, 1982.
 53. Goldman YE: Wag the tail: Structural dynamics of actomyosin. Cell
93:1, 1998.
 54. Leuba SH, Zlatanova J (eds): Biology at the Single Molecule Level.
England, Pergamon Press, 2001.
55. Goldman YE: Kinetics of the actomyosin ATPase in muscle fibers.
Annu Rev Physiol 49:637, 1987.
 56. Dantzig JA, Goldman YE, Millar NC, et al: Reversal of the cross-
bridge force-generating transition by photogeneration of phosphate in
rabbit psoas muscle fibres. J Physiol 451:247, 1992.
 57. Dobbie I, Linari M, Piazzesi G, et al: Elastic bending and active tilting
of myosin heads during muscle contraction. Nature 396:383, 1998.
 58. Forkey JN, Quinlan ME, Shaw MA, et al: Three-dimensional struc-
tural dynamics of myosin V by single-molecule fluorescence polariza-
tion. Nature 422:399, 2003.
 59. Chicurel ME, Singer RH, Meyer CJ, Ingber DE: Integrin binding and
mechanical tension induce movement of mRNA and ribosomes to
focal adhesions. Nature 392:730, 1998.
 60. Kanagawa M, Toda T: The genetic and molecular basis of muscular
dystrophy: Roles of cell-matrix linkage in the pathogenesis. J Hum
Genet 51:915, 2006.
61. Durbeej M, Campbell KP: Muscular dystrophies involving the dys-
trophin-glycoprotein complex: An overview of current mouse mod-
els. Curr Opin Genet Dev 12:349, 2002.
 62. Matsumura K, Ohlendieck K, Ionasescu VV, et al: The role of the
dystrophin-glycoprotein complex in the molecular pathogenesis of
muscular dystrophies. Neuromuscul Disord 3:533, 1993.
 63. Henry MD, Campbell KP: A role for dystroglycan in basement mem-
brane assembly. Cell 95:859, 1998.
 64. Campbell KP, Stull JT: Skeletal muscle basement membrane-sar-
colemma-cytoskeleton interactions minireview series. J Biol Chem
278:12599, 2003.
 65. Wells DJ, Wells KE: Gene transfer studies in animals: What do they
really tell us about the prospects for gene therapy in DMD? Neuromus-
cul Disord 12:S11, 2002.
 66. Bessman SP, Carpenter CL: The creatine-creatine phosphate energy
shuttle. Annu Rev Biochem 54:831, 1985.
 67. Kinosita K Jr, Yasuda R, Noji H: F1-ATPase: A highly efficient rotary
ATP machine. Essays Biochem 35:3, 2000.
 106 DANTZIG  |  Muscle: Anatomy, Physiology, and Biochemistry
 68. Dawson MJ, Gadian DG, Wilkie DR: Muscular fatigue investigated by
phosphorus nuclear magnetic resonance. Nature 274:861, 1978.
 69. Lännergren J, Westerblad H: Force and membrane potential during
and after fatiguing, continuous high-frequency stimulation of single
Xenopus muscle fibres. Acta Physiol Scand 128:359, 1986.
 70. Meyer RA, Brown TR, Kushmerick MJ: Phosphorus nuclear magnetic
resonance of fast- and slow-twitch muscle. Am J Physiol 248:C279,
1985.
 71. Faulkner JA, White TP: Adaptations of skeletal muscle to physical activ-
ity. In Bouchard C, Shephard RJ, Stephens T, et al (eds): Exercise, Fit-
ness, and Health. Champaign, Ill, Human Kinetics, 1990, pp 265-279.
72. Appell HJ: Muscular atrophy following immobilization: A review.
Sports Med 10:42, 1990.
 73. Rubinstein NA, Lyons GE, Kelly AM: Hormonal control of myo-
sin heavy chain genes during development of skeletal muscles. Ciba
Found Symp 138:35, 1988.
 74. Nwoye L, Mommaerts WFHM: The effects of thyroid status on some
properties of rat fast-twitch muscle. J Muscle Res Cell Motil 2:307,
1981.
 75. Florini JR, Ewton DZ, Coolican SA: Growth hormone and the insu-
lin-like growth factor system in myogenesis. Endocrine Rev 17:481,
1996.
 76. Lamberts SW, van den Beld AW, van der Lely A-J: The endocrinol-
ogy of aging. Science 278:419, 1997.
 77. Bower KJ: Anabolic steroid abuse and dependence. Curr Psych Rep
4:377, 2002.
78. Bowers LD: Abuse of performance-enhancing drugs in sport. Ther
Drug Monit 24:178, 2002.
 79. Barton-Davis ER, Shoturma DI, Musaro A, et al: Viral mediated
expression of insulin-like growth factor I blocks the aging-related
loss of skeletal muscle function. Proc Natl Acad Sci U S A 95:15603,
1998.
 80. Bogdanovich S, Krag TOB, Barton ER, et al: Functional improvement
of dystrophic muscle by myostatin blockade. Nature 420:418, 2002.
81. Goldspink G, Harridge SD: Growth factors and muscle ageing. Exp
Gerontol 39:1433, 2004.
 6 Joint Biomechanics:
Key Points
Mechanical loads can damage joint tissues by acute mechani-
cal damage, slowly progressing degeneration, or mechanical
stimulus of enzymatic degradation.
Cartilage responds differently to applied mechanical loads
depending on the health of the tissue.
Mechanical loads influence joint tissue health by regulating
tissue organization and cellular metabolism.
The peak adduction moment during walking is a strong
­predictor of progression of medial compartment knee
­osteoarthritis.
Treatments that reduce adduction moment during walking
provide symptomatic relief.
Human diarthrodial joints must sustain large forces, provide
dynamic joint stability, and permit the movement needed
for normal joint function throughout life. The exceptional
mechanical demand placed on human joints is one of the
primary reasons most individuals incur some type of joint
difficulty during their lifetime. Familiarity with the mechan-
ics occurring at human joints is important for understanding
normal and pathologic joint function.
Joint biomechanics is the study of the interplay between
mechanical loads and joint motions and the relationship
between these loads and the mechanical demands on joint
tissues. Mechanical loads can damage joint tissues through
acute interactions, such as a traumatic knee ligament tear
secondary to an acute overload, or by chronic repetitive
loading causing cumulative microscopic degradation in the
tissue matrix. The resulting tissue damage is typically associ-
ated with a biologic response from the tissue and changes
in tissue morphology and mechanical properties. These tis-
sue modifications alter the joint mechanics during ambula-
tion and potentially can initiate a cascade of self-consistent
events that lead to clinical symptoms and influence treat-
ment outcome. Understanding this complex relationship
between joint tissue structure, biology, and joint mechanics
is essential for properly understanding and treating many
joint pathologies.
The interplay between mechanics and biology is most
evident when considering the mechanics of ambulation
at an in vivo joint level.1 This interplay is easily visualized
at the knee joint, which is used throughout this chapter
to illustrate many of the concepts presented, although the
underlying principles can be extended to most diarthrodial
joints. During ambulatory movement, the forces created by
muscle activity and limb segment inertial properties cause
complex joint motions and tissue strains. During activities
The Role of Mechanics
in Joint Pathology
PAUL L. BRIANT  •
THOMAS P. ANDRIACCHI
such as walking, the motions at the knee involve much more
than rotation around a single axis, and the forces developed
within the knee can reach several times the body weight of
the individual.2 These macroscopic joint loads must be sus-
tained by the tissue matrix and are ultimately transmitted to
the cells, which respond to the mechanical load by altering
their metabolic rates of matrix synthesis and degradation,3,4
changing the tissue mechanical properties.
The biologic response of joint tissue to mechanical loads
means that mechanics can play a large role in the initia-
tion and the progression of many joint diseases. Osteoar-
thritis provides a common example in which mechanics
are clearly implicated in pathology and clinical presenta-
tion. Although the cartilage degeneration associated with
osteoarthritis has been commonly considered to result from
“use-related” degeneration at the joint (“wear and tear”),
such degeneration is more likely caused by a tissue biologic
response to mechanical load, rather than direct mechanical
wear.5 In healthy joints, articular cartilage tends to be thick-
est in the regions of highest load, suggesting that healthy
cartilage responds positively to increased loading.1 These
regions of highest load also tend to degrade most quickly
when thinning has begun, however, suggesting that load is
detrimental to osteoarthritic cartilage.1 The mechanisms
causing this switch in load response are currently unknown;
however, clinical treatments, such as high tibial osteotomy,6
that shift the load distribution within the knee, have been
shown to produce symptomatic relief.
This chapter addresses some of the fundamental prin-
ciples of joint biomechanics. The focus is primarily on
joint-level forces, motions, and structures, but how these
joint-level phenomena are translated down to the tissue and
cellular levels also is discussed.
JOINT ANATOMY IN BIOMECHANICAL
TERMS
A joint is defined as a location where two bones meet and
are connected by soft tissue. The most common type of joint
in the body is a diarthodial (synovial) joint. Diarthodial
joints allow a large range of different types of motions and
include joints such as the knee, wrist, and shoulder. The
other types of joints in the body are fibrous joints, which
are connected by dense fibrous tissue and allow virtually
no relative movement, and cartilaginous joints, which are
connected entirely by cartilage and allow a small amount
of movement.
The knee represents a typical diarthrodial joint, compris-
ing ligaments, muscles and tendons, articular cartilage, joint
capsule, and synovial fluid. The ligaments connect the two
bones directly and, along with the bony geometry, provide
107
 108 BRIANT  |  Joint Biomechanics: The Role of Mechanics in Joint Pathology

passive support and stability for the joint during motion.
The articular cartilage covers the ends of each bone and
is a smooth, fluid-filled tissue that provides a low-friction
surface for smooth joint motion. The synovial fluid brings
nutrients to the articular cartilage and aids in lubricating
the joint.7 The muscles and tendons actively move the joint
and provide a large amount of active stability during physi-
however, the secondary motions (internal-external rotation
and anterior-posterior translation) also are active and are
important for normal knee function.10,11
The range of motion of each DOF is constrained by
the passive mechanical stiffness of the joint tissues, and
35 Flexion/Extension Rotation

Flexion (+)
ologic motion.8,9 This active stability is important because
the passive structures alone are often insufficient to provide 30
adequate joint stability during physiologic motion. In addi-
tion, the knee contains the meniscus, which is a fibrocarti- 25
lage structure that improves joint congruity.
20

JOINT MOTION

(°)
15
All joints have six independent movements, or degrees of
freedom (DOF). Three of these DOF are translations along 10
the anterior-posterior, medial-lateral, and inferior-superior
axes defined at the joint (Fig. 6-1), whereas the other three 5

Extension (-)
DOF are rotations around each axis. Because of the deform-
able nature of joint soft tissues and the inherent laxity 0
within joints, movement occurs in all six DOF during physi- 10 20 30 40 50 60 70 80 90 100
ologic motion (although most joints have a limited number -5
of DOF with a large range of motion). Figure 6-2 shows plots
of the flexion-extension rotation, anterior-posterior trans- 3.0
Anterior/Posterior Translation
lation, and internal-external rotation at the knee during a
single walking cycle captured for a normal individual dur-
Posterior

2.5
ing gait analysis. The primary motion is flexion-extension;
2.0

1.5

1.0
Inferior-superior
(cm)

translation 0.5

0
10 20 30 40 50 60 70 80 90 100
-0.5
Anterior

Internal-external -1.0
rotation
Internal/External Rotation
-1.5

6
External (+)

Varus-valgus
rotation Flexion-extension 4
rotation

2
Anterior-posterior
Medial-lateral
translation
translation 0
(°)

10 20 30 40 50 60 70 80 90 100
-2

-4
Internal (-)

-6

-8 Percent Stance Phase (%)

Figure 6-1  The six degrees of freedom of the knee joint. Rotation
­(flexion-extension) around the medial-lateral axis is the primary degree Figure 6-2  Plots of flexion-extension rotation, anterior-posterior trans-
of freedom; however, the other degrees of freedom also are important lation, and internal-external rotation degrees of freedom of the tibia with
for normal physiologic motion. respect to the femur during the stance phase of walking.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
d F
c
Figure 6-3  The small anterior displacement (d) results in a large change
in contact location (c), owing to the curvature of the knee.
e­ xcessive motion beyond the natural range of motion for a
joint is a common cause of acute joint pathology (i.e., liga-
ment tear). Subtle, long-term changes in joint motion also
are important in joint pathology, especially chronic diseases
such as osteoarthritis. As a result of the curvature of the
knee geometry, small changes in joint kinematics, especially
the secondary motions,12,13 can greatly alter the cartilage
contact locations between the femur and the tibia (Fig.
6-3). This shift in contact alters the mechanical loads applied
to the cartilage and over time may elicit a biologic response
of the chondrocytes leading to tissue degeneration.14,15
JOINT LOADS
During the above-described physiologic joint motions,
forces are developed at the joints, which must be sustained
by the joint tissues. Tissues such as ligaments and tendons
undergo primarily tensile forces, whereas cartilage under-
goes primarily compressive forces. The forces and motions
are generated by muscle forces, inertial (mass) forces of
the body segments, external forces (e.g., a foot contacting
the ground), and friction forces. Joint loads during ambu-
lation in the lower limbs can significantly exceed body
weight by several orders as a result of upward accelerations
against gravity and muscle cocontraction.2 Muscle cocon-
traction occurs when antagonistic muscle groups create
equal force in opposite directions, resulting in no joint
movement, but high joint forces. Cocontraction occurs
frequently during ambulation and provides increased
active joint stability.
Each of the six kinematic DOF has a loading DOF associ-
ated with it. The three translational loads are referred to as
forces and consist simply of a load causing an object to trans-
late (move in a straight line). The loads causing rotational
motion are called moments (torques). A planar moment is
calculated as the product of the force magnitude causing
the rotation times the perpendicular distance between the
line of the force and the center of rotation. The moment
created at the knee by an external horizontal force at the
ground is calculated as the magnitude of the force times the
109
M = Fxd
Figure 6-4  An adduction moment (M) at the knee is produced by the
product of the horizontal component of the ground reaction force (F)
multiplied by the perpendicular distance (d) from its line of action to the
knee center.
p­ erpendicular distance from the knee to the line of action
of the force (Fig. 6-4).
The net joint loads for all six DOF are determined through
experimental gait analysis. An analysis of the joint loads can
provide insight into the role of mechanics in disease ini-
tiation and progression. Studies have shown the adduction
moment at the knee during walking to be a strong predic-
tor for the progression of medial compartment knee osteo-
arthritis.16 The adduction moment is the moment around
the anterior-posterior axis of the knee, and an increase in
adduction moment produces a more varus alignment and
increases the load on the medial compartment of the knee
(Fig. 6-5). Osteoarthritis patients with a higher adduction
moment during walking tended to have thinner cartilage
on the medial compartment relative to the lateral compart-
ment, suggesting that load is detrimental to osteoarthritic
cartilage.1 For healthy patients, an increased adduction
moment has been associated with thicker medial compart-
ment knee cartilage. Healthy cartilage seems to respond dif-
ferently to load than osteoarthritic cartilage, although the
mechanisms causing this switch in load response are cur-
rently unknown.1,16 Mechanical loads play a complex role
in joint disease initiation and progression.
Most loads applied to joint tissues are almost entirely
compressive loads (cartilage), or tensile loads (tendon/liga-
ment). There can be a small amount of friction applied to
the tissues, however, as a result of sliding motion at joints.
Healthy cartilage is very smooth, resulting in very little shear-
ing loads applied to the surface, despite the large amount
of sliding that can occur. It has been suggested, however,
that as the cartilage degenerates during osteoarthritis, the
cartilage surface roughens owing to fibrillation, increasing
the friction at the surface.1 This increase in friction would
alter the mechanical loads applied to the chondrocytes and
elicit a change in cell metabolism. Shear loads applied to
chondrocytes in culture have caused an increase in enzymes
 110 BRIANT  |  Joint Biomechanics: The Role of Mechanics in Joint Pathology
Center
of mass
Adduction
moment
Ground reaction
A force
Figure 6-5  A, The adduction moment at the knee tends to cause the knee to be more varus (bow-legged). The moment is caused by the offset
­ etween the center of mass of the individual and the reaction force at the ground. B, An increase in adduction moment causes an increase in load on
b
the medial compartment.
known to degrade the cartilage matrix,4,14 which may be
responsible for the negative correlation between load and
cartilage thickness in osteoarthritic cartilage.1
JOINT BIOMECHANICS AT A TISSUE LEVEL
The joint loads calculated in the preceding discussion are
the net loads that must occur at the joint to produce the
motion of the adjoining limb segments. These loads are
applied directly to the joint tissues, which deform according
to their mechanical properties. The deformation of the joint
tissues determines the loads applied to individual matrix
elements and the cells within the tissue, both of which may
influence the health of the tissue.
BASIC TISSUE MECHANICS
When a force is applied to a tissue, it deforms over time
by either elongating or compressing. The deformation that
occurs depends on the area the force is applied over and
the size of the tissue. For this reason, the loads applied to
tissues are best described by the quantity stress (force per
unit area), whereas the deformation is described by strain
(deformation per unit length). The ratio between the applied
Medial load
Lateral load
B Aduction moment
stress to resulting strain (force to deformation) is called the
tissue stiffness. Stiffness is an important structural property
for joint tissues and provides a measure of how much a tissue
would deform under a given force. The higher the stiffness
of a tissue, the less it would deform under the same load.
The stiffness depends on internal properties of the tissue,
including the tissue composition, and the organization of
the constituents. A tissue with a highly organized collagen
matrix is likely to have a higher tensile stiffness than a tissue
with a more random collagen organization.
The tissue mechanical properties also are inhomoge-
neous, meaning they vary from point to point within the
tissue,17,18 and anisotropic, meaning the properties vary
with direction. Such inhomogeneity and anisotropy are due
to the spatial variations of the tissue constituents and may
reflect an adaptation of the tissue to the local mechanical
environment.17,19,20 The collagen matrix organization is
one of the most important factors governing the responses
of joint tissues to mechanical load.18 The organization of the
collagen seems to be highly associated with the mechanical
loads applied to the tissue, especially the tensile load.17 Lig-
aments and tendons undergo almost entirely tensile forces
along the length of their main axis and, correspondingly,
have a highly aligned collagen matrix along the axis. This
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
gives them a very high tensile stiffness along their axis, but
a very low stiffness if they are loaded perpendicular to their
axis.
The collagen organization in cartilage also seems to be
associated with the tensile loads, although the load distribu-
tion and extracellular matrix organization are more complex
and not as well understood. In general, the collagen fibers in
cartilage are predominantly tangential to the surface in the
superficial zone, but transition to be perpendicular to the
surface in the deep layer (Fig. 6-6).5 Topographic analyses of
tibial plateau cartilage have shown, however, that the colla-
gen matrix in the superficial zone varies from highly aligned
in the peripheral regions to more randomly organized in
the central region (Fig. 6-7).17,21 This varied organization
may be due to the nonuniform mechanical loads within the
cartilage. The compressive loads applied to the tibial carti-
lage have been shown to be highest near the center of the
plateau, and lower near the periphery.22 A simple noncon-
forming contact analysis of the knee joint surfaces suggests
that under such loading, the stress in the superficial zone is
compressive in the central, high-load region of the cartilage,
but transitions to tensile near the periphery.23,24 Because it
is known that the primary function of collagen in cartilage
is to resist tensile forces,18,19 the higher tensile loads in the
peripheral region may be responsible for the more organized
collagen. Both of these examples show the apparent condi-
tioning of joint tissues to mechanical load,1,5 and highlight
the importance of mechanical load in the development and
maintenance of healthy joints.5
In addition to being inhomogeneous and anisotropic,
joint tissues are viscoelastic, meaning their stiffness depends
on the rate of applied loading. This viscoelasticity is due
primarily to the fluid flow that occurs within tissues under
load. In addition to collagen, which is the primary constitu-
ent of most joint tissues, cartilage contains proteoglycans,
Articular surface
Superficial
zone
Transitional
zone
Deep zone
Figure 6-6  The collagen fiber arrangement in normal articular carti-
lage. The fibers transition from being parallel to the articular surface in
the tangential zone to perpendicular to the surface in the radial zone.
111
which draw water into the tissue because of their strong
electrostatic charge. This water creates an osmotic gradient,
causing the tissue to swell, which is resisted by the colla-
gen matrix.25 When the cartilage is loaded compressively,
the superficial zone collagen matrix is thought to collapse,
effectively sealing the water inside the cartilage, causing the
water to bear much of the compressive load, rather than the
collagen matrix.26 Because water is nearly incompressible,
this process gives cartilage a very high dynamic (short-term)
compressive stiffness. Over time, however, the water is able
to “seep out,” effectively decreasing steady-state (long-term)
stiffness.
JOINT TISSUE DEGENERATION
The effect of mechanics on joint tissues is important to
understand because most joint diseases result in degradation
of the joint tissue matrix. Damage to any joint tissue matrix
causes a biologic response, whether the triggering event is
acute or chronic, and potentially can lead to further tissue
degeneration, potentially at an anatomically distinct loca-
tion. During anterior cruciate ligament rupture, subluxation
at the knee often occurs, damaging the underlying bone and
causing the response of acute bone marrow edema under the
lateral tibial plateau.27 Chronic collagen fibrillation is a more
subtle change, but also can alter the mechanics of cartilage
by allowing fluid to escape when the cartilage is loaded and
increasing the friction at the surface.1 Such changes poten-
tially can cause long-term metabolic changes leading to
chronic joint disease.28 In addition, proinflammatory cyto-
kines, such as interleukin-1 and tumor necrosis factor-α,
cause the release of matrix metalloproteinases 1 and 13 in
cartilage,29 which break down the matrix further. Nonme-
chanical factors such as joint inflammation can cause tis-
sue degeneration and degrade the ability of the cartilage to
sustain mechanical loads, ultimately leading to more rapid
degradation.
JOINT BIOMECHANICS
AT A CELLULAR LEVEL
CELLULAR LOADS AND DEFORMATIONS
The cells in joint structures are responsible for maintain-
ing the extracellular matrix through an active balance of
tissue production and resorption. This balance is regulated
partly by the mechanical loads applied to the cells, through
the process of mechanotransduction. The loads applied to
the cells are generated by deformation of the extracellular
matrix, interstitial fluid pressurization, and interstitial fluid
flow.
Understanding the role of the cells in the initiation and
progression of joint pathologies is challenging because nei-
ther the loads applied to the cells in vivo nor the fundamen-
tal mechanisms by which cells respond to load are currently
known. Determining the in vivo loads applied to the cells
requires bridging many length scales, from the macroscopic
joint loads, down to the tissue loads, and finally to the
cells. One study has attempted to determine chondrocyte
deformation in situ by tracking the cells during compres-
sion of cartilage explants30; however, knowing the cellular
deformation does not elucidate all of the loads applied to
 112 BRIANT  |  Joint Biomechanics: The Role of Mechanics in Joint Pathology
Figure 6-7  A and B, The superficial zone collagen
in the region adjacent to the direct load (A) is thicker
and substantially more organized, with more parallel B
­collagen fibers than the directly loaded central region
(B) with only pockets of parallel fibers.
the cells. To determine how cells respond to load, culture
experiments can be used to apply specific loads to cells and
measure the metabolic response patterns.3,4 Future studies
are needed to determine the cellular deformations and cel-
lular responses to load in more physiologic conditions.
ROLE OF CELLS IN JOINT PATHOLOGY
It is now recognized that cellular pathways comprising local
tissue fibroblasts, chondrocytes, and osteoclasts/osteoblasts
and infiltrating leukocyte populations are crucial in the
pathogenesis of a variety of articular conditions (precise
details are provided in chapters detailing the pathogenesis
of distinct conditions and are not directly relevant to this
discussion). Most acute joint pathologies, such as a ligament
rupture or strain, are caused by direct mechanical overload
and do not involve cells in the short term, although it is
increasingly recognized that even in this acute event there
is a contribution from local tissue mesenchymal cells and
invading cells of the innate immune response as the lesion
progresses. Changes that occur over a long time, such as
in rheumatoid arthritis or osteoarthritis associated with
Superficial
zone
thickness
Superficial
zone
thickness
increased tissue laxity, may be caused, however, by direct
mechanical damage to the extracellular matrix or through
enzymatic degradation as a result of changes in cellular
metabolism (e.g., the release of matrix metalloproteinases
by synovial fibroblasts). Distinguishing between these two
effects is important for determining the underlying cau-
sality of the various components of the pathologic lesion.
Determining the pathways by which cells respond to their
mechanical environment would enhance the development
of new treatments for joint pathologies, including tissue-
engineered approaches. The latter, if achieved, would repre-
sent a novel complementary approach to the management
of complex articular disorders.
IMPORTANCE TO RHEUMATOLOGISTS
Joint mechanics play an important role in the initiation and
the progression of many joint pathologies. One of the fun-
damental aspects of joint biomechanics is the relationship
across scales, from the macroscopic joint motions and loads
to the microscopic matrix and cellular responses. Owing to
the close coupling of these components, changes to any one
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
of them would alter the overall joint mechanics and joint
function.
In addition to helping understand the cause of many joint
diseases, joint biomechanics are important because many
treatments involve altering the mechanical loads applied
to joint tissues to relieve symptoms. Examples include high
tibial osteotomy21 or modified footwear orthotics, which
are designed to reduce load on the medial compartment to
slow the progression of medial compartment knee osteoar-
thritis. Although it has been shown in clinical trials that
such interventions are often successful,21 the fundamental
mechanisms causing the symptomatic relief on a tissue and
cellular level are currently unknown. A proper understand-
ing of the role of mechanics, ranging from the joint level
to the cellular level, would allow for the development of
improved treatment methods and techniques.
REFERENCES
1. Andriacchi TP, Mundermann A, Smith RL, et al: A framework
for the in vivo pathomechanics of osteoarthritis at the knee. Ann
Biomed Eng 32:447-457, 2004.
2. Schipplein OD, Andriacchi TP: Interaction between active and pas-
sive knee stabilizers during level walking. J Orthop Res 9:113-119,
1991.
3. Das P, Schurman DJ, Smith RL: Nitric oxide and G proteins mediate
the response of bovine articular chondrocytes to fluid-induced shear.
J Orthop Res 15:87-93, 1997.
4. Smith RL, Trindade MC, Ikenoue T, et al: Effects of shear stress on
articular chondrocyte metabolism. Biorheology 37:95-107, 2000.
5. Carter DR, Beaupré GS: Skeletal Function and Form: Mechanobiol-
ogy of Skeletal Development, Aging, and Regeneration. Cambridge,
Cambridge University Press, 2000.
6. Prodromos CC, Andriacchi TP, Galante JO: A relationship
between gait and clinical changes following high tibial osteotomy.
J Bone Joint Surg Am 67:1188-1194, 1985.
7. Jay GD, Haberstroh K, Cha CJ: Comparison of the boundary-
­lubricating ability of bovine synovial fluid, lubricin, and Healon.
J Biomed Mater Res 40:414-418, 1998.
8. Harrington IJ: Static and dynamic loading patterns in knee joints with
deformities. J Bone Joint Surg Am 65:247-259, 1983.
9. Hsieh HH, Walker PS: Stabilizing mechanisms of the loaded and
unloaded knee joint. J Bone Joint Surg Am 58:87-93, 1976.
10. Andriacchi TP, Alexander EJ, Toney MK, et al: A point cluster
method for in vivo motion analysis: Applied to a study of knee kine-
matics. J Biomech Eng 120:743-749, 1998.
11. Lafortune MA, Cavanagh PR, Sommer HJ 3rd, et al: Three-dimen-
sional kinematics of the human knee during walking. J Biomech
25:347-357, 1992.
12. Andriacchi TP, Briant PL, Bevill SL, et al: Rotational changes at the
knee after ACL injury cause cartilage thinning. Clin Orthop 442:
39-44, 2006.
113
13. Andriacchi TP, Dyrby CO: Interactions between kinematics and load-
ing during walking for the normal and ACL deficient knee. J Biomech
38:293-298, 2005.
14. Frank EH, Jin M, Loening AM, et al: A versatile shear and compres-
sion apparatus for mechanical stimulation of tissue culture explants.
J Biomech 33:1523-1527, 2000.
15. Gray ML, Pizzanelli AM, Grodzinsky AJ, et al: Mechanical and phys-
iochemical determinants of the chondrocyte biosynthetic response.
J Orthop Res 6:777-792, 1988.
16. Miyazaki T, Wada M, Kawahara H, et al: Dynamic load at baseline
can predict radiographic disease progression in medial compartment
knee osteoarthritis. Ann Rheum Dis 61:617-622, 2002.
17. Briant PL, Bevill SL, Torzilli PA, et al: Collagen organization in the
superficial layer of articular cartilage relative to the mechanical envi-
ronment. American Society of Mechanical Engineers, Summer Bio-
engineering Conference, Vail, Colorado, 2006, 152691.
18. Laasanen MS, Toyras J, Korhonen RK, et al: Biomechanical properties
of knee articular cartilage. Biorheology 40:133-140, 2003.
19. Akizuki S, Mow VC, Muller F, et al: Tensile properties of human knee
joint cartilage, I: Influence of ionic conditions, weight bearing, and
fibrillation on the tensile modulus. J Orthop Res 4:379-392, 1986.
20. Farquhar T, Dawson PR, Torzilli PA: A microstructural model for
the anisotropic drained stiffness of articular cartilage. J Biomech
Eng 112:414-425, 1990.
21. Appleyard RC, Burkhardt D, Ghosh P, et al: Topographical analy-
sis of the structural, biochemical and dynamic biomechanical prop-
erties of cartilage in an ovine model of osteoarthritis. Osteoarthritis
Cartilage 11:65-77, 2003.
22. Baratz ME, Fu FH, Mengato R: Meniscal tears: The effect of meniscec-
tomy and of repair on intraarticular contact areas and stress in the human
knee: A preliminary report. Am J Sports Med 14:270-275, 1986.
23. Johnson KL: Contact Mechanics. Cambridge, Cambridge University
Press, 1985.
24. Wilson W, Driesson NJB, van Donkelaar CC, et al: Prediction of col-
lagen orientation in articular cartilage by a collagen remodeling algo-
rithm. Osteoarthritis Cartilage 14:1196-1202, 2006.
25. Maroudas AI: Balance between swelling pressure and collagen tension
in normal and degenerate cartilage. Nature 260:808-809, 1976.
26. Soltz MA, Ateshian GA: Experimental verification and theoretical
prediction of cartilage interstitial fluid pressurization at an imper-
meable contact interface in confined compression. J Biomech 31:
927-934, 1998.
27. Johnson DL, Urban WP Jr, Caborn DNM, et al: Articular cartilage
changes seen with magnetic resonance imaging-detected bone bruises
associated with acute anterior cruciate ligament rupture. Am J Sports
Med 26:409-414, 1998.
28. Grodzinsky AJ, Levenston ME, Jin M, et al: Cartilage tissue remod-
eling in response to mechanical forces. Annu Rev Biomed Eng 2:
691-713, 2000.
29. Shi J, Schmitt-Talbot E, DiMattia DA, et al: The differential effects
of IL-1 and TNF-α on proinflammatory cytokine and matrix metal-
loproteinase expression in human chondrosarcoma cells. Inflamm Res
53:377-389, 2004.
30. Guilak F, Ratcliffe A, Mow VC: Chondrocyte deformation and local
tissue strain in articular cartilage: A confocal microscopy study.
J Orthop Res 13:410-421, 1995.
 7 Proteinases and Matrix
Key Points
Proteinases are generally classified into aspartic
proteinases, cysteine proteinases, serine proteinases, and
metalloproteinases according to catalytic mechanism.
Because of the acidic pH optima and intracellular localization
within lysosomes, most of the aspartic proteinases and
cysteine proteinases are involved in intracellular degradation
of extracellular matrix (ECM) components.
Serine proteinases and metalloproteinases are neutral
proteinases and play a central role in extracellular
degradation of ECM macromolecules.
ECM-degrading metalloproteinases are composed of the
MMP (matrix metalloproteinase) and ADAMTS (a disintegrin
and metalloproteinase with thrombospondin motifs) gene
families.
Endogenous proteinase inhibitors are proteinase class
specific, whereas α2-macroglobulin inhibits the activities of all
proteinases.
The activities of ECM-degrading proteinases at the local
tissues are regulated by the balance between the proteinases
and their inhibitors, which may be determined by production
rates of proteinases and inhibitors, their secretion, activation
of proenzymes, and anchoring systems of the activated
proteinases to cell surfaces.
ProMMPs (zymogens of MMPs) are activated via the
extracellular, intracellular, and pericellular pathways
depending on the MMP species.
Aggrecan and type II collagen, two major ECM components,
in articular cartilage may be degraded by differential or
complementary actions of the MMP and ADAMTS species in
arthritides.
In rheumatoid arthritis, articular cartilage is destroyed by
proteinases accumulated in synovial fluid, direct contact of
proteolytic synovium and pannus tissue, and proteinases
derived from chondrocytes, whereas bone is resorbed by
osteoclasts mainly by the action of cathepsin K and MMP-9
under acidic and hypercalcemic conditions in subosteoclastic
compartments.
In osteoarthritis, chondrocyte-derived metalloproteinases,
including the MMP and ADAMTS species, contribute primarily
to the breakdown of articular cartilage.
Extracellular matrix (ECM) plays critical roles in normal
development and function of the organism by interacting
with cells and supporting tissue structures. The in vivo cel-
lular functions regulated by cell-ECM interaction include
proliferation, differentiation, apoptosis, and motility. The
proteolytic turnover and remodeling of ECM is transient
Degradation
Yasunori Okada
and highly controlled under physiologic conditions, and
excessive degradation of ECM components by proteinases
causes tissue destruction in many pathologic conditions.
In rheumatoid arthritis and osteoarthritis, ECM-­degrading
proteinases are elevated without sufficient endogenous
inhibitors, and they are believed to play central roles in the
destruction of articular cartilage and bone on the basis of
a local imbalance between proteinases and inhibitors. This
chapter provides up-to-date information about the ECM-
degrading proteinases and their inhibitors. Developments
in the areas of proteinases and matrix degradation occur-
ring since the seventh edition of this book was published
in 2005 are discussed. Chapters from earlier editions offer a
more comprehensive coverage of the older literature.1,2
EXTRACELLULAR MATRIX–DEGRADING
PROTEINASES
ECM is degraded by endopeptidases (i.e., proteinases) that
act internally on polypeptide chains; little evidence is pres-
ent for the roles of exopeptidases that cleave one or a few
amino acids from the N or C terminus. Proteinases comprise
aspartic proteinases, cysteine proteinases, serine proteinases,
and metalloproteinases, which are classified according to cat-
alytic mechanism. Proteinases from each of the four classes
are involved in the degradation of ECM macromolecules.
ASPARTIC PROTEINASES
Most aspartic proteinases have two aspartic acid residues in
their catalytic sites, where the nucleophile that attacks the
scissile peptide bond is an activated water molecule. Among
the proteinases belonging to this group, cathepsin D is the
major aspartic proteinase involved in ECM degradation
(Table 7-1). It exhibits proteolytic activity against most
substrates, such as aggrecan and collagen telopeptides, with
pH optima between pH 3.5 and 5.0. Because of the acidic
pH optima and intracellular localization within lysosomes,
cathepsin D is probably responsible for intracellular deg-
radation of phagocytosed ECM fragments that previously
were degraded in the extracellular spaces. A study on carti-
lage explant cultures using the aspartic proteinase inhibitor
suggests, however, the possibility that cathepsin D secreted
extracellularly contributes to the degradation of aggrecan in
articular cartilage.3
CYSTEINE PROTEINASES
Cysteine proteinases are endopeptidases in which the
nucleophile of the catalytic site is the sulfhydryl group of a
cysteine residue. The ECM-degrading cysteine ­ proteinases
115
 116 OKada  |  Proteinases and Matrix Degradation

Table 7-1  Proteinases That May Be Involved in Degradation of Extracellular Matrix

Enzyme Molecular Mass (kD) Source Inhibitor
Aspartic Proteinases
Cathepsin D 34 Lysosome Pepstatin
Cysteine Proteinases
Cathepsin B 25 Lysosome Cystatins
Cathepsin L 24 Lysosome Cystatins
Cathepsin S 24 Lysosome Cystatins
Cathepsin K 29 Lysosome Cystatins
Calpain 110 (80 +30) Cytosol Calpastatin
Serine Proteinases
Neutrophil elastase 30 Neutrophils α1-PI
Cathepsin G 30 Neutrophils α1-Antichymotrypsin
Proteinase 3 29 Neutrophils α1-PI, elafin
Plasmin 94 Plasma Aprotinin
Plasma kallikrein 88/85 Plasma Aprotinin
Tissue kallikrein 46 Glandular tissues Aprotinin; kallistatin
tPA 67-72 Endothelial cells; chondrocytes PAI-1; PAI-2
uPA 54/33 Fibroblasts; chondrocytes PAI-1; PAI-2; PN-1
Tryptase 30-35 Mast cells Trypstatin
Chymase 26 Mast cells α1-PI
MMPs
Secreted-type MMPs
Collagenases
Interstitial collagenase (MMP-1) 52/56* Fibroblasts; synovial cells; chondrocytes;   TIMPs
macrophages; endothelial cells; cancer cells
Neutrophil collagenase (MMP-8) 75* Neutrophils TIMPs
Collagenase-3 (MMP-13) 65 Chondrocytes; breast carcinoma cells TIMPs
Gelatinases
Gelatinase A (MMP-2) 72 Fibroblasts; chondrocytes; mesangial cells;   TIMPs; RECK
macrophages; endothelial cells
Gelatinase B (MMP-9) 92* Neutrophils; macrophages; osteoclasts;   TIMPs
trophoblasts; T lymphocytes; cancer cells
Stromelysins
Stromelysin-1 (MMP-3) 57/59* Synovial cells; chondrocytes; fibroblasts TIMPs
Stromelysin-2 (MMP-10) 56 Cancer cells; T lymphocytes TIMPs
Matrilysins
Matrilysin-1 (MMP-7) 28 Cancer cells; chondrocytes; macrophages;   TIMPs
mesangial cells; gland cells
Matrilysin-2 (MMP-26) 28 Placenta; endometrium TIMPs
Furin-activated MMPs
Stromelysin-3 (MMP-11) 58 Cancer stromal cells TIMPs
Epilysin (MMP-28) 56 Epidermis; testis; lung; cancer cells TIMPs
Other secreted-type MMPs
Metalloelastase (MMP-12) 54 Macrophages TIMPs
RASI-1 (MMP-19) 57 Synovial cells; ovary TIMPs
Enamelysin (MMP-20) 54 Enameloblast TIMPs
MMP-21 Unknown Unknown Unknown
MMP-27 Unknown Unknown Unknown
Membrane-anchored MMPs
Type I transmembrane-type MMPs
MT1-MMP (MMP-14) 66 Cancer cells; glioma cells; chondrocytes;   TIMPs except for TIMP-1;
fibroblasts; synovial cells RECK
MT2-MMP (MMP-15) 68 Cancer cells; glioma cells TIMPs except for TIMP-1
MT3-MMP (MMP-16) 64 Glioma cells TIMPs except for TIMP-1
MT5-MMP (MMP-24) 63 Brain; glioma cells Unknown
GPI-linked MMPs
MT4-MMP (MMP-17) Unknown Unknown Unknown
MT6-MMP (MMP-25) Unknown Leukemia cells Unknown
Type II transmembrane-type MMPs
MMP-23 Unknown Ovary; endometrium; testis; prostate Unknown

*Glycosylated form.
MMP-4, MMP-5, and MMP-6 are missing MMPs.
MMP-18 (Xenopus collagenase 4; from Stolow MA, et al: Identification and characterization of a novel collagenase in Xenopus laevis: possible roles during
frog development. Mol Biol Cell 7:1471, 1996), and MMP-22 (chick MMP; from Yang M, Kurkinen M: Cloning and characterization of a novel matrix metallo-
proteinase CMMPJ, CMMP, from chicken embryo fibroblasts. CMMP, Xenopus XMMP, and human MMP19 have a conserved unique cysteine in the catalytic
domain. J Biol Chem 273:17893, 1998) are not mammalian and thus are omitted from this list.
PAI, plasminogen activator inhibitor; PI, proteinase inhibitor, PN, proteinase nexin; RECK, reversion-inducing, cysteine-rich protein with Kazal motifs; TIMP,
tissue inhibitor of metalloproteinases; tPA, tissue-type plasminogen activator; uPA, urokinase-type plasminogen activator.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
include lysosomal cathepsins B, L, S, and K and the calpains
(see Table 7-1). Cathepsins B and L digest the telopeptide
regions of fibrillar collagen types I and II, the nonhelical
regions of collagen types IX and XI, and aggrecan at acidic
pH.1 Cathepsin S has a similar spectrum of substrates
within a broad range of pH values. Cathepsin K, also
called cathepsin O, O2, or X, is a ­collagenolytic cathepsin
that efficiently cleaves type I collagen at the triple helical
regions at pH values between 4.5 and 6.6.4 The proteinase
also degrades gelatin and osteonectin. Because cathepsins
B, L, S, and K are expressed in synovium or articular car-
tilage or both in rheumatoid arthritis and osteoarthritis,
they may be involved in the cartilage destruction through
degradation of the ECM macromolecules. Cathepsin K
also plays a key role in bone resorption in such joint dis-
eases (see later).
Calpains are Ca2+-dependent, papain-like cysteine
proteinases and are ubiquitously distributed among mam-
malian cells. The best-characterized members of the
calpain superfamily are μ-calpain and m-calpain, which
also are called conventional (μ-calpain) and classic
(m-calpain) calpains.5 Calpains are involved in various
pathologic conditions, such as muscle dystrophy, by act-
ing intracellularly. They are present in the extracellular
spaces and in osteoarthritic synovial fluid, and they can
degrade aggrecan.
SERINE PROTEINASES
Serine proteinases require the hydroxyl group of a serine
residue acting as the nucleophile that attacks the peptide
bond. They include the largest number of proteinases
classified to about 40 families. Most can degrade ECM
macromolecules. The major ECM-degrading serine pro-
teinases in joint tissues are subsequently described (see
Table 7-1).
Neutrophil Elastase and Cathepsin G
Neutrophil elastase and cathepsin G are serine proteinases
that are synthesized as precursors in promyelocytes in bone
marrow and subsequently stored in the azurophil granules
of polymorphonuclear leukocytes as active enzymes. Mature
leukocytes do not synthesize elastase, but they mobilize
azurophil granules to the cell surface and release the pro-
teinases in response to various stimuli. Monocytes have low
levels of elastase, but lose the enzyme during the differentia-
tion into macrophages. Neutrophil elastase and cathepsin G
are basic glycoproteins with isoelectric points larger than 9
(neutrophil elastase) and about 12 (cathepsin G). They can
be readily trapped in cartilage matrix that has a negative
charge.
Neutrophil elastase and cathepsin G cleave elastin; the
telopeptide region of fibrillar collagen types I, II, and III;
other collagen types IV, VI, VIII, IX, X, and XI; and other
ECM components, such as fibronectin, laminin, and aggre-
can at neutral pH. These serine proteinases also can be
involved indirectly in the breakdown of ECM by activating
the zymogen of pro-matrix metalloproteinases (proMMPs)6
and by inactivating endogenous proteinase inhibitors, such
as α2-antiplasmin, α1-antichymotrypsin, and tissue inhibi-
tors of metalloproteinases (TIMPs).
117
Mast Cell Chymase and Tryptase
Chymase and tryptase are packaged in secretory granules
together with histamine and other mediators in mast cells,
which are infiltrated in rheumatoid synovium. Chymase is a
chymotrypsin-like proteinase with a broad spectrum of activ-
ity against ECM components such as type VI collagen7 and
aggrecan. It also activates proMMPs, such as proMMP-1,
proMMP-3, and proMMP-9.6 Although prochymase is acti-
vated intracellularly and stored in the granules, the activity
in the granules is limited at low pH and becomes fully active
when released extracellularly. Tryptase is a trypsin-like pro-
teinase that degrades collagen type VI7 and fibronectin; it
also activates proMMP-3.6
Plasmin and Plasminogen Activators
Plasminogen is synthesized in liver and secreted to plasma.
It can bind to fibrin and to cells, and after activation by
plasminogen activators, plasmin readily digests fibrin. Mem-
brane-bound plasmin also degrades many ECM components,
including proteoglycan, fibronectin, type IV collagen, and
laminin.8 Another important function of plasmin is to
initiate the activation of proMMPs,6 activate latent cell-
­associated transforming growth factor (TGF)-β1, and act as
proenzyme convertase. Plasmin is generated through acti-
vation of plasminogen mainly by plasminogen activators,
principally two serine proteinases, tissue-type ­plasminogen
activator (tPA) and urokinase-type plasminogen activator
(uPA).
The tPA is synthesized as a proenzyme of 70 kD and is
secreted into the circulating blood primarily by endothelial
cells, fibroblasts, chondrocytes, and tumor cells.8 In addition
to being a major activator of plasminogen for fibrinolysis,
tPA plays a key role in the clearance of fibrin from the cir-
culation.
The uPA molecule was first purified from urine as a
proenzyme of 54 kD.8 It is converted to the active form of
two chains of 30 kD and 24 kD linked by a disulfide bond.
Another fully active form of 33 kD is generated by plasmin.
Although the expression of uPA is limited to certain cells
such as renal tubules and bladder urothelium under physi-
ologic conditions, it is more widely expressed in various
cells, including invasive cancer cells, migrating keratino-
cytes, and activated leukocytes, in pathologic situations.
Pro-uPA and two-chain uPA bind to a specific uPA receptor,
a single-chain glycoprotein with a glycosylphosphatidylino-
sitol (GPI) moiety expressed on fibroblasts, macrophages,
and tumor cells. Receptor-bound uPA preferentially acti-
vates cell membrane–bound plasminogen into plasmin. Cell
membrane–bound plasmin can activate receptor-bound
pro-uPA. Among its specificities, uPA has a limited action
on fibronectin.
Kallikreins
Two types of kallikreins, plasma and tissue kallikreins, are
known. Plasma kallikrein, with two disulfide-linked chains
(36 kD and 52 kD), is generated from prokallikrein of 88 kD
by coagulation factor XIIa or by kallikrein itself. It activates
kininogens to bradykinin and activates proMMP-1 and
proMMP-3.6 Tissue kallikrein is synthesized in glandular
 118 OKada  |  Proteinases and Matrix Degradation

tissues. It releases Lys-bradykinin from kininogen and acti-
vates proMMP-8.6
METALLOPROTEINASES
Similar to aspartic proteinases, metalloproteinases are endo-
peptidases in which the nucleophilic attack on a peptide
bond is mediated by a water molecule. A divalent metal cat-
ion, usually zinc, activates the water molecule. Among the
metalloproteinase groups, MMPs (matrix metalloprotein-
ases), which are also designated matrixins (a subfamily of
the metzincin superfamily), are key ECM-degrading, zinc-
dependent endopeptidases (Table 7-2; see Table 7-1). More
recently accumulated evidence indicates, however, that
some members of the ADAMTS (a disintegrin and metal-
loproteinase with thrombospondin motifs) family, which is
an MMP-related gene family, also are involved in ECM deg-
radation (Table 7-3).

Table 7-2  Substrates of Human Matrix Metalloproteinases

Enzymes ECM Substrates Non-ECM Substrates
Secreted-type MMPs
Collagenases
Interstitial collagenase (MMP-1) Collagens I, II, III, VII, X; gelatins; aggrecan; link protein; α2-Macroglobulin; α1-PI; α1-antichymotrypsin;  
entactin; tenascin; perlecan IGF-BP-2, -3, -5; pro-IL-1β; CTGF
Neutrophil collagenase (MMP-8) Collagens I, II and III; gelatins; aggrecan; link protein α1-PI
Collagenase-3 (MMP-13) Collagens I, II, III, IV, IX, X, XIV; aggrecan; Fn; tenascin CTGF; pro-TGF-β; α1-antichymotrypsin
Gelatinases
Gelatinase A (MMP-2)  Gelatins; collagens IV, V, VII, XI; Ln; Fn; elastin; aggrecan; Pro-TGF-β; FGF receptor I; MCP-3; IGF-BP-5;  
link protein pro-IL-1β; galectin-3; plasminogen
Gelatinase B (MMP-9) Gelatins; collagens III, IV, V; aggrecan; elastin; entactin;   Pro-TGF-β; IL-2 receptor α; Kit-L; IGF-BP-3;  
link protein pro-IL-1β; α1-PI; galectin-3
Stromelysins
Stromelysin-1 (MMP-3)  Aggrecan; decorin; gelatins; Fn; Ln; collagens III, IV, IX, X; IGF-BP-3; pro-IL-1β; HB-EGF; CTGF; E-cadherin;
  tenascin; link protein; perlecan  α1-­antichymotrypsin; α1-PI; α2-macroglobulin;
plasminogen; uPA
Stromelysin-2 (MMP-10) Aggrecan; Fn; Ln; collagens III, IV, V; link protein Unknown
Matrilysins
Matrilysin-1 (MMP-7)  Aggrecan; gelatins; Fn; Ln; elastin; entactin; collagen IV; Pro-α-defensin; Fas-L; β4 integrin; E-cadherin;  
tenascin; link protein pro-TNF-α; CTGF; HB-EGF
Matrilysin-2 (MMP-26) Gelatin; collagen IV; Fn; fibrinogen α1-PI
Furin-activated MMPs
Stromelysin-3 (MMP-11) Fn; Ln; aggrecan; gelatins α1-PI; α2-macroglobulin; IGF-BP-1
Epilysin (MMP-28) Unknown Unknown
Other Secreted-type MMPs
Metalloelastase (MMP-12) Elastin; Fn; collagen V; osteonectin Plasminogen; apolipoprotein A
RASI-1 (MMP-19)  Collagen IV; gelatin; Fn; tenascin; aggrecan; COMP; Ln; Unknown 
nidogen
Enamelysin (MMP-20) Amelogenin; aggrecan; gelatin; COMP Unknown
MMP-21 Unknown Unknown
MMP-27 Unknown Unknown
Membrane-anchored MMPs
Type I Transmembrane-type MMPs
MT1-MMP (MMP-14) Collagens I, II, III; gelatins; aggrecan; Fn; Ln; fibrin; Ln-5 CD44; tissue transglutaminase
MT2-MMP (MMP-15) Fn; tenascin; nidogen; aggrecan; perlecan; Ln Tissue transglutaminase
MT3-MMP (MMP-16) Collagen III; Fn; gelatin Tissue transglutaminase
MT5-MMP (MMP-24) PG Unknown
GPI-linked MMPs
MT4-MMP (MMP-17) Gelatin; fibrinogen Unknown
MT6-MMP (MMP-25) Gelatin; collagen IV; fibrin; Fn; Ln Unknown
Type II Transmembrane-type MMP
MMP-23 Gelatin Unknown
COMP, cartilage oligomeric matrix protein; CTGF, connective tissue growth factor; ECM, extracellular matrix; Fn, fibronectin; GPI, glycosylphosphatidylino-
sitol; HB-EGF, heparin-binding epidermal growth factor; IGF-BP, insulin-like growth factor binding protein; IL-1, interleukin-1; Ln, laminin; MCP, monocyte
­chemoattractant protein; PG, proteoglycan; PI, proteinase inhibitor; TGF, transforming growth factor; TNF, tumor necrosis factor; uPA, urokinase-type plas-
minogen activator.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE 119

Matrix Metalloproteinases C-terminal hemopexin-like domain, which is connected to

The human MMP family comprises 23 different members,
which have MMP designations (numbered according to a
sequential numbering system) and common names coined
by the authors of the published reports (see Tables 7-1 and
7-2). Based on the biochemical properties provided by the
domain structures and on their substrate specificity, these
family members are classified into two major subgroups:
secreted-type MMPs and membrane-anchored MMPs.
MMP-4, MMP-5, and MMP-6 are excluded from the list
because they are identical to other known MMPs (i.e.,
MMP-3 and MMP-2). MMP-18 and MMP-22 also are miss-
ing in Tables 7-1 and 7-2 because they are assigned to Xeno-
pus collagenase-4 and chicken MMP.
Most secreted-type MMPs, including collagenases, stro­
melysins, and other MMPs, are composed of three basic
domains—the propeptide, catalytic, and hemopexin-like
domains—that are preceded by hydrophobic signal pep-
tides (Fig. 7-1). The N-terminal propeptide domain has
one unpaired cysteine in the conserved sequence of PRC-
GXPD. The cysteine residue in the sequence interacts with
the essential zinc atom in the catalytic domain to prevent it
from binding the catalytic water molecule, maintaining the
proenzyme in an inactive state. The catalytic domain has
the zinc-binding motif HEXGHXXGXXH, in which three
histidines bind to the catalytic zinc atom. The four-blade,
the catalytic domain by a proline-rich hinge region, inter-
acts with ECM components and can play a role in deter-
mining the substrate specificity in some MMPs. Gelatinases
have these domains with additional insertions of colla-
gen-binding type II repeats of fibronectin in the catalytic
domain (see Fig. 7-1); this provides them with collagen-
binding properties. Matrilysins are the shortest, lacking the
hemopexin-like domains. Furin-activated MMPs contain
insertions of a basic motif with a cleavage site by propro-
tein convertases including furin at the end of the propep-
tide domains (see Fig. 7-1).
Membrane-anchored MMPs include three different types
of MMPs—type I transmembrane-type MMPs, GPI-linked
MMPs, and type II transmembrane-type MMP. MT1-,
MT2-, MT3-, and MT5-MMPs have type I transmembrane
domains in the C-terminal region, but MT4- and 6-MMPs
­contain GPI anchors in the C-terminal region without
transmembrane domains (see Fig. 7-1). MMP-23 is unique
in that it has type II transmembrane domain, a cysteine
array, and an immunoglobulin-like domain instead of a
hemopexin-like domain (see Fig. 7-1).
Secreted-type Matrix Metalloproteinases
Collagenases (MMP-1, MMP-8, and MMP-13). The col-
lagenases include MMP-1 (interstitial collagenase, collage-
nase-1), MMP-8 (neutrophil collagenase, collagenase-2),

Table 7-3  Members of the ADAMTS Gene Family

Demonstration of Functions and Biochemical
ADAMTS Other Names Proteinase Activity* Features Tissue and Cell Expression
ADAMTS1 C3-C5; METH1; KIAA1346 + Digestion of aggrecan and   Kidney; heart; cartilage
versican; binding to heparin
ADAMTS2 Procollagen N-proteinase; + Processing of collagen I and II Skin; tendon
hPCPNI; PCINP N-propeptides
ADAMTS3 KIAA0366 + Processing of collagen   Brain
N-propeptides
ADAMTS4 KIAA0688; aggrecanase-1; + Digestion of aggrecan,   Brain; heart; cartilage
ADMP-1 brevican, and versican
ADAMTS5 ADAMTS11; aggrecanase-2; + Digestion of aggrecan Uterus; placenta; cartilage
ADMP-2
ADAMTS6 — − — Placenta
ADAMTS7 — − — Various tissues
ADAMTS8 METH-2 + Digestion of aggrecan;   Lung; heart
inhibition of angiogenesis
ADAMTS9 KIAA1312 + Digestion of aggrecan Cartilage
ADAMTS10 — − — —
ADAMTS12 — − — Lung (fetus)
ADAMTS13 VWFCP; C9orf8 + Cleavage of von Willebrand factor Liver; prostate; brain
ADAMTS14 — + Processing of collagen   Brain; uterus
N-propeptides
ADAMTS15 — + Digestion of aggrecan Liver (fetus); kidney (fetus)
ADAMTS16 — − — Prostate; brain; uterus
ADAMTS17 FLJ32769; LOC123271 − Prostate; brain; liver —
ADAMTS18 ADAMTS21; HGNC:16662 − Prostate; brain —
ADAMTS19 — − — Lung (fetus)
ADAMTS20 — − — Brain; testis

*Proteinase activities are shown in 10 members of the ADAMTS family, but not in 9 other members.
 120 OKada  |  Proteinases and Matrix Degradation

Secreted-type MMP Membrane-anchored MMP

N N
Collagenases
C Stromelysins Tm C Type I
Zn Other MMPs F Zn

N MT1, 2, 3, 5-MMPs

Gelatinases N
C
Zn C
GPI
F Zn

Zn Matrilysins MT4, 6-MMPs

C

N
Tm N Type II

F Zn Furin-activated F Zn
C
C MMPs

MMP-23

ADAMTS Membrane-type ADAM

N N

D Ts S C D C E Tm C
F Zn F Zn

Figure 7-1  Domain structures of two types of matrix metalloproteinases (MMPs) (secreted-type MMP and membrane-anchored MMP) and two types
of a disintegrin and metalloproteinases (ADAMs) (a disintegrin and metalloproteinase with thrombospondin motifs [ADAMTS] and membrane-type
ADAM). The typical domain structure of most secreted-type MMPs (collagenases, stromelysins, and other MMPs) is composed of a prodomain, catalytic
domain, hinge, and hemopexin-like domain. Gelatinases (MMP-2 and MMP-9) have additional insertions of collagen-binding type II repeats of fibro-
nectin in the catalytic domain, whereas matrilysins (MMP-7 and MMP-26) lack a hemopexin-like domain. Furin-activated MMPs (MMP-11 and MMP-28)
contain an RKRR sequence (furin recognition site = F) at the end of the propeptide. Membrane-anchored MMPs are composed of type I transmembrane-
type MMPs (MT1-, MT2-, MT3-, MT5-MMPs), GPI-linked MMPs (MT4, MT6-MMPs), and type II transmembrane-type MMP (MMP-23). All of them have furin
recognition sites. ADAMTS has a prodomain, a furin recognition site (F), a catalytic domain, a hinge, a disintegrin domain (D), thrombospondin motifs
(Ts), and a spacer domain (S). Membrane-type ADAM is composed of a prodomain, furin recognition site (F), catalytic domain, hinge, disintegrin domain
(D), cysteine-rich domain (C), EGF-like domain (E), and transmembrane domain (Tm).

and MMP-13 (collagenase-3). These MMPs attack triple
helical regions of interstitial collagen types I, II, and III at
a specific single site following a glycine residue Gly (Ile or
Leu)-(Ala or Leu), located about three fourths of the dis-
tance from the N terminus. This cleavage generates frag-
ments approximately three fourths and one fourth of the
size of the collagen molecules. A biochemical study has
­disclosed the molecular mechanism of the cleavage: MMP-
1 unwinds the triple helical structure by interacting with
the α2(I) chain of type I collagen and cleaves the three α
chains in succession.9 MMP-13 is unique in that it cleaves
α chains of type II collagen at two sites of the Gly906-Leu907
and Gly909-Gln910 bonds.10 All of these collagenases de-
grade the interstitial collagens, but their specific activities
against the collagens are different; MMP-1, MMP-8, and
MMP-13 preferentially digest collagen types III, I, and II.10,11
­Although rodents such as mice were originally thought to
have only two collagenases (MMP-8 and MMP-13) and to
lack the MMP-1 gene, rodent homologues of the human
MMP-1 gene were cloned and named mouse collagenase A
and B (Mcol-A and Mcol-B).12
In addition to the interstitial fibrillar collagens, MMP-1,
MMP-8, and MMP-13 degrade many other ECM macromole-
cules. MMP-1 digests entactin, collagen X, gelatins, perlecan,
aggrecan, and cartilage link protein (see Table 7-2). MMP-8
digests aggrecan, gelatins, and cartilage link protein (see Table
7-2). MMP-13 hydrolyzes aggrecan; types IV, IX, X, and XIV
collagens; fibronectin; and tenascin.1 Non-ECM substrates of
MMP-1, MMP-8, and MMP-13 include α2-macroglobulin,
α1-antiproteinase inhibitor, α1-antichymotrypsin, and insu-
lin-like growth factor binding protein (IGF-BP)-2 and IGF-
BP-3 (see Table 7-2).
Gelatinases (MMP-2 and MMP-9). MMP-2 (gelatinase
A) and MMP-9 (gelatinase B) belong to the gelatinase
subgroup. Both MMPs readily digest gelatins and cleave
­collagen types IV and V.13,14 Elastin, aggrecan, and cartilage
link protein also are substrates of the gelatinases. Although
MMP-2 and MMP-9 share such substrates, they have differ-
ent activities on several ECM macromolecules. MMP-2, but
not MMP-9, digests fibronectin and laminin,13 and type III
collagen and α2 chains of type I collagen are degraded only
by MMP-9.14 The gelatinases also process directly TGF-β
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
into an active ligand (see Table 7-2). MMP-2 and MMP-
9 cleave fibroblast growth factor receptor type I and inter-
leukin (IL)-2 receptor type α (see Table 7-2). MMP-9 also
releases soluble Kit-ligand.15 MMP-2 processes monocyte
chemoattractant protein (MCP)-3 into an MCP-3 fragment
deleting the N-terminal four amino acids, which can bind
to CC-chemokine receptors and act as a general chemokine
antagonist.16
Stromelysins (MMP-3 and MMP-10). The subgroup
of stromelysins consists of MMP-3 (stromelysin-1) and
MMP-10 (stromelysin-2). They share 78% identity in ami-
no acid sequence and have similar enzymatic properties.17
The enzymes hydrolyze numerous ECM macromolecules,
including aggrecan, fibronectin, laminin, and collagen IV
(see Table 7-2).18 Collagen types III, IX, and X and telo-
peptides of collagen types I, II, and XI also are digested by
MMP-3.19 In addition to the ECM components, MMP-3
is active on IGF-BP-3, IL-1β, heparin-binding epidermal
growth factor (HB-EGF), connective tissue growth fac-
tor (CTGF), E-cadherin, α1-antichymotrypsin, and α1-
proteinase inhibitor (see Table 7-2). MMP-3 also activates
many proMMPs.6 A similar activator function has been
identified for MMP-10.20
Matrilysins (MMP-7 and MMP-26). Matrilysins include
MMP-7 (matrilysin-1) and MMP-26 (matrilysin-2), which
are the smallest of the MMPs, having only the propeptide
and catalytic domains. The substrate specificity of MMP-7
is similar to that of stromelysins, digesting numerous ECM
components, including aggrecan; gelatins; fibronectin; lam-
inin; elastin; entactin; collagen types III, IV, V, IX, X, and
XI; fibrin/fibrinogen; vitronectin; tenascin; and link protein
(see Table 7-2). Although these substrates overlap with the
substrates of other MMPs, the specific activity of MMP-7 to
most substrates is the highest among the MMPs.21,22 Non-
ECM molecules, such as α-defensin, Fas ligand, β4 integrin,
E-cadherin, plasminogen, tumor necrosis factor (TNF)-α,
and CTGF, also are the substrates for MMP-7 (see Table
7-2). MMP-26 degrades gelatin, type IV collagen, fibronec-
tin, fibrinogen, and α1-proteinase inhibitor,23-25 but infor-
mation about other substrates is still limited.
Furin-activated Matrix Metalloproteinases (MMP-11 and
MMP-28). MMP-11 (stromelysin-3) and MMP-28 (epily-
sin) contain an RKRR sequence at the end of the propep-
tide, which is a unique motif for intracellular processing of
proproteins to mature molecules by furin and other propro-
tein convertases. ProMMP-11 is activated intracellularly by
the action of furin.26 MMP-11 shows only weak ­proteolytic
activity against gelatin, laminin, fibronectin, and aggre-
can,27 but it has respectable catalytic action in digesting α1-
proteinase inhibitor, α2-macroglobulin, and IGF-BP-1 (see
Table 7-2).28,29 MMP-28 can degrade casein, but its natural
substrates are unknown.30
Other Secreted-type Matrix Metalloproteinases (MMP-
12, MMP-19, MMP-20, MMP-21, and MMP-27). MMP-
12 (metalloelastase),31 MMP-19 (RASI-1),32 MMP-20
(enamelysin),33 MMP-21,25 and MMP-27 have structural
characteristics similar to collagenases and stromelysins.
These MMPs are not classified into the above-mentioned
subgroups, however, because their substrates and other
biochemical characters are not fully examined at present.
Overall information on the substrate specificity of MMP-
12,31 MMP-19,34,35 and MMP-2033,35 suggests that they
121
are stromelysin-like proteinases. MMP-1231 digests elas-
tin, fibronectin, collagen V, osteonectin, and plasminogen
(see Table 7-2). MMP-19, which was originally reported
as MMP-18 but renamed as MMP-19, cleaves type IV col-
lagen, laminin, fibronectin, gelatin, tenascin, entactin, fi-
brin/fibrinogen, aggrecan, and cartilage oligomeric matrix
protein (COMP) (see Table 7-2).34,35 MMP-20 also digests
amelogenin, aggrecan, and COMP.35 Substrates of MMP-21
and MMP-27 are unknown, however.
Membrane-anchored Matrix Metalloproteinases. Type I
transmembrane-type MMPs include MT1-MMP (MMP-
14),36 MT2-MMP (MMP-15),37 MT3-MMP (MMP-16),38
and MT5-MMP (MMP-24).39 All of these MT-MMPs can
activate proMMP-2, but MT1-MMP may play a major role
in the activation of proMMP-2 in various tissues (see later).
Besides the activator function, however, MT1-MMP digests
the triple helical portions of interstitial collagen types I, II,
and III and other ECM components, including fibronectin,
laminin, aggrecan, and gelatin (see Table 7-2).40 MT2-
MMP also digests fibronectin, tenascin, nidogen, aggrecan,
perlecan, and laminin.41 MT3-MMP cleaves collagen type
III, fibronectin, and gelatins.42 MT4-MMP (MMP-17)43 and
MT6-MMP (MMP-25)44 are GPI-linked MMPs. MT4-MMP
and MT6-MMP can digest gelatin and fibrin/fibrinogen
(see Table 7-2).44-46 MMP-23 (cysteine array-MMP, MIFR)
is a type II transmembane-type MMP47; almost identical
genes are cloned, called MMP-23A and MMP-23B. MMP-23
digests gelatin,48 but no information about other substrates
is available (see Table 7-2). A unique aspect of MMP-23
is that this MMP is expressed in only female and male re-
productive organs, such as endometrium, ovary, testis, and
prostate,48 but its functions are not well established.
ADAM Family
Members of the ADAM (a disintegrin and metalloprotein-
ase) gene family, which also are called mammalian reproly-
sins, are classified into two groups based on the C-terminal
structural differences of the molecules (see Fig. 7-1): mem-
brane-type ADAM with transmembrane domain (ADAM)
and secreted-type ADAM with thrombospondin motifs
(ADAMTS). The active sites in the catalytic domains of
most members of both groups contain a common sequence
of HEXGHXXGXXHD with the “Met-turn,” which also is
present in MMP members.
ADAMTS subgroup includes 19 members. Although
information about substrates and biologic functions is still
limited, ADAMTS1, ADAMTS2, ADAMTS3, ADAMTS4,
ADAMTS5, ADAMTS8, ADAMTS9, ADAMTS14,
and ADAMTS15 all are ECM-degrading proteinases (see
Table 7-3). ADAMTS1,49 ADAMTS4,50 ADAMTS5,51
ADAMTS8, ADAMTS9, and ADAMTS15 can preferen-
tially cleave aggrecan at the five Glu-X bonds, including the
Glu373-Ala374 bond (the aggrecanase site). Because of aggre-
can-degrading activity, ADAMTS4 and ADAMTS5 are
named aggrecanase-1 and aggrecanase-250,51; versican also is
digested by these proteinases,52 and brevican is cleaved by
ADAMTS4 (see Table 7-3).53 The C-terminus-truncated
ADAMTS4 also degrades fibromodulin and decorin.54
ADAMTS2 and ADAMTS3 ­ process the N-terminal pro-
peptides of type I and II collagens and are named procollagen
 122 OKada  |  Proteinases and Matrix Degradation
N-proteinase. Activity of procollagen N-proteinase also is
known with ADAMTS14. ADAMTS13 is a von Willebrand
factor–cleaving proteinase, and its mutation causes thrombotic
thrombocytopenic purpura. Proteinase activities of other
ADAMTS species are still unknown.
Two thirds of the membrane-type ADAM molecules are
catalytically inactive nonproteolytic homologues (Table
7-4). Among the ADAMs, ADAM8, ADAM9, ADAM10,
ADAM12, ADAM15, ADAM17, ADAM19, and ADAM28
are shown to have proteinase activity (see Table 7-4). Although
ADAM10 and ADAM15 degrade type IV collagen, the main
substrates of these ADAMs are various membrane proteins,
which include precursors of cytokines and growth factors
such as TNF-α, HB-EGF and neuregulin, IGF-BPs, receptors
such as p75 TNF receptor, IL-1 receptor II, and other mem-
brane proteins related to development such as Notch ligand
and ephrin (see Table 7-4).55-60 According to these data, a
major function of the ADAMs is shedding of the membrane
proteins. ADAM17 cleaves a proform of TNF-α at the physi-
ologic processing site into the soluble form of TNF-α and is
called TNF-α-conversing enzyme. ADAM17 also is involved
in release of L-selectin, TGF-α, and p75 TNF ­ receptor.55
ADAM9, ADAM12, and ADAM17 can shed HB-EGF from
its precursor. ADAM12 and ADAM28 cleave IGF-BP-3 and
IGF-BP-5.60,61 CD23 is shed by ADAM8, ADAM15, and
ADAM28.62 Other functions of ADAMs include binding
to integrins, cell-cell interaction, cell ­ migration, and signal
transduction (see Table 7-4).63
ENDOGENOUS PROTEINASE INHIBITORS
Endogenous proteinase inhibitors control the activities
of proteinases in vivo. The inhibitors are derived from
plasma or cells in the local tissues. Plasma contains ­several
­proteinase inhibitors, and about 10% of all the plasma pro-
teins are proteinase inhibitors. Most are proteinase class
specific, but α2-macroglobulin inhibits the activities of pro-
teinases from all four groups. Major endogenous inhibitors
of the ECM-degrading proteinases are listed in Table 7-5.
α2-MACROGLOBULIN
The α2-macroglobulin molecule is a large plasma glycopro-
tein of 725 kD, which consists of four identical subunits
of 185 kD that are linked in pairs by disulfide bonds. The
pairs assemble noncovalently. Almost all active protein-
ases, regardless of the proteinase classes, bind and attack
the so-called bait region, located near the center of the
subunit. After cleaving within the bait region, the protein-
ase is physically trapped within the molecule by inducing
a conformational change of the inhibitor, resulting in a
proteinase/α2-macroglobulin complex. Although the pro-
teinase in the complex remains active against very small
substrates, it is trapped by the arms of the α2-macroglobu-
lin from degrading larger proteins. Besides the function as
a proteinase inhibitor, α2-macroglobulin may act as a car-
rier protein because it also binds to numerous growth factors
and cytokines, such as platelet-derived growth factor, basic
fibroblast growth factor, TGF-β, insulin, and IL-1β.
The α2-macroglobulin molecule is synthesized mainly
in liver, but also locally by macrophages, fibroblasts, and
­adrenocortical cells. Concentration of the inhibitor in
plasma is 250 mg/dL. Because of its large molecular weight,
it is not present in noninflammatory synovial fluid. During
synovial inflammation, α2-macroglobulin penetrates into
the joint cavity. Rheumatoid synovial fluid has about the
same concentration of the inhibitor as plasma.
INHIBITORS OF SERINE PROTEINASES
The primary inhibitors of serine proteinases include the
members of the serpin (serine proteinase inhibitor) gene
family, Kunitz-type inhibitors, and others (see Table 7-5).
The serpins are glycoproteins of 50 to 100 kD and share
homology with human α1-proteinase inhibitor.64 The major
serpins involved in the regulation of ECM-degrading serine
proteinases are α1-proteinase inhibitor, α1-antichymotryp-
sin, α2-antiplasmin, plasminogen activator inhibitors (PAI-
1 and PAI-2), protein C inhibitor (PAI-3), C1-inhibitor,
kallistatin, and proteinase nexin-1 (PN-1). The main pro-
teinases inhibited by these molecules are listed in Table
7-5. Although PAI-1 and PAI-2 inhibit tPA and uPA, the
inhibition by PAI-1 and PAI-2 is more effective to tPA and
uPA, respectively.
Kunitz-type inhibitors include aprotinin, trypstatin, and
PN-2, which is identical to a β-amyloid protein precursor.
Secretory leukocyte proteinase inhibitor, which inhibits
neutrophil elastase and cathepsin G, is present in many
secretory and inflammatory fluids and in cartilage. Elafin is a
serine proteinase inhibitor with 38% identity with the sec-
ond domain of secretory leukocyte proteinase inhibitor; it
inhibits neutrophil elastase and proteinase 3.
INHIBITORS OF CYSTEINE PROTEINASES
The members of the cystatin superfamily and calpastatin
belong to the family of inhibitors of ECM-degrading
cysteine proteinases (see Table 7-5). Cystatins capable of
inhibiting lysosomal cysteine proteinases consist of three
groups. Subgroup 1 comprises stefins A and B. Each has
a molecular mass of 11 kD, and the stefins reside within
cells. Subgroup 2 comprises cystatin C and S, each with
a molecular mass of 13 kD. They occur at relatively high
concentrations in cerebrospinal fluid and saliva. Subgroup
3 comprises the kininogens. Kininogens that participate
in blood ­ coagulation and inflammation also are inhibitors
of cysteine proteinases. Calpains are not inhibited by cys-
tatins, but are inhibited by calpastatin (120 kD), which is a
cytosolic-specific inhibitor of calpain.
TISSUE INHIBITORS OF METALLOPROTEINASES
TIMPs are a gene family consisting of four different mem-
bers with approximately 40% to 50% sequence identity
(i.e., TIMP-1, TIMP-2, TIMP-3, and TIMP-4), which have
molecular masses ranging from 21 to 28 kD in humans.65-69
Virtually all TIMPs inhibit the activities of MMPs by bind-
ing in a 1:1 molar ratio to form tight, noncovalent com-
plexes65 except that TIMP-1 does not inhibit efficiently
MT-MMPs.41,42,70 TIMPs contain 12 highly conserved cys-
teine residues that form six intrachain disulfide bonds, which
are essential for maintaining the correct ternary structure of
the molecule67,71 and stable inhibitor activity.65 The TIMP
molecules have two structurally distinct subdomains: an
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE 123

Table 7-4  Members of the ADAM Gene Family

Proteinase-type (P) or
Non–Proteinase-type Tissue and Cell
ADAM Other Names (NP) Functions and Biochemical Features Expression
ADAM1 PH-30α; Fertilin-α NP Sperm/egg binding/fusion; binding to integrin  Sperm
α9β1
ADAM2 PH-30β; Fertilin-β NP Sperm/egg binding/fusion; binding to integrin Sperm
α4β1, α6β1, and α9β1
ADAM3 Cyritestin; tMDC; NP Binding to integrin α4β1, α6β1, and α9β1 Sperm
CYRN
ADAM4 tMDC V NP — Testis
ADAM5 tMDC II NP — Testis
ADAM6 tMDC IV NP — Testis
ADAM7 EAP I; GP-83 NP Binding to integrin α4β1, α4β7, and α9β1 Testis
ADAM8 MS2 (CD156) P Neutrophil infiltration; shedding of CD23 Macrophages;  
neutrophils
ADAM9 MDC9; MCMP; P Shedding of HB-EGF; TNF-p75 receptor; cleavage of Various tissues
­Meltrin-γ APP; digestion of fibronectin and gelatin; ­binding
to integrin α2β1, α6β1, α6β4, α9β1, and αVβ5
ADAM10 MDAM; Kuzbanian P Release of TNF-α; digestion of collagen IV, gelatin, Kidney; brain;
and myelin basic protein; cleavage of Delta,   ­chondrocytes
APP, L1, and CD44; shedding of HB-EGF; presence
of RRKR sequence
ADAM11 MDC NP Tumor suppressor gene (?) Brain
ADAM12 Meltrin-α; MCMP; P Muscle formation; presence of RRKR sequence; Osteoblasts; muscle cells;
MLTN; MLTNA ­binding to integrin α4β1 and α9β1; digestion   chondrocytes; placenta
of IGF-BP-3 and –5; shedding of HB-EGF;  
digestion of collagen IV, gelatin, and fibronectin
ADAM13 xMDC13a;   NP Movement of neural crest Xenopus laevis
xADAM13a
ADAM14 ADM-1 NP — Caenorhabditis elegans
ADAM15 Metargidin; MDC15; P Expression in arteriosclerosis; binding to integrin Smooth muscle cells;
AD56; CR II-7 αvβ3, α5β1, and α9β1; digestion of collagen IV chondrocytes; endothe-
and gelatin; shedding of CD23 lial cells; osteoclasts
ADAM16 xMDC16 NP — X. laevis
ADAM17 TACE; cSVP P Shedding of TNF-α, TGF-β, TNF-p75 receptor,   Macrophages; various  
RANKL, and HB-EGF; presence of RRKR   tissues; carcinoma
sequence; cleavage of APP, Notch, L-selectin,   tissue
and CD44; binding to integrin α5β1
ADAM18 tMDC III NP — Testis
ADAM19 Meltrin-β; FKSG34 P Formation of neuron; digestion of neuregulin; Testis
­binding to integrin α4β1 and α5β1
ADAM20 — P Formation of sperm Testis
ADAM21 — P — Testis
ADAM22 MDC2 NP — Brain
ADAM23 MDC3 NP Binding to integrin αvβ3 Brain; heart
ADAM24 Testinase-1 NP Sperm/egg binding/fusion Testis
ADAM25 Testinase-2 NP — Testis
ADAM26 Testinase-3 NP — Testis
ADAM28 e-MDC II; MDC-Lm; P Digestion of myelin basic protein and IGF-BP-3; Testis; lung; lymphocytes;
MDC-Ls shedding of CD23; binding to integrin α4β1,   pancreas; uterus
α4β7, and α9β1
ADAM29 svph 1 NP — Testis
ADAM30 svph 4 P — Testis
ADAM32 AJ131563 NP — Testis
ADAM33 — P Mutation in bronchial asthma patients; binding to Lung (fibroblasts, smooth
integrin α4β1, α5β1, and α9β1 muscle cells)
ADAM34 Testinase-4 NP — Testis
APP, amyloid precursor protein; HB-EGF, heparin-binding epidermal growth factor; IGF-BP, insulin-like growth factor binding protein; RANKL, receptor activator
of nuclear factor кB ligand; TGF, transforming growth factor; TNF, tumor necrosis factor.
 124 OKada  |  Proteinases and Matrix Degradation

Table 7-5  Endogenous Inhibitors of Extracellular Matrix–Degrading Proteinases

Inhibitor Molecular Mass (kD) Source Target Enzyme
α2-Macroglobulin 725 Plasma (liver); macrophages; fibroblasts Most proteinases from all classes
Inhibitors of Serine Proteinase
Serpins
α1-Proteinase inhibitor 52 Plasma; macrophages Neutrophil elastase, cathepsin G,
proteinase 3
α1-Antichymotrypsin 58 Plasma Cathepsin G; chymotrypsin; chy-
mase; tissue kallikrein
α2-Antiplasmin 67 Plasma Plasmin
Proteinase nexin-1 45 Fibroblasts Thrombin; uPA; tPA; plasmin;
trypsin; trypsin-like serine
proteinase
PAI-1 45 Endothelial cells; fibroblasts; platelets; tPA; uPA
plasma
PAI-2 47 Plasma; macrophages uPA; tPA
Protein C inhibitor 57 Plasma; urine Active protein C; tPA; uPA; tissue
kallikrein
C1-inhibitor 96 Plasma Plasma kallikrein; C1 esterase
Kallistatin 92 Plasma; liver; stomach; kidney; pancreas Tissue kallikrein
Kunins
Aprotinin 7 Mast cells Plasmin; kallikrein
Trypstatin 6 Mast cells Tryptase
Proteinase nexin-2   100 Fibroblasts EGF binding protein, NGF-γ, tryp-
(β-amyloid protein precursor) sin, chymotrypsin, factor XIa
Others
SLPI 15 Bronchial secretions; seminal plasma; Neutrophil elastase; cathepsin G;
cartilage chymotrypsin; trypsin
Elafin 7 Horny layers of skin Neutrophil elastase; proteinase 3
Inhibitors of Cysteine Proteinase
Stefin A 11 Cytosol Cysteine proteinases
Stefin B 11 Cytosol Cysteine proteinases
Cystatin C 13 Body fluids Cysteine proteinases
Cystatin S 13 Seminal plasma; tears; saliva Cysteine proteinases
Kininogens 50-78/108-120 Plasma Cysteine proteinases
Calpastatin 120 Cytosol Calpains
Metalloproteinase Inhibitors
TIMP-1 28 Connective tissue cells; macrophages MMPs
TIMP-2 22 Connective tissue cells; macrophages MMPs
TIMP-3 21/24* Fibroblasts; synovial cells MMPs; ADAMs; ADAMTS
TIMP-4 21 Heart; brain; testis MMPs
RECK Unknown Many tissue cells; fibroblasts MMP-2; MT1-MMP
*Glycosylated form.
EGF, epidermal growth factor; NGF, nerve growth factor; PA, plasminogen activator; PAI, plasminogen activator inhibitor; RECK, reversion-inducing, cysteine-
rich protein with Kazal motifs; SLPI, secretory leukocyte proteinase inhibitor; TIMP, tissue inhibitor of metalloproteinases; tPA, tissue-type plasminogen activator;
uPA, urokinase-type plasminogen activator.

N-terminal subdomain that consists of loops 1 through
3 and a C-terminal subdomain that consists of loops 4
through 6. The N-terminal subdomain of each TIMP mol-
ecule contains the inhibitory activity for MMPs.65 Stud-
ies on the crystal structures of the MMP/TIMP complexes
show that the wedge-shaped TIMPs bind with their edge
into the entire length of the active-site cleft of their cog-
nate MMPs.67 High affinity and efficient inhibitor activity
of TIMP-2 to MT1-MMP are explained by the interaction
between a quite long hairpin loop of TIMP-2 and a loop
over the rim of the active-site cleft of MT1-MMP.72
TIMP-1 and TIMP-2 are unique in that they make
the complexes with proMMP-9 and proMMP-2 (i.e., the
proMMP-9/TIMP-1 and proMMP-2/TIMP-2 complexes).
Similar complex formation also is known between TIMP-4
and proMMP-2. Because the complexes are made through
the interaction between their C-termini,65 TIMPs in the
complexes retain inhibitor activity against MMPs. The
activation of proMMP-9 and proMMP-2 is suppressed in
the complex forms; the complex formation may be a safety
device for these gelatinases.14 The proMMP-2/TIMP-2
complex is useful for the efficient activation of proMMP-2
by MT1-MMP on the cell membranes because MT1-MMP
captures proMMP-2 to the cell membranes through the tri-
molecular complex formation between the catalytic domain
of MT1-MMP and the N-terminal domain of TIMP-2 (see
later).73,74
Besides the inhibition and interactions of TIMPs to
MMPs, TIMP-3, among the TIMPs, most efficiently inhib-
its the activities of ADAM10, ADAM12, ADAM17,
ADAM28, and ADAM33,75 although ADAM8, ADAM9,
and ADAM19 are not inhibited by TIMPs. Because TIMP-3
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
also efficiently inhibits the aggrecan-degrading activity of
ADAMTS4 and ADAMTS5, TIMP-3 may be a common
tissue inhibitor of the ADAM members. The N-­terminal
subdomain of TIMP-3 is critical to the inhibition of the
activities of ADAM members and MMPs, but the inhibition
mechanism seems to be different.76 TIMPs are multifunc-
tional proteins with more diverse actions than MMP/ADAM
inhibitors, including growth factor activity, antiangiogenic
activity, and regulatory activity of apoptosis.69
Another new MMP inhibitor is RECK (reversion-induc-
ing, cysteine-rich protein with Kazal motifs).77 RECK is
a GPI-linked glycoprotein harboring three inhibitor-like
domains and inhibits the activities of at least MMP-2,
MMP-9, and MT1-MMP. Although this inhibitor seems to
play a key role in the angiogenic processes in vivo, its bio-
chemical mechanism as an MMP inhibitor and functions in
pathologic conditions such as arthritides remain unknown.
REGULATION OF PROTEINASE ACTIVITY
The activities of ECM-degrading proteinases in tissues are
regulated by the balance between the proteinases and their
inhibitors. The balance at the local tissues depends on sev-
eral factors, including production rates of proteinases and
inhibitors, their secretion, activation of proenzymes, and
anchoring systems of the activated proteinases to cell sur-
faces. Production levels of the proteinases and inhibitors
within the cells are controlled mainly by their gene expres-
sion. Activation processes of proMMPs and membrane
anchoring of their activities have been established by exten-
sive experimental work.
GENE EXPRESSION OF PROTEINASES
AND INHIBITORS
Matrix Metalloproteinases and Tissue Inhibitors
of Metalloproteinases
Normal cells except for inflammatory cells do not produce
MMPs or TIMPs in the tissues under physiologic conditions,
but their expression is stimulated by many factors under
pathologic conditions. Neutrophils and macrophages syn-
thesize MMP-8 and MMP-9 during the differentiation and
store them within the granules of the differentiated cells.
Tumor cells express many MMPs, such as MMP-1, MMP-7,
MMP-9, MMP-10, and MT1-MMP, and TIMP-1 predomi-
nantly by oncogenic transformation. The gene expression of
MMPs and TIMPs in the tissue cells other than inflamma-
tory cells and tumor cells is regulated by numerous factors,
however, including cytokines, growth factors, and chemical
and physical stimuli.
Much information is available for regulators of MMP-
1 and MMP-3, which are coordinately expressed in many
cell types after stimulation with cytokines and growth fac-
tors, factors acting at the cell surface, and chemical agents
(Table 7-6). The induced production of MMP-1 and MMP-3
is suppressed by retinoic acid, TGF-β, and glucocorticoid.
The gene expression of MMP-7 and MMP-9 is regulated by
similar factors, but the regulation is stricter, and fewer fac-
tors modulate the expression (see Table 7-6). MT1-MMP
expression is upregulated by phorbormyristate acetate (12-
O-tetradecanoylphorbol-13-acetate), concanavalin A, basic
125
fibroblast growth factor, and TNF-α, and it is downregulated
by glucocorticoids in various cells. TNF-α and IL-1α stimu-
late osteoarthritic chondrocytes to express the MT1-MMP
gene.78 In contrast to these MMPs, MMP-2 and TIMP-2 are
unique in that factors capable of enhancing the production
of MMP-1, MMP-3, and TIMP-1 are inactive.
TIMP-1 expression is enhanced or suppressed in response
to many factors, including cytokines, growth factors, and
oncogenic transformation (see Table 7-6). Effects of these
stimulatory factors are common to the gene expression of
MMPs, but they are regulated independently. TGF-β, reti-
noic acid, progesterone, and estrogen enhance TIMP-1
expression in fibroblasts, but they suppress the expression of
MMP-1 and MMP-3. Although information about stimulat-
ing and suppressive factors of TIMP-1, TIMP-2, and TIMP-
3 is available (see Table 7-6), factors controlling the gene
expression of TIMP-4 are not well known. Previous studies
have identified the elements in the promoters of MMPs and
TIMPs,2 which are related to or responsible for the stimula-
tion or suppression of the gene expression with various fac-
tors. Regulation of the gene expression is generally explained
by the structural characteristics of the promoters.2
Serine Proteinases and Their Inhibitors
Neutrophil elastase, cathepsin G, chymase, and tryptase
are stored within the secretory granules and secreted into
the extracellular milieu after activation of neutrophils and
mast cells. The expression of these serine proteinases is con-
trolled mainly by the cellular differentiation. Precursors of
plasmin and plasma kallikrein are constitutively synthesized
predominantly in liver, circulate in blood as zymogen forms
(i.e., plasminogen and prekallikrein), and reach the inflamed
tissues by being released from blood vessels. The proteinase
activities in the tissues are controlled mainly through acti-
vation of the proenzymes by activators. The uPA and tPA
molecules, activators of plasminogen, are synthesized by
tissue cells, and their gene expression is regulated by many
factors (Table 7-7). The uPA synthesis is upregulated in
many normal cell types and in transformed cells by agents
that increase intracellular cyclic adenosine monophosphate
(cAMP) levels (e.g., calcitonin, vasopressin, cholera toxin,
cAMP analogues), growth factors (e.g., EGF, platelet-
derived growth factor, vascular endothelial growth factor);
cytokines (IL-1, TNF-α), and phorbol esters, whereas gluco-
corticoid decreases the expression.8 The expression of tPA
is regulated by similar factors (see Table 7-7). In endothe-
lial cells, proteinases are enhancers; thrombin and plasmin
stimulate the production of tPA.8 PAI-1 and PAI-2 also are
regulated by common factors, many of which also enhance
the production of uPA and tPA (see Table 7-7). Most serpins
are constitutively produced in liver and secreted to plasma.
Lysosomal Cysteine and Aspartic Proteinases
The expression of lysosomal cysteine proteinases, cathep-
sins B, L, and K, is generally constitutive, but cellular trans-
formation is often associated with increased synthesis of
cathepsins B and L. Cathepsin B transcription varies with
cell type and the state of differentiation of tumor cells; it
is increased in chondrocytes by IL-1. Malignant transfor-
mation, tumor promoters, and growth factors stimulate the
 126 OKada  |  Proteinases and Matrix Degradation

Table 7-6  Factors That Modulate Synthesis of Matrix Metalloproteinases and Tissue Inhibitors  
of Metalloproteinases
Enzyme or TIMP Stimulating Factor* Suppressive Factor
MMP-1 Cytokines and growth factors: IL-1; TNF-α; EGF; PDGF; bFGF; Retinoic acids; glucocorticoids; estrogen; progesterone;  
VEGF; NGF; TGF-α; IFN-α; IFN-β; IFN-γ; leukoregulin; relaxin TGF-β; transmembrane neural cell adhesion molecule;
cAMP; INF-γ; adenovirus E1A
Factors acting at cell surface: calcium ionophore A23187;
cell fusion; collagen; concanavalin A; integrin receptor
antibody; crystals of urate, hydroxyapatite, and calcium
pyrophosphate; SPARC (osteonectin/BM 40);  
iron; extracellular matrix metalloproteinase inducer
(EMMPRIN/CD147/basigin/M6 antigen); phagocytosis
Chemical agents: cAMP; colchicine; cytochalasins B and D;
LPS; pentoxifylline; TPA; calmodulin inhibitors; serotonin;
1,25-(OH)2 vitamin D3; platelet-activating factor; serum
amyloid A; β-microglobulin
Physical factors: heat shock; ultraviolet irradiation
Others: viral transformation; oncogenes; autocrine agents;
aging of fibroblasts
MMP-2 TGF-β; concanavalin A; H-ras transformation; extracellular Adenovirus E1A
matrix metalloproteinase inducer (EMMPRIN/CD147/ 
basigin/M6 antigen)
MMP-3 IL-1; TNF-α; EGF; concanavalin A; SPARC (osteonectin/BM   Retinoic acids; glucocorticoids; estrogen; progesterone; 
40); LPS; TPA; extracellular matrix metalloproteinase TGF-β; adenovirus E1A
­inducer (EMMPRIN/CD147/basigin/M6 antigen); viral
­transformation; oncogenes; integrin receptor antibody;
heat shock; calcium ionophore A23187; cytochalasin B
MMP-7 IL-1; TNF-α; EGF; TPA; LPS Unknown
MMP-8 TNF-α; TPA; IL-1 Unknown
MMP-9 IL-1; TNF-α; EGF; TGF-β; TPA; H-ras; v-Src; SPARC   Retinoic acids; adenovirus E1A
(osteonectin/BM40)
MMP-10 TPA; A23187; TGF-β; EGF Unknown
MMP-11 Retinoic acids bFGF
MMP-13 bFGF; TNF-α; TGF-β Unknown
MT1-MMP Concanavalin A; TPA; bFGF; TNF-α; IL-1α Glucocorticoids
TIMP-1 IL-1; IL-6; IL-11; TPA; TGF-β; TNF-α; retinoic acids; LPS; Extracellular matrix; cytochalasins
­progesterone; estrogen; oncogenic transformation;  
viral infection
TIMP-2 Progesterone TGF-β; LPS
TIMP-3 EGF; TGF-β; TPA; TNF-α; glucocorticoids; oncostatin M Unknown
*Factors regulating gene expression of other MMPs excluded from this table and TIMP-4 are unknown.
bFGF, basic fibroblast growth factor; cAMP, cyclic adenosine monophosphate; EGF, epidermal growth factor; IFN, interferon; IL, interleukin; LPS, lipopoly-
saccharide; NGF, nerve growth factor; PDGF, platelet-derived growth factor; TGF, transforming growth factor; TNF, tumor necrosis factor; TPA, 12-O-tetradec-
anoylphorbol-13-acetate; VEGF, vascular endothelial growth factor.

synthesis of cathepsin L. Cathepsin K gene expression in
monocyte-macrophage lineage depends on the cellular dif-
ferentiation to osteoclasts, but all-trans retinoic acid upregu-
lates the expression in rabbit osteoclasts. Lysosomal aspartic
proteinase, cathepsin D, is constitutively expressed in almost
all cells, although estradiol, calcitriol, and retinoic acid can
regulate the expression.
ACTIVATION MECHANISMS OF THE ZYMOGENS
OF METALLOPROTEINASES
All of the MMPs are synthesized as inactive zymogens (pro­
MMPs), and activation of proMMPs is prerequisite to their
functioning in vivo. ProMMPs are kept inactive by an inter-
action between a cysteine-sulfhydryl group in the conserved
propeptide sequence PRCGXPD and the zinc ion bound to
the catalytic domain, preventing the formation of a water-
zinc complex that is essential to the enzymatic reaction.
Activation requires proteolytic removal of the propeptide
domain. There are three pathways of the proMMP activa-
tion—extracellular, intracellular, and pericellular (Fig. 7-2).
Extracellular Activation
Extracellular activation, which is applicable to many
secreted MMPs (e.g., proMMP-1, proMMP-3, proMMP-
7, proMMP-8, proMMP-9, proMMP-10, proMMP-12, and
proMMP-13), is initiated through the disruption of the Cys-
Zn2+ interaction by treatment with nonproteolytic agents or
proteinases and completed by autocatalytic processing.6,69
Nonproteolytic activators used in vitro include thiol-modi-
fying reagents (e.g., mercurial compounds, iodoacetamide,
N-ethylmaleimide, oxidized glutathione), hypochlorous
acid, sodium dodecyl sulfate, chaotropic agents, and physi-
cal factors (heat and acid exposure).6 Most of these factors,
especially 4-aminophenylmercuric acetate (APMA), enable
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE 127

proMMP molecules to generate a short-lived intermediate, and leading to generation of active MMPs starting with Tyr
which is formed by removal of a part of propeptide by an or Phe at the N terminus. Such a process may not be essen-
intramolecular reaction.6 The fully activated form is made tial, however, for proMMP-9 activation by APMA because
by an intermolecular autocatalysis cleaving three amino a fully active form retaining the PRCGVPD sequence is
acids downstream from the conserved sequence PRCGXPD generated by the cleavage of the Ala74-Met75 bond upstream
of the conserved sequence.14 Concerning proMMP-9 acti-
Table 7-7  Factors That Regulate Expression   vation during cerebral ischemia in vivo, nitric oxide is
of Plasminogen Activators and Their Inhibitors reported to activate proMMP-9 by S-nitrosylation.79
A similar stepwise activation is proposed for the proteo-
Enzyme lytic activation of proMMPs. Proteinases initially attack
or Inhibitor Stimulatory Factor Suppressive Factor
the proteinase-susceptible bait regions in the propeptides
uPA TPA; IL-1; INF-γ; EGF;   Glucocorticoids; and generate proteolytically active intermediates through
PDGF; bFGF; VEGF;   TGF-β
TGF-β; cholera toxin;
destabilization of the Cys-Zn2+ interaction.6 In the second
cAMP; estrogen;   step, the final activation site is autolytically catalyzed by
calcitonin; vasopres- the active intermediate instead of the trigger proteinases,
sin; disruption of and active MMPs without propeptides are made. In many
E-cadherin–dependent cases, the bait region sequences in the propeptide dictate
cell-cell adhesion
which proteinases can become an activator of a particu-
tPA TPA; EGF; bFGF; VEGF; TNF-α lar MMP.6 Potential activators of proMMPs are listed in
retinoic acids; glu-
cocorticoids; cAMP; Table 7-8. Plasmin may play a major role in the activation
thrombin; plasmin; of proMMP-3 and proMMP-10 in vivo because treatment
follicle-stimulating of these proMMPs with plasmin leads to full activation in
hormone; luteinizing vitro.80 ProMMP-1 activation by plasmin alone results in
hormone; gonado-
tropin-releasing
only about 25% of the potential MMP-1 activity, how-
hormone ever, and full activation requires the subsequent cleavage
PAI-1 IL-1; TNF-α; TGF-β; cAMP
of the Gln80-Phe81 bond by active MMP-3, MMP-7, or
bFGF; VEGF; TPA; MMP-10.6,22 MMP-3 and MMP-10 can directly activate
glucocorticoids proMMP-7,22 proMMP-8, proMMP-9,14,20 and proMMP-
PAI-2 TPA; LPS; TNF-α; colony- Glucocorticoids 1311 into fully active forms. This intermolecular activation
stimulating factor; cascade of MMPs may be important to in vivo activation
cholera toxin; dengue of proMMPs.
virus
PN-1 TPA; EGF; thrombin Unknown
Intracellular Activation
bFGF, fibroblast growth factor; cAMP, cyclic adenosine monophosphate;
EGF, epidermal growth factor; IFN, interferon; IL, interleukin; LPS, lipopolysac- Because proMT-MMPs proMMP-23, proMMP-11, and
charide; PAI, plasminogen activator inhibitor; PDGF, platelet-derived growth proMMP-28 have basic motifs containing an RXKR
factor; PN, proteinase nexin; TGF, transforming growth factor; TNF, tumor
necrosis factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; tPA, tissue-type
sequence at the end of the propeptide domains, proprotein
plasminogen activator; uPA, urokinase-type plasminogen activator; VEGF, convertases, such as furin, a processing enzyme in the trans-
vascular endothelial growth factor. Golgi apparatus, are considered to activate these ­proMMPs

Active MMP-3 Active MMP-2

Zn Zn

Extracellular
activation
Active MMP-11
Zn Figure 7-2  Activation mechanisms
Ct Zn
of proMMPs. Most secreted-type
Zn proMMPs, such as proMMP-3, are ac-
Zn tivated extracellularly by many pro-
Pericellular teinases (extracellular activation).
activation Furin-activated secreted proMMPs,
including proMMP-11, and proMT-
MMPs, such as proMT1-MMP, are
intracellularly activated through
Active MT1-MMP removal of the propeptides (arrow-
heads) by the action of proprotein
Zn
Zn convertases such as furin (intracellu-
Ct Zn
TIMP-2 lar activation). ProMMP-2 is activated
on the cell membrane by MT1-MMP;
this activation requires the trimo-
Zn Intracellular F
F lecular complex of MT1-MMP/TIMP-
Zn activation Zn
2/proMMP-2 and dimerization of
ProMMP-2 MT1-MMP (pericellular activation).
ProMMP-3
ProMT1-MMP ProMMP-11 Ct, C-terminal domain of TIMP-2; F,
furin recognition site.
 128 OKada  |  Proteinases and Matrix Degradation
Table 7-8  Activators of Pro-Matrix Metalloproteinases
ProMMP Activator
ProMMP-1 Trypsin (partial); plasmin (partial); plasma
kallikrein (partial); chymase (partial);
MMP-3; MMP-7; MMP-10; MMP-11
ProMMP-2 MT1-MMP; MT2-MMP; MT3-MMP; MT5-
MMP
ProMMP-3 Plasmin; plasma kallikrein; trypsin;  
tryptase; chymase; cathepsin G;   through screening proMMP-7-binding molecules by a yeast
chymotrypsin; neutrophil elastase;
thermolysin
ProMMP-7 MMP-3; MMP-10 (partial); trypsin; plasmin
(partial); neutrophil elastase (partial)
ProMMP-8 MMP-3; MMP-10; tissue kallikrein; neutro-
phil elastase; cathepsin G; trypsin
ProMMP-9 MMP-3; MMP-2; MMP-7; MMP-10 (partial);
MMP-13; trypsin; chymotrypsin; cathep-
sin G; tissue kallikrein
ProMMP-10 Plasmin; trypsin; chymotrypsin
ProMMP-11 Furin
ProMMP-13 MMP-2; MMP-3; MT1-MMP; plasmin
ProMT1-MMP Furin
intracellularly (see Fig. 7-2). Intracellular activation of
proMMP-11 and proMT1-MMP by furin is shown.26,81 After
the activation, MMP-11 is secreted from the cells, and MT1-
MMP is expressed on the cell membranes. Because other
proMT-MMPs, proMMP-23 and proMMP-28 also have the
motif, furin is presumably responsible for the intracellular
activation of these proMMPs.
Pericellular Activation
ProMMP-2 is unique in that it is activated pericellularly
by MT1-MMP, MT2-MMP, MT3-MMP, MT5-MMP, and
MT6-MMP, but not by ordinary MMP-activatable endo-
peptidases.13 MT4-MMP does not activate proMMP-2.
This pericellular activation has been extensively studied
with MT1-MMP and found to occur in a two-step manner.
MT1-MMP cleaves the Asn37-Leu38 bond in the propep-
tide of proMMP-2, generating an intermediate form that is
converted to a fully activated enzyme by an intermolecular
autocatalytic mechanism.70 TIMP-2 is essential to the effi-
cient pericellular activation of proMMP-2 by MT1-MMP.
The N-terminal inhibitor and C-terminal tail domains of
TIMP-2 bind to the catalytic domain of MT1-MMP and
the C-terminal hemopexin-like domain of proMMP-2,
forming a trimolecular complex of MT1-MMP/TIMP-2/
proMMP-2 on the cell membranes (see Fig. 7-2). Captur-
ing proMMP-2 by the trimolecular complex formation on
the cell membranes facilitates proMMP-2 activation by
increasing a local concentration of proMMP-2 and present-
ing it to the near, noninhibited MT1-MMP.73 Dimerization
of MT1-MMP through an interaction of the C-terminal
hemopexin-like domain is required for the efficient activa-
tion of proMMP-2 by MT1-MMP.82 MT1-MMP initiates
proMMP-2 ­activation by attacking a part of the proMMP-2
propeptide, and another already activated MMP-2 finally
activates proMMP-2 by removing a residual portion of
the propeptide. Integrins such as αvβ3 may be involved in
the process as an additional receptor for transferring acti-
vated MMP-2 to the integrin.83 MT2-MMP, MT3-MMP, or
MT5-MMP can activate proMMP-2 in the transfected
cells,37-39,84 but the activation mechanisms for these MT-
MMPs are not well understood. MT1-MMP also activates
proMMP-13 on the cell surface,85 and this activation does
not seem to require TIMP-2.86
Pericellular activation of proMMP-7 has been discovered
two-hybrid system.87 ProMMP-7 is captured on the cell
membrane by the interaction of the proMMP-7 propeptide
with the C-terminal extracellular loop of CD151, a member
of the transmembrane 4 superfamily, and is pericellularly
activated.87 This new pericellular activation of proMMP-7
requires a substrate of MMP-7. Integrins such as α3β1 and
α6β4, α chains of which interact with CD151, also may be
involved in the activation. Although the precise molecular
mechanisms of this pericellular activation system, includ-
ing the activator itself, are still unclear, proMMP-7 and
CD151 are overexpressed in osteoarthritic chondrocytes,
and proMMP-7 is activated by the interaction with CD151
in articular cartilage of osteoarthritis.88
PERICELLULAR DOCKING OF MATRIX
METALLOPROTEINASES
Discovery of membrane-anchored MMPs (i.e., MT1-,
MT2-, MT3-, MT4-, MT5-, MT6-MMPs and MMP-
23) and subsequent studies of proMMP-2 activation by
MT1-MMP have established pericellular actions of these
MMPs including MMP-2. Secreted MMPs were originally
thought to digest ECM macromolecules extracellularly
after the activation, but more recent studies have indi-
cated the possibility that they also may function on the
cell membranes through their cell surface docking.74,89,90
Besides the proMMP-2/TIMP-2/MT1-MMP system, sev-
eral secreted MMPs are reported to interact with cell
membrane proteins, which include α2 chains of integrin
α2β1 and CD147 (EMMPRIN) for MMP-1,91 αvβ3 integ-
rin and caveolin-1 for MMP-2,83,92 CD44 heparan sulfate
proteoglycan and cholesterol sulfate on the cell surface for
MMP-7,93,94 and CD44 for MMP-9.95 Because all of these
cell membrane proteins bind to active forms of the MMPs,
their proteolytic activities can be used on the cell surfaces
to digest ECM and non-ECM molecules located close to
the cell membranes. The importance of pericellular dock-
ing of secreted MMPs in arthritic tissues should be shown
by further work.
JOINT DESTRUCTION AND PROTEINASES
DEGRADATION OF EXTRACELLULAR MATRIX
IN ARTICULAR CARTILAGE
In most joint diseases, such as rheumatoid arthritis and
osteoarthritis, articular cartilage is the major target tissue
for destruction, and excessive degradation of cartilage ECM
by proteinases is a key process in the destruction. Histo-
logically, depletion of proteoglycans from articular cartilage
(degradation of proteoglycans) is a common initial change
in these joint diseases, and subsequently collagen fibrils are
degraded, leading to fibrillation and laceration secondary to
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
destruction of the arcade structures of collagen fibrils in the
articular cartilage (Fig. 7-3).
Aggrecan, a major proteoglycan in cartilage, is suscep-
tible to degradation by many proteinases, including MMPs,
ADAMTS species, neutrophil elastase, cathepsin G, and
cathepsin B. Because most of these proteinases mainly
cleave the peptide bonds located in the interglobular G1-
G2 domain, the major glycosaminoglycan-bearing aggrecan
fragments are detached from the hyaluronan attachment site
(G1 domain) after the cleavage and released from the car-
tilgae matrix. The two major aggrecan fragments with the
N-terminal sequences starting from Phe342 or Ala374 of the
core protein are detected in joint fluids from patients with
various inflammatory arthritides and osteoarthritis.96 Many
MMPs, including MMP-1, MMP-2, MMP-3, MMP-7, MMP-
8, MMP-9, MMP-13, and MT1-MMP, preferentially cleave
the Asn341-Phe342 bond (the MMP site).21 ADAMTS spe-
cies, including ADAMTS1,49 ADAMTS4,50 ADAMTS5,51
ADAMTS8, ADAMTS9, and ADAMTS15, clip the Glu373-
Ala374 bond (the aggrecanase site) in addition to other sites
in the G2-G3 domains. Members of the MMP and ADAMTS
families may play central roles in the aggrecan degradation in
arthritides. Based on the data that synthetic MMP inhibitors
do not efficiently prevent aggrecan degradation in articular
cartilage, ADAMTS species have been focused as aggre-
can-degrading proteinases in arthritides. It is not settled,
however, whether MMP and ADAMTS species play differ-
ential or complementary roles in the aggrecan degradation.
Decorin, a leucine-rich repeat proteoglycan, also is digested
by MMP-2, MMP-3, and MMP-797 and ADAMTS4.54 Infor-
mation about proteinases responsible for the degradation
of other proteoglycans, including fibromodulin, ­ lumican,
bi­glycan, PRELP (arginine-rich end leucine repeat protein),
chondroadherin, and syndecan present in articular cartilage
is limited, although fibromodulin is cleaved by C-terminus-
truncated ADAMTS4.54
Fibrillar interstitial collagens (i.e., types I, II, and III
collagens) are extremely resistant to most proteinases
because of their triple-helical structures. In general, clas-
sic collagenases, including MMP-1, MMP-8, and MMP-13,
are responsible for degradation of these collagens. Among
them, MMP-13 may be most important for the degradation
of cartilage collagen because it preferentially digests type II
collagen.11 MT1-MMP degrades types I, II, and III colla-
gens.40 Type III collagen also is susceptible to degradation by
MMP-3,80 MMP-9,14 MT3-MMP,42 and neutrophil elastase.
Cleavage of the telopeptides by the telopeptidase activity of
MMP-3,19 MMP-9,14 neutrophil elastase, cathepsin G, and
cysteine proteinase cathepsins is important for depolymer-
ization of the cross-linked collagens.1 When the collagen
molecules are cleaved, the helical structures are unwound at
37°C (i.e., body temperature) and become denatured into
gelatins, which are digested into smaller peptides by gelatin-
ases (MMP-2 and MMP-9)13,14 and other nonspecific tissue
proteinases. Type V collagen is readily digested by MMP-213
and MMP-9.14 In contrast, type VI collagen is resistant to
most MMPs, including MMP-1, MMP-2, MMP-3, MMP-
7, MMP-9, MMP-10, and MT1-MMP, but is susceptible to
neutrophil elastase, cathepsin G, chymase, and tryptase.7
Type IX collagen is degraded by MMP-3.19 Type X collagen
is susceptible to MMP-1 and MMP-2, and type XI collagen
is degraded by MMP-2.
129
Cartilage
destruction
in rheumatoid
arthritis
Synovium
Normal (a) Proteinases (b) Proteolytic
cartilage in synovial fluid synovium
and pannus
I
II
(c) Proteinases
III from
chondrocytes
Pannus
IV
Figure 7-3  Structure of normal articular cartilage and its destruction by
proteinases in rheumatoid arthritis. Normal articular cartilage is divided
into four zones (I, II, III, and IV). Collagen fibrils are aligned parallel to the
articular surface in the superficial zone, and they blend with radial fibers
and form plates or sheets sweeping vertically through the middle zone,
showing arcade structures that originate from the calcified zone (IV). In
rheumatoid arthritis, synovial tissue cells and inflammatory cells pro-
duce various proteinases, most of which are secreted into synovial fluid.
These proteinases in synovial fluid attack the surface of the articular car-
tilage from the synovial fluid (a). At the periphery of the articular surface,
proteolytic synovium degrades cartilage by direct contact, and pannus
­tissue covers and invades cartilage (b). Chondrocytes, which secrete pro-
teinases by stimulation with various cytokines and growth factors, also
are implicated in cartilage destruction (c).
Fibronectin is degraded by many MMPs, including MMP-
2, MMP-3, MMP-7, MMP-10, MMP-11, MMP-13, MMP-
19, MT1-MMP, MT2-MMP, MT3-MMP, and other serine
proteinases. Link protein also is susceptible to many pro-
teinases, such as MMP-1, MMP-2, MMP-3, MMP-7, MMP-
8, MMP-9, MMP-10, neutrophil elastase, and cathepsin G.
COMP is digested by MMP-19 and MMP-20.35 Proteinases
capable of digesting cartilage matrix protein and cartilage
intermediate layer protein are unknown, however.
CARTILAGE DESTRUCTION BY PROTEINASES
IN RHEUMATOID ARTHRITIS
Articular cartilage in rheumatoid arthritis is destroyed by
proteinases in three pathways: (1) destruction from sur-
faces of articular cartilage by proteinases present in synovial
fluid, (2) destruction through direct contact of proteolytic
synovium or pannus tissue or both to articular cartilage, and
(3) intrinsic destruction by proteinases derived from chon-
drocytes (see Fig. 7-3).
Rheumatoid arthritis is characterized by chronic prolif-
erative synovitis, which shows hyperplasia of the synovial
lining cells, inflammatory cell infiltration, and angiogen-
esis in the sublining cell layer. Hyperplastic synovial lining
cells overproduce MMP-1, MMP-3, MMP-9, MT1-MMP,
and ADAMTS4 and TIMP-1 and TIMP-3.1,98-100 Sublining
fibroblasts produce MMP-2 and TIMP-2.1 Polymorpho-
nuclear leukocytes infiltrated in the synovium and joint
cavity contain MMP-8 in the specific granules and MMP-9
 130 OKada  |  Proteinases and Matrix Degradation

and neutrophil elastase, cathepsin G, and proteinase 3 in after degradation of cartilage ECM, death of chondrocytes
the azurophil granules. They are released from cells during occurs, leading to further progressive cartilage destruction.
phagocytosis of tissue debris and immune complexes. Other
inflammatory cells in the synovium include macrophages,
MONITORING OF RHEUMATOID SYNOVITIS
lymphocytes, and mast cells. Macrophages produce MMP-
BY SERUM MMP-3 LEVELS
1, MMP-9, TIMP-1, and TIMP-2. uPA and cathepsins B,
L, and D also are secreted from activated macrophages. The data that rheumatoid synovium produces and secretes
T lymphocytes in the synovium synthesize MMP-9. Chymase many proteinases into the extracellular milieu suggest that
and tryptase are degranulated from mast cells in response to measuring of the proteinases in joint fluid or serum samples is
activation by immune complexes. Endothelial cells express a useful method to monitor the activity of rheumatoid syno-
many MMPs, including MMP-1, MMP-2, MMP-3, MMP-9, vitis. Among the proteinases, measuring of serum MMP-3
and MT1-MMP; tPA; and their inhibitors. These protein- levels has been established and used as a monitoring system
ases may be involved in tissue remodeling during angiogen- for rheumatoid synovitis.103,104 Serum MMP-3 concentra-
esis in the synovium instead of cartilage destruction. tions are known to increase in rheumatoid patients even at
All of these proteinases and inhibitors produced by syno- the early stage of the disease and become an indicator of joint
vial tissue cells and inflammatory cells seem to be secreted destruction in patients with early rheumatoid arthritis.103
into the synovial fluid and attack the surfaces of articular MMP-3 levels also reflect the effectiveness of antirheumatic
cartilage when active proteinases overwhelm inhibitors. therapy, such as anti–TNF-α antibody treatment105 and
MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, TIMP-1, and arthroplasty of major joints such as knee and hip joints.104
TIMP-2 are detectable in rheumatoid synovial fluids, and the Although the concentrations increase in some patients with
molar ratios of MMPs to TIMPs correlate with metallopro- systemic lupus erythematosus, connective tissue diseases, or
teinase activity, which is detectable in rheumatoid synovial glomerulonephritis and secondary to corticosteroid therapy
fluids.101 MMPs are thought to be in favor of proteinase in in patients with rheumatoid arthritis, determination of
rheumatoid synovial fluids. The cartilage in the central part serum MMP-3 levels is a simple and noninvasive method for
of the articular surface shows surface irregularity (fibrilla- monitoring synovial inflammation and pathologic processes
tion) and proteoglycan depletion without being covered by underlying joint destruction in rheumatoid arthritis.104
pannus tissue even in the early stage of rheumatoid arthritis
(Fig. 7-4). This cartilage degradation may be ascribed to the BONE RESORPTION IN RHEUMATOID ARTHRITIS
proteolytic damage by the action of the proteinases present
in synovial fluid (see Fig. 7-3). Bone is resorbed by osteoclasts even in the early stage of
Articular cartilage at the margins of the articular surface, rheumatoid arthritis. This is commonly observed at the
to which synovial tissue can directly attach, is progressively bare zone, where pannus-like granulation tissue invades the
degraded even in the early stage (see Fig. 7-4). Because bone marrow and destroys subchondral bone. Activated
rheumatoid synovial lining cells exhibit strong gelatinolytic osteoclasts attach to only mineralized bone matrix, and this
activity, which is probably generated through activation of
proMMP-2 by the action of MT1-MMP,98 direct contact of
the proteolytically active synovial tissue to articular carti-
lage is a destruction pathway of the cartilage (see Fig. 7-3).
Although rheumatoid synovium contains high concentra-
tions of active proteinases, the synovium can avoid the
attack by MMPs because type VI collagen, a major compo-
nent in the lining cell layer,102 is resistant to the activities of
MMP-1, MMP-2, MMP-3, and MT1-MMP.40,102 Pannus tis-
sue is a connective tissue growing from the marginal transi-
tional zone on the surface of the partially degraded articular
cartilage. It is unknown whether pannus tissue formation is
a sign of active destruction or repair of the articular carti-
lage, but the balance of data favors the former. Immunolo-
calization of MMP-1 and phagocytosis of collagen fibrils by
pannus cells at sites of pannus-cartilage junction may sug-
gest a role of the tissue in the cartilage destruction.
In addition to the extrinsic pathway for the cartilage
damage, cartilage may be destroyed by proteinases derived
from chondrocytes (see Fig. 7-3). Chondrocytes under
stimulation are capable of expressing various proteinases,
including MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, Synovium
MMP-13, MT1-MMP, MT3-MMP, ADAM9, ADAM10,
ADAM17, ADAMTS4, and other classes of proteinases. In Figure 7-4  Destruction of articular cartilage of the proximal inter-
rheumatoid arthritic cartilage, MMP-1, MMP-2, MMP-3, phalangeal joint obtained by autopsy from an early-stage rheumatoid 
patient. Articular cartilage shows fibrillation and proteoglycan deple-
MMP-7, MMP-9, MMP-13, and MT1-MMP are expressed tion at the central part of the articular surface (small arrows) and marked
in the chondrocytes located in the proteoglycan-depleted ­destruction at the marginal area, which contacts synovium (large arrows).
zone. When large areas of the cartilage surface are ulcerated Alcian blue staining for proteoglycans.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
cell-matrix contact is carried out between αvβ3 integrin of
osteoclasts and Arg-Gly-Asp (RGD) sequence of osteopon-
tin in the matrix. ECM degradation of the mineralized bone
is possible only after demineralization of the bone matrix
by proton secreted by osteoclasts because proteinases can-
not permeate the matrix components in the mineralized tis-
sues. Matrix degradation by osteoclasts is performed in the
­subosteoclastic compartments, which have acidic (pH 4 to 5)
and hypercalcemic (40 to 50 mM Ca2+) conditions.106
The major component of the ECM proteins in mature
bone is insoluble, highly cross-linked type I collagen,
although type III and V collagens also are present. Other
minor components in bone matrix are leucine-rich repeat
proteoglycans (decorin and biglycan) and glycoproteins
such as osteopontin, osteonectin (SPARC), osteocalcin
(bone Gla-protein), and thrombospondin. Among colla-
genolytic cysteine proteinases, including cathepsins B, K, L,
and S, cathepsin K is considered to be most important for
bone resorption because of its collagenolytic activity with a
broad pH optimum and selective expression in osteoclasts
and giant cells of giant cell tumors.1 Mutations of human
cathepsin K are responsible for pyknodysostosis, an auto-
somal recessive osteochondrodysplasia characterized by
osteopetrosis and short stature.107 Cathepsin K–deficient
mice have a similar phenotype. Despite the importance of
cathepsin K in bone resorption, osteoclastic bone resorp-
tion cannot be completely inhibited by cysteine protein-
ase inhibitors; it is inhibited to a similar degree by MMP
inhibitors.106 MMP-9 is highly expressed in osteoclasts in
normal and rheumatoid bones108 and giant cells of giant
cell tumors. MMP-9 has telopeptidase activity against sol-
uble and insoluble type I collagen and strong gelatinolytic
activity.14,108 ProMMP-9 is activated by acid exposure, and
when activated, it is proteolytically active under acidic and
hypercalcemic conditions.108 MMP-9-deficient mice show a
transient disturbance of growth plate development. Cathep-
sin K and MMP-9 may be involved in bone resorption in
rheumatoid arthritis. Although MT1-MMP is reportedly
expressed in osteoclasts in rheumatoid arthritis,109 evidence
of the direct involvement in osteoclastic bone resorption
is limited. A more recent study shows that prostate can-
cer–induced osteolysis is carried out via the solubilization of
receptor activator of nuclear factor кB ligand by the action
of MMP-7 expressed by osteoclasts in rodents.110 No data
are available, however, for the involvement of MMP-7 in
the osteoclastic bone resorption in rheumatoid arthritis.
CARTILAGE DESTRUCTION BY PROTEINASES
IN OSTEOARTHRITIS
In osteoarthritis, no prominent inflammatory changes
occur in the synovium during early stages of the disease,
but elevated production of enzymes by the chondrocytes
themselves contributes to the breakdown of cartilage. Many
MMPs, including MMP-1,111 MMP-2,78,112 MMP-3,113
MMP-7,114 MMP-8,111 MMP-9,112 MMP-13,10,111 and
MT1-MMP,78 are expressed in osteoarthritic cartilage.
MMP-3, MMP-7, and MT1-MMP are immunolocalized to
chondrocytes in the proteoglycan-depleted zone of osteo-
arthritic cartilage, and the levels of their staining correlate
directly with the histologic Mankin score.78,113,114 Among
the classic collagenolytic MMPs (i.e., MMP-1, MMP-8, and
131
MMP-13), MMP-13 may be most important for degradation
of the cartilage collagen because of preferential digestion of
type II collagen over type I and III collagens.10,11 Because
MT1-MMP efficiently activates proMMP-2 within the
osteoarthritic cartilage,78 and it can activate proMMP-13,86
MT1-MMP may play a key role in cartilage degradation
through activation of proMMP-2 and proMMP-13 and its
own proteolytic activity against cartilage ECM. Considering
intermolecular activation cascade for proMMPs by active
MMPs, another key MMP in osteoarthritic cartilage tis-
sue is MMP-3, which can activate proMMP-1, proMMP-7,
proMMP-8, proMMP-9, and proMMP-13. MMP-3 not only
digests many cartilage ECM components such as aggrecan,
type IX collagen, and link protein, but also activates those
proMMPs. ProMMP-7 is activated pericellularly after being
anchored via the complex formation with CD151 in osteo-
arthritic chondrocytes.88 Because CD151 immunoreactivity
directly correlates with the Mankin score and the degree of
chondrocyte cloning, and MMP-7 not only digests cartilage
ECM, but also sheds precursors of growth factors such as
HB-EGF, MMP-7 may be involved in cartilage destruction
or chondrocyte cloning or both.88
Other proteinases implicated in cartilage destruction in
osteoarthritis are members of the ADAMTS family. Chon-
drocytes are known to express ADAMTS1, ADAMTS4,
and ADAMTS5. Based on the data that the cartilage
destruction in experimental osteoarthritis is prevented in
ADAMTS5 knockout mice, but not ADAMTS4 knock-
out mice, ADAMTS5 is considered to play a key role in
the cartilage destruction in mice.115 Information about
the expression of ADAMTS species and regulation of the
activities in human osteoarthritic cartilage is still limited,
however. Members of the membrane-type ADAM family
(i.e., ADAM10, ADAM12, ADAM15, and ADAM17) are
expressed in osteoarthritic cartilage, but the functions of
these ADAMs in osteoarthritic cartilage are unknown.
REFERENCES
  1. Okada Y: Proteinases and matrix degradation. In Ruddy S, Harris
ED Jr, Sledge CB (eds): Kelley’s Textbook of Rheumatology, 6th ed.
Philadelphia, WB Saunders, 2001, pp 55-72.
  2. Okada Y: Proteinases and matrix degradation. In Harris ED Jr, Budd
RC, Firestein GS, et al (eds): Kelley’s Textbook of Rheumatology,
7th ed. Philadelphia, WB Saunders, 2005, pp 63-81.
  3. Handley CJ, Mok MT, Ilic MZ, et al: Cathepsin D cleaves aggrecan at
unique sites within the interglobular domain and chondroitin sulfate
attachment regions that are also cleaved when cartilage is maintained
at acid pH. Matrix Biol 20:543-553, 2001.
  4. Bromme D, Okamoto K, Wang BB, et al: Human cathepsin O2, a
matrix protein-degrading cysteine protease expressed in osteoclasts:
Functional expression of human cathepsin O2 in Spodoptera frugi-
perda and characterization of the enzyme. J Biol Chem 271:2126-
2132, 1996.
  5. Sorimachi H, Suzuki K: The structure of calpain. J Biochem (Tokyo)
129:653-664, 2001.
  6. Nagase H: Activation mechanisms of matrix metalloproteinases.
Biol Chem 378:151-160, 1997.
  7. Kielty CM, Lees M, Shuttleworth CA, et al: Catabolism of intact
type VI collagen microfibrils: Susceptibility to degradation by serine
proteinases. Biochem Biophys Res Commun 191:1230-1236, 1993.
  8. Saksela O, Rifkin DB: Cell-associated plasminogen activation:
Regulation and physiological functions. Annu Rev Cell Biol 4:93-
126, 1988.
  9. Chung L, Dinakarpandian D, Yoshida N, et al: Collagenase unwinds
triple-helical collagen prior to peptide bond hydrolysis. EMBO J
23:3020-3030, 2004.
 132 OKada  |  Proteinases and Matrix Degradation
10. Mitchell PG, Magna HA, Reeves LM, et al: Cloning, expression, and
type II collagenolytic activity of matrix metalloproteinase-13 from
human osteoarthritic cartilage. J Clin Invest 97:761-768, 1996.
11. Knauper V, Lopez-Otin C, Smith B, et al: Biochemical characteriza-
tion of human collagenase-3. J Biol Chem 271:1544-1550, 1996.
12. Balbin M, Fueyo A, Knauper V, et al: Identification and enzymatic
characterization of two diverging murine counterparts of human
interstitial collagenase (MMP-1) expressed at sites of embryo implan-
tation. J Biol Chem 276:10253-10262, 2001.
13. Okada Y, Morodomi T, Enghild JJ, et al: Matrix metalloproteinase 2
from human rheumatoid synovial fibroblasts: Purification and acti-
vation of the precursor and enzymic properties. Eur J Biochem 194:
721-730, 1990.
14. Okada Y, Gonoji Y, Naka K, et al: Matrix metalloproteinase 9 (92-
kDa gelatinase/type IV collagenase) from HT 1080 human fibrosar-
coma cells: Purification and activation of the precursor and enzymic
properties. J Biol Chem 267:21712-21719, 1992.
15. Heissig B, Hattori K, Dias S, et al: Recruitment of stem and pro-
genitor cells from the bone marrow niche requires MMP-9 mediated
release of kit-ligand. Cell 109:625-637, 2002.
16. McQuibban GA, Gong JH, Tam EM, et al: Inflammation dampened
by gelatinase A cleavage of monocyte chemoattractant protein-3.
Science 289:1202-1206, 2000.
17. Nagase H, Ogata Y, Suzuki K, et al: Substrate specificities and activa-
tion mechanisms of matrix metalloproteinases. Biochem Soc Trans
19:715-718, 1991.
18. Okada Y, Nagase H, Harris ED Jr: A metalloproteinase from human
rheumatoid synovial fibroblasts that digests connective tissue
matrix components: Purification and characterization. J Biol Chem
261:14245-14255, 1986.
19. Wu J, Lark MW, Chun LE, et al: Sites of stromelysin cleavage
in collagen types II, IX, X, and XI of cartilage. J Biol Chem 266:
5625-5628, 1991.
20. Nakamura H, Fujii Y, Ohuchi E, et al: Activation of the precursor of
human stromelysin 2 and its interactions with other matrix metal-
loproteinases. Eur J Biochem 253:67-75, 1998.
21. Fosang AJ, Neame PJ, Last K, et al: The interglobular domain of
cartilage aggrecan is cleaved by PUMP, gelatinases, and cathepsin B.
J Biol Chem 267:19470-19474, 1992.
22. Imai K, Yokohama Y, Nakanishi I, et al: Matrix metalloproteinase 7
(matrilysin) from human rectal carcinoma cells: Activation of the
precursor, interaction with other matrix metalloproteinases and enzy-
mic properties. J Biol Chem 270:6691-6697, 1995.
23. Uria JA, Lopez-Otin C: Matrilysin-2, a new matrix metalloprotein-
ase expressed in human tumors and showing the minimal domain
organization required for secretion, latency, and activity. Cancer Res
60:4745-4751, 2000.
24. Park HI, Ni J, Gerkema FE, et al: Identification and characterization
of human endometase (matrix metalloproteinase-26) from endome-
trial tumor. J Biol Chem 275:20540-20544, 2000.
25. Marchenko GN, Ratnikov BI, Rozanov DV, et al: Characterization
of matrix metalloproteinase-26, a novel metalloproteinase widely
expr­essed in cancer cells of epithelial origin. Biochem J 356:705-718,
2001.
26. Pei D, Weiss SJ: Furin-dependent intracellular activation of the
human stromelysin-3 zymogen. Nature 375:244-247, 1995.
27. Noel A, Santavicca M, Stoll I, et al: Identification of structural deter-
minants controlling human and mouse stromelysin-3 proteolytic
activities. J Biol Chem 270:22866-22872, 1995.
28. Pei D, Majmudar G, Weiss SJ: Hydrolytic inactivation of a breast
carcinoma cell-derived serpin by human stromelysin-3. J Biol Chem
269:25849-25855, 1994.
29. Manes S, Mira E, Barbacid MM, et al: Identification of insulin-like
growth factor-binding protein-1 as a potential physiological substrate
for human stromelysin-3. J Biol Chem 272:25706-25712, 1997.
30. Lohi J, Wilson CL, Roby JD, et al: Epilysin, a novel human matrix
metalloproteinase (MMP-28) expressed in testis and keratinocytes
and in response to injury. J Biol Chem 276:10134-10144, 2001.
31. Shapiro SD, Kobayashi DK, Ley TJ: Cloning and characterization of
a unique elastolytic metalloproteinase produced by human alveolar
macrophages. J Biol Chem 268:23824-23829, 1993.
32. Pendas AM, Knauper V, Puente XS, et al: Identification and char-
acterization of a novel human matrix metalloproteinase with unique
structural characteristics, chromosomal location, and tissue distribu-
tion. J Biol Chem 272:4281-4286, 1997.
33. Llano E, Pendas AM, Knauper V, et al: Identification and structural
and functional characterization of human enamelysin (MMP-20).
Biochemistry 36:15101-15108, 1997.
34. Stracke JO, Hutton M, Stewart M, et al: Biochemical character-
ization of the catalytic domain of human matrix metalloproteinase
19: Evidence for a role as a potent basement membrane degrading
enzyme. J Biol Chem 275:14809-14816, 2000.
35. Stracke JO, Fosang AJ, Last K, et al: Matrix metalloproteinases
19 and 20 cleave aggrecan and cartilage oligomeric matrix protein
(COMP). FEBS Lett 478:52-56, 2000.
36. Sato H, Takino T, Okada Y, et al: A matrix metalloproteinase
expressed on the surface of invasive tumour cells. Nature 370:61-65,
1994.
37. Will H, Hinzmann B: cDNA sequence and mRNA tissue distribution
of a novel human matrix metalloproteinase with a potential trans-
membrane segment. Eur J Biochem 231:602-608, 1995.
38. Takino T, Sato H, Shinagawa A, et al: Identification of the second
membrane-type matrix metalloproteinase (MT- MMP-2) gene from a
human placenta cDNA library: MT-MMPs form a unique membrane-
type subclass in the MMP family. J Biol Chem 270:23013-23020,
1995.
39. Pei D: Identification and characterization of the fifth membrane-type
matrix metalloproteinase MT5-MMP. J Biol Chem 274:8925-8932,
1999.
40. Ohuchi E, Imai K, Fujii Y, et al: Membrane type 1 matrix metallo-
proteinase digests interstitial collagens and other extracellular matrix
macromolecules. J Biol Chem 272:2446-2451, 1997.
41. d’Ortho MP, Will H, Atkinson S, et al: Membrane-type matrix
metalloproteinases 1 and 2 exhibit broad-spectrum proteolytic capac-
ities comparable to many matrix metalloproteinases. Eur J Biochem
250:751-757, 1997.
42. Shimada T, Nakamura H, Ohuchi E, et al: Characterization of a trun-
cated recombinant form of human membrane type 3 matrix metal-
loproteinase. Eur J Biochem 262:907-914, 1999.
43. Itoh Y, Kajita M, Kinoh H, et al: Membrane type 4 matrix metallo-
proteinase (MT4-MMP, MMP-17) is a glycosylphosphatidylinositol-
anchored proteinase. J Biol Chem 274:34260-34266, 1999.
44. Pei D: Leukolysin/MMP25/MT6-MMP: A novel matrix metallo-
proteinase specifically expressed in the leukocyte lineage. Cell Res
9:291-303, 1999.
45. Wang Y, Johnson AR, Ye QZ, et al: Catalytic activities and substrate
specificity of the human membrane type 4 matrix metalloproteinase
catalytic domain. J Biol Chem 274:33043-33049, 1999.
46. English WR, Puente XS, Freije JM, et al: Membrane type 4 matrix
metalloproteinase (MMP17) has tumor necrosis factor-alpha con-
vertase activity but does not activate pro-MMP2. J Biol Chem
275:14046-14055, 2000.
47. Pei D, Kang T, Qi H: Cysteine array matrix metalloproteinase (CA-
MMP)/MMP-23 is a type II transmembrane matrix metalloprotein-
ase regulated by a single cleavage for both secretion and activation.
J Biol Chem 275:33988-33997, 2000.
48. Velasco G, Pendas AM, Fueyo A, et al: Cloning and characterization
of human MMP-23, a new matrix metalloproteinase predominantly
expressed in reproductive tissues and lacking conserved domains in
other family members. J Biol Chem 274:4570-4576, 1999.
49. Rodriguez-Manzaneque JC, Westling J, Thai SN, et al: ADAMTS1
cleaves aggrecan at multiple sites and is differentially inhibited
by metalloproteinase inhibitors. Biochem Biophys Res Commun
293:501-508, 2002.
50. Tortorella MD, Burn TC, Pratta MA, et al: Purification and cloning
of aggrecanase-1: A member of the ADAMTS family of proteins. Sci-
ence 284:1664-1666, 1999.
51. Abbaszade I, Liu RQ, Yang F, et al: Cloning and characterization
of ADAMTS11, an aggrecanase from the ADAMTS family. J Biol
Chem 274:23443-23450, 1999.
52. Sandy JD, Westling J, Kenagy RD, et al: Versican V1 proteolysis in
human aorta in vivo occurs at the Glu441-Ala442 bond, a site that is
cleaved by recombinant ADAMTS-1 and ADAMTS-4. J Biol Chem
276:13372-13378, 2001.
53. Nakamura H, Fujii Y, Inoki I, et al: Brevican is degraded by matrix
metalloproteinases and aggrecanase-1 (ADAMTS4) at different sites.
J Biol Chem 275:38885-38890, 2000.
54. Kashiwagi M, Enghild JJ, Gendron C, et al: Altered proteolytic activ-
ities of ADAMTS-4 expressed by C-terminal processing. J Biol Chem
279:10109-10119, 2004.
 PART 1  |  STRUCTURE AND FUNCTION OF BONE, JOINTS, AND CONNECTIVE TISSUE
55. Peschon JJ, Slack JL, Reddy P, et al: An essential role for ectodo-
main shedding in mammalian development. Science 282:1281-1284,
1998.
56. Sunnarborg SW, Hinkle CL, Stevenson M, et al: Tumor necrosis
factor-alpha converting enzyme (TACE) regulates epidermal growth
factor receptor ligand availability. J Biol Chem 277:12838-12845,
2002.
57. Black RA: Tumor necrosis factor-alpha converting enzyme. Int J Bio-
chem Cell Biol 34:1-5, 2002.
58. Yan Y, Shirakabe K, Werb Z: The metalloprotease Kuzbanian
(ADAM10) mediates the transactivation of EGF receptor by G pro-
tein-coupled receptors. J Cell Biol 158:221-226, 2002.
59. Asakura M, Kitakaze M, Takashima S, et al: Cardiac hypertrophy
is inhibited by antagonism of ADAM12 processing of HB-EGF:
Metalloproteinase inhibitors as a new therapy. Nat Med 8:35-40,
2002.
60. Mochizuki S, Shimoda M, Shiomi T, et al: ADAM28 is activated by
MMP-7 (matrilysin-1) and cleaves insulin-like growth factor binding
protein-3. Biochem Biophys Res Commun 315:79-84, 2004.
61. Loechel F, Fox JW, Murphy G, et al: ADAM 12-S cleaves IGFBP-
3 and IGFBP-5 and is inhibited by TIMP-3. Biochem Biophys Res
Commun 278:511-515, 2000.
62. Fourie AM, Coles F, Moreno V, et al: Catalytic activity of ADAM8,
ADAM15, and MDC-L (ADAM28) on synthetic peptide substrates
and in ectodomain cleavage of CD23. J Biol Chem 278:30469-30477,
2003.
63. Reiss K, Ludwig A, Saftig P: Breaking up the tie: Disintegrin-like
metalloproteinases as regulators of cell migration in inflammation
and invasion. Pharmacol Ther 111:985-1006, 2006.
64. Potempa J, Korzus E, Travis J: The serpin superfamily of protein-
ase inhibitors: Structure, function, and regulation. J Biol Chem
269:15957-15960, 1994.
65. Murphy G, Willenbrock F: Tissue inhibitors of matrix metalloen-
dopeptidases. Methods Enzymol 248:496-510, 1995.
66. Gomez DE, Alonso DF, Yoshiji H, et al: Tissue inhibitors of metallo-
proteinases: Structure, regulation and biological functions. Eur J Cell
Biol 74:111-122, 1997.
67. Bode W, Fernandez-Catalan C, Tschesche H, et al: Structural prop-
erties of matrix metalloproteinases. Cell Mol Life Sci 55:639-652,
1999.
68. Brew K, Dinakarpandian D, Nagase H: Tissue inhibitors of metal-
loproteinases: Evolution, structure and function. Biochim Biophys
Acta 1477:267-283, 2000.
69. Visse R, Nagase H: Matrix metalloproteinases and tissue inhibitors of
metalloproteinases: Structure, function, and biochemistry. Circ Res
92:827-839, 2003.
70. Will H, Atkinson SJ, Butler GS, et al: The soluble catalytic domain
of membrane type 1 matrix metalloproteinase cleaves the propeptide
of progelatinase A and initiates autoproteolytic activation: Regula-
tion by TIMP-2 and TIMP-3. J Biol Chem 271:17119-17123, 1996.
71. Gomis-Ruth FX, Maskos K, Betz M, et al: Mechanism of inhibition
of the human matrix metalloproteinase stromelysin-1 by TIMP-1.
Nature 389:77-81, 1997.
72. Fernandez-Catalan C, Bode W, Huber R, et al: Crystal structure of
the complex formed by the membrane type 1-matrix metalloprotein-
ase with the tissue inhibitor of metalloproteinases-2, the soluble pro-
gelatinase A receptor. EMBO J 17:5238-5248, 1998.
73. Kinoshita T, Sato H, Okada A, et al: TIMP-2 promotes activation
of progelatinase A by membrane-type 1 matrix metalloproteinase
immobilized on agarose beads. J Biol Chem 273:16098-16103, 1998.
74. Seiki M: The cell surface: The stage for matrix metalloproteinase
regulation of migration. Curr Opin Cell Biol 14:624-632, 2002.
75. Huovila AP, Turner AJ, Pelto-Huikko M, et al: Shedding light on
ADAM metalloproteinases. Trends Biochem Sci 30:413-422, 2005.
76. Wei S, Kashiwagi M, Kota S, et al: Reactive site mutations in tissue
inhibitor of metalloproteinase-3 disrupt inhibition of matrix metal-
loproteinases but not tumor necrosis factor-alpha-converting enzyme.
J Biol Chem 280:32877-32882, 2005.
77. Oh J, Takahashi R, Kondo S, et al: The membrane-anchored MMP
inhibitor RECK is a key regulator of extracellular matrix integrity and
angiogenesis. Cell 107:789-800, 2001.
78. Imai K, Ohta S, Matsumoto T, et al: Expression of membrane-type 1
matrix metalloproteinase and activation of progelatinase A in human
osteoarthritic cartilage. Am J Pathol 151:245-256, 1997.
133
79. Gu Z, Kaul M, Yan B, et al: S-nitrosylation of matrix metallopro­
teinases: Signaling pathway to neuronal cell death. Science 297:
1186-1190, 2002.
80. Nagase H: Human stromelysins 1 and 2. Methods Enzymol 248:
449-470, 1995.
81. Sato H, Kinoshita T, Takino T, et al: Activation of a recombi-
nant membrane type 1-matrix metalloproteinase (MT1-MMP) by
furin and its interaction with tissue inhibitor of metalloproteinases
(TIMP)-2. FEBS Lett 393:101-104, 1996.
82. Itoh Y, Takamura A, Ito N, et al: Homophilic complex formation of
MT1-MMP facilitates proMMP-2 activation on the cell surface and
promotes tumor cell invasion. EMBO J 20:4782-4793, 2001.
83. Brooks PC, Stromblad S, Sanders LC, et al: Localization of matrix
metalloproteinase MMP-2 to the surface of invasive cells by interac-
tion with integrin alpha v beta 3. Cell 85:683-693, 1996.
84. Morrison CJ, Butler GS, Bigg HF, et al: Cellular activation of MMP-
2 (gelatinase A) by MT2-MMP occurs via a TIMP-2-independent
pathway. J Biol Chem 276:47402-47410, 2001.
85. Cowell S, Knauper V, Stewart ML, et al: Induction of matrix metal-
loproteinase activation cascades based on membrane-type 1 matrix
metalloproteinase: associated activation of gelatinase A, gelatinase B
and collagenase 3. Biochem J 331:453-458, 1998.
86. Knauper V, Bailey L, Worley JR, et al: Cellular activation of proMMP-
13 by MT1-MMP depends on the C-terminal domain of MMP-13.
FEBS Lett 532:127-130, 2002.
87. Shiomi T, Inoki I, Kataoka F, et al: Pericellular activation of
proMMP-7 (promatrilysin-1) through interaction with CD151. Lab
Invest 85:1489-1506, 2005.
88. Fujita Y, Shiomi T, Yanagimoto S, et al: Tetraspanin CD151 is
expressed in osteoarthritic cartilage and is involved in pericellular
activation of pro-matrix metalloproteinase 7 in osteoarthritic chon-
drocytes. Arthritis Rheum 54:3233-3243, 2006.
89. Sternlicht MD, Werb Z: How matrix metalloproteinases regulate cell
behavior. Annu Rev Cell Dev Biol 17:463-516, 2001.
90. Egeblad M, Werb Z: New functions for the matrix metalloproteinases
in cancer progression. Nat Rev Cancer 2:161-174, 2002.
91. Guo H, Li R, Zucker S, et al: EMMPRIN (CD147), an inducer of
matrix metalloproteinase synthesis, also binds interstitial collagenase
to the tumor cell surface. Cancer Res 60:888-891, 2000.
92. Puyraimond A, Fridman R, Lemesle M, et al: MMP-2 colocalizes with
caveolae on the surface of endothelial cells. Exp Cell Res 262:28-36,
2001.
93. Yu WH, Woessner JF Jr, McNeish JD, et al: CD44 anchors the assem-
bly of matrilysin/MMP-7 with heparin-binding epidermal growth
factor precursor and ErbB4 and regulates female reproductive organ
remodeling. Genes Dev 16:307-323, 2002.
94. Yamamoto K, Higashi S, Kioi M, et al: Binding of active matrilysin
to cell surface cholesterol sulfate is essential for its membrane-asso-
ciated proteolytic action and induction of homotypic cell adhesion.
J Biol Chem 281:9170-9180, 2006.
95. Yu Q, Stamenkovic I: Cell surface-localized matrix metalloprotein-
ase-9 proteolytically activates TGF-beta and promotes tumor inva-
sion and angiogenesis. Genes Dev 14:163-176, 2000.
96. Fosang AJ, Last K, Maciewicz RA: Aggrecan is degraded by matrix
metalloproteinases in human arthritis: Evidence that matrix metal-
loproteinase and aggrecanase activities can be independent. J Clin
Invest 98:2292-2299, 1996.
97. Imai K, Hiramatsu A, Fukushima D, et al: Degradation of decorin by
matrix metalloproteinases: Identification of the cleavage sites, kinetic
analyses and transforming growth factor-beta1 release. Biochem J
322:809-814, 1997.
98. Yamanaka H, Makino K, Takizawa M, et al: Expression and tissue
localization of membrane-types 1, 2, and 3 matrix metalloproteinases
in rheumatoid synovium. Lab Invest 80:677-687, 2000.
99. Yamanishi Y, Boyle DL, Clark M, et al: Expression and regulation
of aggrecanase in arthritis: The role of TGF-beta. J Immunol 168:
1405-1412, 2002.
100. Takizawa M, Ohuchi E, Yamanaka H, et al: Production of tissue
inhibitor of metalloproteinases 3 is selectively enhanced by calcium
pentosan polysulfate in human rheumatoid synovial fibroblasts.
Arthritis Rheum 43:812-820, 2000.
101. Yoshihara Y, Nakamura H, Obata K, et al: Matrix metalloprotein-
ases and tissue inhibitors of metalloproteinases in synovial fluids
from patients with rheumatoid arthritis or osteoarthritis. Ann
Rheum Dis 59:455-461, 2000.
 134 OKada  |  Proteinases and Matrix Degradation
102. Okada Y, Naka K, Minamoto T, et al: Localization of type VI col-
lagen in the lining cell layer of normal and rheumatoid synovium.
Lab Invest 63:647-656, 1990.
103. Yamanaka H, Matsuda Y, Tanaka M, et al: Serum matrix metallo-
proteinase 3 as a predictor of the degree of joint destruction during
the six months after measurement, in patients with early rheumatoid
arthritis. Arthritis Rheum 43:852-858, 2000.
104. Kobayashi A, Naito S, Enomoto H, et al: Serum MMP-3 levels of
matrix metalloproteinase-3 (stromelysin 1) for monitoring synovitis
in rheumatoid arthritis. Arch Pathol Lab Med 131:563-570, 2007.
105. Catrina AI, Lampa J, Ernestam S, et al: Anti-tumour necrosis fac-
tor (TNF)-alpha therapy (etanercept) down-regulates serum matrix
metalloproteinase (MMP)-3 and MMP-1 in rheumatoid arthritis.
Rheumatology (Oxford) 41:484-489, 2002.
106. Delaisse J, Vaes G: Mechanism of mineral solubilization and matrix
degradation in osteoclastic bone resorption. In Rifkin BR, Gay CV
(eds): Biology and Physiology of the Osteoclast. Boca Raton, CRC
Press, 1992, pp 289-314.
107. Gelb BD, Shi GP, Chapman HA, et al: Pycnodysostosis, a lysosomal
disease caused by cathepsin K deficiency. Science 273:1236-1238,
1996.
108. Okada Y, Naka K, Kawamura K, et al: Localization of matrix
metalloproteinase 9 (92-kilodalton gelatinase/type IV collagenase =
gelatinase B) in osteoclasts: Implications for bone resorption. Lab
Invest 72:311-322, 1995.
109. Pap T, Shigeyama Y, Kuchen S, et al: Differential expression pattern
of membrane-type matrix metalloproteinases in rheumatoid arthritis.
Arthritis Rheum 43:1226-1232, 2000.
110. Lynch CC, Hikosaka A, Acuff HB, et al: MMP-7 promotes prostate
cancer-induced osteolysis via the solubilization of RANKL. Cancer
Cell 7:485-496, 2005.
111. Shlopov BV, Lie WR, Mainardi CL, et al: Osteoarthritic lesions:
Involvement of three different collagenases. Arthritis Rheum
40:2065-2074, 1997.
112. Mohtai M, Smith RL, Schurman DJ, et al: Expression of 92-kD type
IV collagenase/gelatinase (gelatinase B) in osteoarthritic cartilage
and its induction in normal human articular cartilage by interleukin
1. J Clin Invest 92:179-185, 1993.
113. Okada Y, Shinmei M, Tanaka O, et al: Localization of matrix metal-
loproteinase 3 (stromelysin) in osteoarthritic cartilage and synovium.
Lab Invest 66:680-690, 1992.
114. Ohta S, Imai K, Yamashita K, et al: Expression of matrix metallo-
proteinase 7 (matrilysin) in human osteoarthritic cartilage. Lab
Invest 78:79-87, 1998.
115. Glasson SS, Askew R, Sheppard B, et al: Deletion of active
ADAMTS5 prevents cartilage degradation in a murine model of
osteoarthritis. Nature 434:644-648, 2005.
 Part CELLS INVOLVED IN AUTOIMMUNE
2 DISEASES AND INFLAMMATION
8 Mononuclear
Key Points
Macrophages play an important role in tissue homeostasis
and in cellular and humoral innate and adaptive immunity.
Macrophages, dendritic cells, and osteoclasts derive from
blood monocytes.
Tissue macrophages display marked phenotypic
­heterogeneity, depending on their local microenvironment.
Distinct subsets of monocytes are recruited to inflammatory
sites in tissues.
Microbes and cytokines (e.g., interferon-γ, interleukin [IL]-4,
IL-10) regulate macrophage activation selectively.
Macrophages express a range of opsonic and nonopsonic
receptors to recognize and take up foreign and altered host
components.
Toll-like membrane and NOD-like cytosolic receptors induce
alterations in macrophage gene expression and secretion of
inflammatory mediators.
Macrophages and their products contribute to tissue damage
and repair and chronic inflammation and autoimmunity.
Mononuclear phagocytes are widely distributed, biosyntheti-
cally active cells derived from hematopoietic precursors that
circulate as monocytes and enter tissues constitutively and
in response to inflammatory stimuli. In connective tissue
and bone, they play a major role in homeostasis, growth, and
remodeling, as mature macrophages and osteoclasts (Fig. 8-1).
Myeloid dendritic cells (DCs) represent a distinct form of
differentiation, specialized to maintain immune tolerance or
to induce humoral and cellular immunity through B lympho-
cytes and T lymphocytes. Through their recognition, antigen-
presenting, regulatory, and effector mechanisms, mononuclear
phagocytes contribute to a range of inflammatory, infectious,
autoimmune, metabolic, and degenerative rheumatic ­diseases,
providing targets for therapeutic intervention.
Growth of knowledge in mononuclear phagocyte biology
has developed in close association with understanding of
pathogenesis and treatment of chronic arthritis. (See Table 8-1
Phagocytes
in Rheumatic
Diseases
SIAMON GORDON
for a selective review of historical milestones.) Relevant
examples include the development of steroidal and nonste-
roidal anti-inflammatory agents and of anti–tumor necrosis
factor (TNF)-α monoclonal antibodies.1 Genetic lesions
in macrophage colony-stimulating factor (M-CSF) cause
osteoclast deficiency and osteopetrosis in mouse models,
whereas human mutations in cytosolic Nucleotide Oligo-
merisation Domain (NOD)-like receptors (NLRs) result in
interleukin (IL)-1β overproduction and hyperinflammatory
syndromes,2 often associated with persistent joint disease.
Immune complex deposition and complement activation
combine with Toll-like receptor (TLR) recognition to
induce effector pathways of tissue destruction and repair.
This chapter reviews general aspects of mononuclear
phagocyte differentiation, recruitment, and activation, inte-
grating the properties of different cellular subtypes. Studies
on the biology of macrophages, DCs, and osteoclasts have
diverged to the extent that common features have been
overlooked in the understandable search for specificity. This
review is an attempt, in part, to reintegrate these differen-
tiated sublineages. General features that are especially rel-
evant to physiologic and pathologic consequences of their
presence in bone, joints, and connective tissues are discussed,
and gaps in our knowledge are pointed out. The emphasis is
on studies on humans, where available, with reference to
murine models where applicable. The subject area encom-
passes innate3,4 and acquired immunity, autoimmunity, and
“osteoimmunology,”5 a reflection of local specialization of
mononuclear phagocyte and lymphocyte biology. Relevant
topics discussed elsewhere in this textbook include innate
immunity (see Chapter 16), cytokines and chemokines (see
Chapter 23), osteoclast functions (see Chapter 4) and anti-
TNF-α therapy (see Chapter 58). For further details in con-
nection with macrophage6-10 and DC biology,11 see reviews
cited in this chapter.
OVERVIEW
Circulating monocytes give rise to tissue macrophages,
which display considerable phenotypic microheterogene-
ity in different organs.12 Similarly, and with some overlap
in properties, myeloid DCs are present as heterogeneous
135
 136 GORDON  |  Mononuclear Phagocytes in Rheumatic Diseases
N vated T lymphocytes and their products, such as interferon-γ,
Ly
M
Figure 8-1  Thin section of a bone marrow–derived mouse macro-
phage. Cells were cultured for 7 days, fixed, and processed for conven-
tional electron microscopy. Ly, lysosome; M, mitochondrion; N, nucleus.
Bar = 2 μm. (Courtesy of Chantal de Chastellier, Centre d’Immunologie de
Marseille-Luminy, Marseille, France).
sentinel cells at mucosal and cutaneous surfaces,13-16 under-
going a complex maturation process as they migrate to lym-
phoid organs after capture of antigens. Plasmacytoid DCs
may represent a distinct sublineage, specialized to produce
high levels of type I interferon in response to viral stimuli.17
Mononuclear precursors in the blood give rise to multinu-
cleated osteoclasts, specialized to resorb bone.18 Circulating
monocytes are themselves heterogeneous, using distinct
chemokine and adhesion receptors to give rise to different
tissue mononuclear phagocytes.19 These cells differ in life
span depending on the recruitment stimuli and on local fac-
tors in their environment, and express diverse plasma mem-
brane receptors, making it possible for them to interact with
many different cell types and with microbial and modified
host components. Phagocytosis is a hallmark of the ability
of macrophages and DCs to engulf particulates, including
foreign materials, bacteria,20 and dying host cells generated
by apoptosis or necrosis.21
These particulates are recognized by nonopsonic recep-
tors, including scavenger and lectin-like receptors, or after
opsonization with antibodies or complement or both that
enhance uptake via Fc and complement receptors. In addi-
tion, other humoral proteins, such as Pentraxins, interact
with their target ligands,22 bridging them to less well-defined
macrophage receptors, which regulate early cellular responses
during innate immunity. TLRs play an important role in
sensing the nature of the captured cargo, often interacting
with the extensive repertoire of non-TLR receptors (NTRs).
An extended family of nod-like receptors (NLRs) sense
various cytosolic ligands, resulting in a complex assembly of
­proteins (inflammasomes), caspase activation, and release of
IL-1β.23 Uptake of particulate and soluble antigen, directly
or by “cross-priming,”24 induces DC maturation, ­processing,
and association with major ­ histocompatibility complex
(MHC) II molecules, and presentation of peptides to naive
CD4 T lymphocytes. Endogenously generated or foreign
peptides generated during biosynthesis of virus glycopro-
teins associate with MHC I molecules for surface recogni-
tion by cytotoxic T cells (mainly CD8). DCs and possibly
macrophages regulate T cell activation or tolerance through
additional costimulatory surface antigens and cytokines,
depending partly on concomitant TLR stimulation. Acti-
IL-4/IL-13, and IL-10, regulate the effector functions of mono-
nuclear phagocytes.
Digestion of macromolecules occurs in the vacuolar
compartment involving dynamic membrane traffic and
interactions with the cytoskeleton.25 Complex intracellu-
lar pathways transduce surface and vacuolar-derived stimuli
to initiate formation of intracellular protein signaling com-
plexes. Transcription factors translocate to the nucleus and
form activation or inhibitory chromatin-binding complexes
that regulate gene expression and biosynthesis of secretory
­products, such as TNF-α. Other, low-molecular-weight
metabolites are generated by nontranscriptional mecha-
nisms, yielding inflammatory mediators and antimicrobial
activities. Osteoclasts are able to secrete Hydrochloric  Acid
(HCL) and potent proteolytic enzymes into sealed-off local-
ized areas of bone, to which they adhere tightly through spe-
cialized podosomes and actin rings.
The secretory activities of mononuclear phagocytes influ-
ence a range of cellular and extracellular targets to main-
tain tissue homeostasis, but also are responsible for tissue
destruction. Their trophic actions,26 through cellular con-
tact with other stromal cells and extracellular matrix and
secretion, regulate tissue catabolism, cell growth, angiogen-
esis, and repair. In addition to local effects, macrophages
contribute to systemic integration of proinflammatory and
anti-­inflammatory effects, acting on the central nervous sys-
tem, endocrine organs, liver, and energy stores.
Overall, the activation phenotype of mononuclear phago­
cytes is modulated by extrinsic factors (e.g., cytokines and
hormones), by balance of surface receptors with activating/
inhibitory cytoplasmic motifs, by cytosolic regulators such
as the suppressors of cytokine synthesis (SOCS)27 proteins,
by phosphorylation/dephosphorylation of signaling mol-
ecules, and by assembly of transcription factor complexes
on chromatin. Microarray analysis of macrophages has
made it possible to distinguish characteristic signatures of
gene expression after innate activation via TLRs, deactiva-
tion via glucocorticoids, or modulation via cytokines. As a
result, the phenotype of tissue macrophages is markedly het-
erogeneous, making for complexity of function in different
sites in health and disease, but also providing opportunities
for novel state-specific or tissue-specific targeting by drugs.
LIFE HISTORY AND HETEROGENEITY
(MACROPHAGES, DENDRITIC
CELLS, AND OSTEOCLASTS)
In an adult, all three sublineages of mononuclear phago-
cytes originate from CD34+ committed progenitor cells in
the bone marrow (Fig. 8-2). These diverge from lymphoid
cells and subsequently from polymorphonuclear leukocytes,
although many genes are still expressed, but not translated,
in both types of phagocytic cell. In vitro, bone marrow and
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION 137

Table 8-1  Milestones in the History of Mononuclear Phagocytes

Subject Comment Investigators
Phagocytosis Cellular defenses in innate immunity Metchnikoff
Opsonization Humoral interactions Wright
Clearance Reticuloendothelial system Aschoff
Monocytic origin Circulating precursors,  Gowans; Ebert and Florey;  
heterogeneity Ziegler-Heitbrock; Geissmann et al
Mononuclear phagocyte   Integrative classification of diverse tissue macrophages Van Furth et al
system
Marginal zone spleen Capture capsulated bacteria Humphrey; Kraal
Osteoclasts Mononuclear origin Loutit
Dendritic cells Unique antigen-presenting cells Steinman and Cohn
Intracellular pathogens Bacille Calmette-Guérin, Toxoplasma, Listeria, Mackaness; Jones and Hirsch; North;
Legionella pneumophila Portnoy; Horwitz
Glucocorticoids and   An important, still neglected aspect of   Thompson and Van Furth
mucopolysaccharides anti-inflammatory actions
Cell biology Culture mouse/human macrophages Cohn
Endocytosis Membrane flow Steinman; Unanue
Lysosomes Fusion and acidification, inhibition Mycobacterium D’Arcy Hart
­tuberculosis (MTB): Vacuolar system, antigen degradation
Cytoskeleton Abundant Stossel
Adhesion receptors Migration Springer; Arnaout
Fc and C receptors Receptor function V. Nussenzweig; Rabinovitch; Silverstein
Opsonic phagocytosis Cloning Mellman; Unkeless; Ravetch; Aderem
Secretion Acid hydrolases Allison; Davies
Lysozyme, neutral proteinases Gordon; Werb
Reactive oxygen and nitrogen radicals Karnovsky; Babior; Rossi; Nathan; Segal
Cytokines Macrophage colony-stimulating factor growth   Stanley; Wiktor-Jedrzejczak;
factor osteopetrosis
Granulocyte-macrophage colony-stimulating factor Metcalf
Tumor necrosis factor-α role in host defense,   Cerami 
granuloma formation Kindler; Vassalli
Interferon-γ classic macrophage activation Dalton; Gray
Interleukin-4/13 alternative activation Mantovani
Inborn errors Dominant mutations leading to immunodeficiency Newport; Levine; Casanova
Gene expression PU-1 McKercher; Singh; Hume
Signaling Stats Schreiber
N-ramp Iron exchange in vacuole and genetic resistance   Skamene; Gros; Bradley; Blackwell
to intracellular pathogens
Toll-like receptors (mammalian) Lipopolysaccharide mutant; CD14   Beutler; Medzhitov; Akira; Wright
(lipopolysaccharide-binding protein receptor)
Scavenger receptors Foam cell formation Brown and Goldstein
Atherogenesis Krieger; Kodama
Mannose receptor C-type lectin, endocytosis, receptor recycling Stahl
β-Glucan receptors Fungal recognition by Dectin-1 Brown
NOD-like receptors Hyperinflammatory syndromes, inflammasomes Tschopp
Note. This is a highly selective, personal list. No attempt has been made to ascribe priority, and the author apologizes to all omitted. For references, see general reviews.

blood monocytes can be stimulated by growth factors28 to
generate macrophages (M-CSF or granulocyte-macrophage
colony-stimulating factor [GM-CSF]), DCs (GM-CSF, with
or without IL-4), or osteoclasts (M-CSF and Rank ligand).
Some of these growth factors also are essential in vivo (e.g.,
osteopetrotic, M-CSF-deficient mice lack many, but not
all, populations of tissue macrophages and osteoclasts).29,30
Monocytopoiesis is less well understood in humans and
may depend on M-CSF and GM-CSF. Trophic interac-
tions between hematopoietic precursors and stromal cells
in the bone marrow (mesenchymal and hematopoietic in
origin) are mediated by cell contact, via surface receptors,26
and by soluble factors (e.g., c-kit and its ligand and IL-1).
­Transcription factors that are essential for monocyte/macro-
phage production and related cells include Pu-1,31,32 other
ets family members, maf,33 and mi, implicated in microph-
thalmia.
Although bone marrow precursors proliferate vigorously
as they differentiate in the presence of M-CSF or GM-CSF,
monocytes become refractory to these growth stimuli (e.g.,
as to the growth-promoting actions of IL-3, IL-4, or IL-13).
Restriction of DNA synthesis is associated with chromatin
 138 GORDON  |  Mononuclear Phagocytes in Rheumatic Diseases
Bone marrow
Progenitor
Monoblast
Figure 8-2  Differentiation and dis-
tribution of mononuclear phagocytes.
Distinct subpopulations of circulating 
monocytes are thought to give rise
to resident tissue macrophages (MØ),
dendritic cells (DC), and osteoclasts
compared with cells recruited by an Promonocytes
inflammatory stimulus. Further phe-
notypic heterogeneity arises from
microenvironmental stimuli, such as
cytokines and microbial products. For
further details of morphologic and
other properties of cells in different
tissues, see Gordon and colleagues49
and Gordon and Hughes.12 Monocytes
condensation, whereas RNA and protein synthesis persists
and can be modulated by a variety of stimuli, as discussed
further subsequently.
Tissue macrophages, DCs, and osteoclasts all derive
from circulating monocytes, although these already may
display heterogeneity (see Fig. 8-2). Cells are recruited
constitutively to peripheral sites in the steady state. Addi-
tional monocytes can be recruited to local sites in response
to infectious, inflammatory, and metabolic stimuli. Such
“elicited” cells display distinct properties from the “resi-
dent” cells, which in the case of macrophages and DCs
also display marked diversity, depending on their location.
A considerable body of evidence, although incomplete,
indicates that different subsets of macrophages and DCs
originate from distinct monocyte populations in peripheral
blood.34-37 Apart from marker antigens (e.g., CD14, the
receptor for lipopolysaccharide-binding proteins, and Gr-1,
a mouse Ly-6 antigen expressed by polymorphonuclear
neutrophils and by some monocytes), levels of chemokine
receptors for fractalkine (CX3CR1) and CCL2 (MCP1)
seem to distinguish monocyte subsets that give rise to
inflammatory and resident macrophages (see Fig. 8-2).
Resident macrophages and immature DCs are present in
many lymphohematopoietic and nonlymphoid organs;
selected markers and properties are listed in Table 8-2. The
CD68 antigen, a late endosomal mucin-like glycoprotein
related to the LAMP family, is the most broadly expressed
marker for all mononuclear phagocytes, although its func-
tion is still obscure.
Osteoclasts and resident macrophages are found on the
surface of bone and display distinct phenotypes. Although
antigenic markers for human and mouse mononuclear phago-
cytes are useful for phenotypic analysis, no single marker is
definitive in their classification, and it is impossible, in most
Peripheral blood Tissues
Resident tissue MØ?
CCR2-
e.g., splenic MØ, Kupffer cells,
CD62L-
alveolar MØ, microglia, osteoclasts
X3CR1high-
Resident tissue DC?
Inflammation
CD14++ Pathogen
MØ
clearance
CCR2+ Resolution of
CD62L+ inflammation
CX3CR1low
DC
Antigen
presentation
tissues, to distinguish locally activated resident cells from
“elicited,” newly recruited, and locally activated monocytes.
This problem is due partially to rapid modulation of antigen
expression in blood monocytes when they enter a particular
tissue microenvironment. Species differences in anatomy
and of marker expression are a further confounding factor.
Apart from the expression of chemokine receptors,38
adhesion molecules play a role in selective monocyte “hom-
ing,” but their differential expression by ­ macrophages,
DCs, and osteoclasts is still less well defined than for lympho-
cyte subpopulations. These include various heterodimeric
integrins implicated in adhesion to endothelium, to extra-
cellular matrix, and to bone. There is considerable scope for
characterization of additional receptors and markers—one
example is the EGF-TM7 family of leukocyte receptors illus-
trated in Figure 8-3.39 Although the F4/80 antigen has been
extremely useful as a differentiation marker in the mouse,
the human counterpart (EMR1) has not been of compa-
rable value, showing a predilection for eosinophils. EMR2,
a related human myeloid cell receptor, is a useful marker,
however, expressed by many tissue mononuclear phagocytes;
it binds chondroitin sulfate proteoglycans broadly present
in connective tissue and has been implicated in leukocyte
adhesion, migration, and activation.40
In joints, there is a resident synovial macrophage popula-
tion, and recruited monocytes and macrophages are promi-
nent in inflammatory, autoimmune, and infectious diseases,
together with DCs and other myeloid and lymphoid cells.
To my knowledge, these resident and recruited mononuclear
phagocytes have not yet been attributed to particular mono-
cyte subsets. These may not be the same monocyte subpopu-
lations that give rise to resident and recruited mononuclear
phagocytes in other tissues and in response to different
pathologies.41
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION 139

Table 8-2  Selected Properties of Mononuclear F4/80

­Phagocytes and Related Cells EMR1 1 EMR2 1 CD97 1

Monocytes/Macrophages: Antigen Marker 2 2 2

F4/80 (mouse) 3 3 3
EMR2-EGF module containing receptor (human)
CD68 4 4 4
Complement receptor 3 (CR3) (CD11b)
Sialoadhesin (Siglec-1) 5 5 5
Scavenger receptors:
Scavenger receptor A (SR-A) 6
Macrophage receptor collagenous domain (MARCO)
Mannose receptor
Macrophage colony-stimulating factor receptor
(M-CSFR)
Other properties
Opsonic phagocytosis
Lysozyme secretion
Abundant acid hydrolases
Myeloid Dendritic Cells: Antigens
Major histocompatibility complex class II
Costimulatory molecules
CD11c Figure 8-3  Myeloid cell antigens of the EGF-TM7 family. These G pro-
CD8α+/− tein coupled receptor(GPCR) related receptors have a large extracellular
DEC205 domain consisting of multiple epidermal growth factor (EGF) modules.
Dendritic Cell-specific ICAM-grabbing non-integrin (DC-SIGN) The F4/80 antigen, an excellent marker for mouse macrophages, has
Dendritic Cell-Lysosome associated membrane Protein (DC-LAMP) been implicated in peripheral tolerance.127 The human orthologue EMR1
Other properties is a marker for human eosinophils. EMR2 is not expressed in the mouse,
Activation of naive CD4 T lymphocytes but is present on human monocytes, macrophages, immature myeloid
Plasmacytoid Dendritic Cells: Antigens dendritic cells, and activated polymorphonuclear neutrophils. It is a use-
ful surface marker for macrophages in tissues, including rheumatoid ar-
CD123 thritic joints. CD97, expressed on myeloid cells and selected nonmyeloid
B220 cells, is a receptor for the complement regulatory protein, CD55.128 EMR2
Lectin-like receptors (Siglec H) and CD97 bind chondroitin sulfate B.40
Other properties
Type I interferon production cells are distinct from, but share endocytic properties with,
In vitro growth by flt-3 ligand
sinusoidal endothelial cells. The constitutive exit through
Osteoclasts vascular endothelium to become tissue macrophages and
CD68 DCs is not understood, whereas induced mobilization is well
Tartrate Resistant Acid Phosphatase (TRAP) characterized, sharing many features with that of polymor-
Calcitonin receptor phonuclear neutrophils.
αvβ3
Vacuolar H+ ATPase Figure 8-4 summarizes stages and molecules implicated
Proteinase K in monocyte egress.43,44 Although most monocytes exit the
Resorption of living bone microvasculature by diapedesis between endothelial cells,
Note. Marker expression varies, depending on cell localization, maturation, there is evidence for an alternative transcytotic mecha-
and activation. Some markers also are present on other myeloid cells (e.g., nism, as for lymphocytes.45 The roles of L-selectin, β2 and
polymorphonuclear neutrophils) and selected endothelial cells. Structures other integrins,46 the immunoglobulin superfamily mol-
and functions of receptor antigens are described elsewhere in this chapter. ecule CD31, and CD99 have been established by analysis
of genetic deficiencies in humans and mice,47 and by use
MOBILIZATION OF MONONUCLEAR of monoclonal antibodies against these48 and other defined
adhesion molecules, such as VCAM and VLA-4. Endothe-
PHAGOCYTES lial cell ligands implicated in monocyte adhesion include
Similar to other white blood cells, mononuclear phagocytes fractalkine, a tethered chemokine; other chemokines may
are distributed in intravascular and extravascular compart- be presented by glycosaminoglycans.
ments, sharing common mechanisms of mobilization, but The subsequent migration and fate of mononuclear
also displaying distinct features, among themselves and phagocytes in tissues differ strikingly. Although many resi-
compared with other cell types. Exit from the bone marrow dent macrophage and DC subpopulations in tissues are
is controlled constitutively and on demand, but this pro- well characterized,12,49 the origins of several functionally
cess is not well understood except for the role of chemokine specialized cell types related to this overall lineage remain
receptors, such as CCR2 (ligands MCP1 through MCP4).42 unclear.50,51 Macrophages become sessile, but can be induced
What determines differentiation into the mature macro- by inflammatory stimuli to migrate to draining lymph nodes
phages that form part of the stromal microenvironment in and remain there, without re-entering the circulation.
bone marrow is unknown; one possibility is re-entry of cir- Immature DCs respond to antigenic and inflammatory stim-
culating monocytes from blood to bone, as for ­osteoclasts. uli by migration to draining lymphoid tissues, transporting
Apart from the circulating pool of monocytes, many mature antigens for presentation to lymphocytes (Fig. 8-5). DC
macrophages adopt a sinusoidal distribution in liver (Kupffer maturation is accompanied by major changes in DC proper-
cells) and in selected lymphoid and endocrine organs. These ties (Fig. 8-6 and Table 8-3). DCs become highly motile and
 140 GORDON  |  Mononuclear Phagocytes in Rheumatic Diseases

Firm adhesion
Rolling
CD62L shedding Increase in CD11b

Extravasation

CD54 CD29
PSGL1
CD62P
JAM
CD62L
Chemokines (e.g., CCR2/MCP-1)
CD11b
CD11a Chemokine receptor (e.g., CCL2)

N-Glycans CD31/PECAM

CD99 CD49d

Figure 8-4  Stages and molecular interactions implicated in monocyte recruitment by inflammatory stimuli. Diapedesis of monocytes shares features
with that of polymorphonuclear neutrophils.

T cells

Dendritic cells

Follicular
DCs
Peripheral
tissues

Afferent
B B lymphatics

Blood B Blood
B
T

Bone marrow
T

Figure 8-5  Positioning of dendritic cells (DCs) within lymphoid tissues. Blood-derived monocytic precursors enter skin and mucosal peripheral tis-
sues and differentiate into Langerhans cells within epithelia and into other immature DCs. When established, Langerhans cells turn over independently
from bone marrow–derived cells58 (e.g., after ultraviolet irradiation). On exposure to stimuli (foreign antigens, local infections) and constitutively (after
uptake of apoptotic cells, such as in the gastrointestinal tract), DCs undergo maturation, upregulate CCR7, and enter afferent lymphatics and second-
ary lymphoid tissues, where they interact with CD4 T lymphocytes. CD4 T lymphocytes become activated, interacting with B cells or CD8 T lympho-
cytes, and re-enter blood, homing to peripheral sites. DCs also are able to interact with innate lymphocytes and natural killer cells. Follicular DCs in 
B cell areas have a distinct, poorly defined bone marrow origin, express novel antigenic markers, and are able to capture immune complexes through
complement activation.50 Plasmacytoid DCs have a distinct interfollicular location and express markers of a myeloid and a lymphoid nature. (Courtesy of
R. Steinman.)
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION 141

Immature DC Mature DC Table 8-3  Maturation Markers for Human  

(steady state) (infection) Monocyte-Derived ­Dendritic Cells
Immature Mature
Enhancer/Costimulatory Molecules
CD80 (B7-1) Low High
CD86 (B7-2) Low High
Microbial products CD83 De novo
Cytokines EMR2 (CD312) High Moderate/low
CD40 ligation
Innate lymphocytes, Antigen Uptake
Etc FcγRII High Low
Signaling
CD40 Low High
CXCR4 Low High
CCR5 High Low
Antigen T cell immunity CCR6 High Low
capture CCR7 Low High
Cytokines, chemokines
Antigen Presentation
B7 and TNF costimulators
HLA-DR (major histocompatibility Moderate High
Figure 8-6  Dendritic cell (DC) maturation. In the steady state, imma- complex class II)
ture DCs are actively endocytic, capturing antigens efficiently through HLA-DQ (major histocompatibility Moderate High
pattern recognition receptors. In response to a range of exogenous and complex class II)
endogenous stimuli, they translocate major histocompatibility complex CD1a High Low
class II to the surface, express a range of costimulatory molecules and Others
secretory products, and become efficient antigen-presenting cells. As a
result, DCs control adaptive immunity or induce tolerance. TNF, tumor DC-SIGN (CD209) High Low
necrosis factor. (Courtesy of R. Steinman.) CD14 High/moder- Negative
ate
CD123 (IL-3R) Low High
express a range of chemokine and adhesion receptors. Entry
Note. In addition to the listed antigens and antigen-presenting cell functions,
of myeloid DCs and tissue macrophages into lymphatics is dendritic cell production of, and response to, other growth factors, chemo-
less well understood, but may involve interactions with the kines and cytokines, microbial products, and immune complexes varies during
mannose receptor. As a result, DCs can present exogenous maturation and activation in vitro. Validation in vivo is ­incomplete.
antigens and self-antigens to CD4 T lymphocytes, to acti-
vate or tolerize their responses. Although in vitro systems
are widely used to generate DCs by cultivation of monocytes through a common receptor chain,57 and GM-CSF promote
or bone marrow precursors in cytokine-supplemented media macrophage fusion. Foreign surfaces including biomaterials
(GM-CSF, with or without IL-4), their properties may not also can play a role in granuloma formation, and chemokine
correspond to those of DC populations in vivo. receptors have been implicated in the induction of foreign
Mononuclear phagocytes use integrins and receptors for body–induced giant cells.
extracellular matrix such as CD44, regulating the dynamics The surface molecules involved in these examples of
of their adhesion and migration. Osteoclast adhesion to bone macrophage homokaryon formation and the functional sig-
depends on αvβ3 and possibly other adhesion ­ molecules nificance of induced fusion are still poorly understood. By
implicated in podosome attachment. Plasma membrane contrast, multinucleation in osteoclasts is a physiologic pro-
receptors such as CD97 and EMR2, members of the EGF- cess, dependent on M-CSF and RANK ligand, and several
TM7 family, and TREM-1,52 an immunoglobulin super- plasma membrane (CD44, CD9, TREM-2, DC-STAMP)
family molecule with an ITAM-based activation motif (see and intracellular (c-src, c-fos) molecules have been impli-
later), regulate myeloid cell effector functions and adhesion cated in this differentiation process. Multinucleation is
and migration in extravascular tissue compartments. thought to favor efficient localized resorption of bone. Osteo­
Monocytes recruited to tissues by poorly degradable for- clasts become polarized for secretion and display highly
eign materials or by selected microbial pathogens or parasites active plasma membrane ruffling. Interactions with osteo-
form granulomata, organized structures rich in macrophages, blasts; local cytokines, such as osteoprotegerin; and circu-
incorporating other myeloid and lymphoid cells and fibro- lating hormones, including calcitonin, parathormone, and
blasts and extracellular matrix. Pathogen-induced granu- vitamin D metabolites, regulate their gene expression and
loma formation53 depends on adhesion molecules such as function, as discussed in Chapter 4. Vitamin D receptors
CR3, a β2 integrin, TNF-α, and chemokine receptors such also modulate the function of macrophages and DCs.
as CCR4; granuloma macrophages express abundant secre- The turnover of different mononuclear phagocytes var-
tory products such as lysozyme54 and proinflammatory cyto- ies, depending on their activation status and tissue localiza-
kines. Characteristic morphologic evidence of macrophage tion.58 Resident macrophages can live for weeks or months,
differentiation in granulomata includes epithelioid cell and whereas inflammation reduces survival to hours or days.
multinucleated giant cell formation, such as with Mycobac- DCs are relatively short-lived cells; osteoclast turnover in
terium tuberculosis infection. Giant cells arise by monocyte vivo has not been studied in detail. The role of apoptosis
recruitment and macrophage fusion, rather than by impaired in mononuclear phagocyte turnover is poorly characterized,
cytokinesis.55 Cytokines such as IL-4 and IL-13,56 acting in contrast to in polymorphonuclear neutrophils, but cell
 142 GORDON  |  Mononuclear Phagocytes in Rheumatic Diseases

IL-1R TLR1-TLR2 TLR7, TLR8

or TLR5 or TLR2-TLR6 TLR3 TLR4 or TLR9

Plasma membrane

TIR domain

MyD88 MAL

TRIF TRAM

NF-κB NF-κB NF-κB NF-κB NF-κB NF-κB

IRF3 IRF5 IRF3 IRF5
IRF5 IRF7
IRF7 IRFI (in mDCs)

SARM

Figure 8-7  Overview of transcription factor activation through TIR domain–containing adapters for the TLR/IL-1R superfamily. Each adapter is dif-
ferentially used by receptor complexes to regulate transcription factor activation positively. The exception is SARM (sterile α- and armadillo motif– 
containing protein), which inhibits TRIF (Toll/IL-1R [TIR] domain–containing adapter protein inducing interferon [IFN]-β)-mediated transcription factor
activation. IL-1R, interleukin-1 receptor; IRF, IFN regulatory factor; MAL, MyD88 (myeloid differentiation primary response gene 88) adapter-like protein;
mDC, myeloid dendritic cell; NF B, nuclear factor B; TLR, Toll-like receptor; TRAM, TRIF-related adapter molecule. (From O’Neill LA, Bowie AG: The family
of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat Rev Immunol 7:353-364, 2007.)

survival is regulated by growth factors such as M-CSF and
interactions with neighboring cells and pathogens.
RECOGNITION
In the past decade, emphasis on the problem of immune
recognition has shifted to a considerable extent from
somatically rearranged receptors for peptide, on lym-
phocytes, to germline-encoded receptors on myeloid
antigen-presenting cells (APCs).59-61 Previous stud-
ies on myeloid cells focused on well-known opsonic
receptors for antibody (Fc receptors) and complement
(complement receptor). The concept of direct pattern
recognition receptors for conserved structures on microbes
provided an impetus to studies on innate immunity, rein-
forced by the discovery of TLRs. More recently, the inves-
tigation of numerous NTRs, including a range of lectin-like
recognition molecules and of scavenger receptors, mark-
edly enhanced knowledge of the recognition repertoire.62
Finally, came the discovery of cytosolic recognition pro-
teins, the NLR family,23 implicated in genetic and micro-
bial-induced hyperinflammatory syndromes and in Crohn’s
disease.
These molecules are mainly, although not exclusively,
expressed by macrophages and DCs and play an impor-
tant role in immune responses to foreign or modified host
ligands, resulting in inflammation and autoimmunity. Given
the complexity of microbial and cellular particulates com-
pared with discrete soluble ligands, different receptors
collaborate and synergize with one another to activate or
inhibit subsequent APC responses. The original distinction
drawn between pattern recognition receptors for exogenous,
so-called pathogen-associated molecular patterns and for
endogenous, host-derived ligands has been eroded to some
extent because individual receptors are able to bind both
types of ligand,63 although their distinct cellular expression
and signaling responses may account for discrimination by
host APCs. In particular, the recognition and uptake of
apoptotic cells results in downregulation of macrophage
effector molecules,64 as opposed to the proinflammatory
effects induced by microbial ligands. The signaling path-
ways induced by other, modified host-derived ligands, such
as oxidized lipoproteins and hyaluronates, are not well-
characterized.
This section summarizes selected receptor structures,
indicates some of their ligands, and addresses briefly signal-
ing and antigen processing pathways. Further details are
provided in Chapter 16, which also illustrates interactions
between humoral and cellular arms of the innate response.
TOLL-LIKE RECEPTORS
Key features of TLRs are illustrated in Figure 8-7.65-67 TLRs
consist of homodimers or heterodimers of transmembrane
glycoproteins containing extracellular leucine-rich repeats
(LRR) and cytoplasmic TIR domains, similar to the intra-
cellular domain of the IL-1 receptor, which contains extra-
cellular immunoglobulin superfamily domains. TLRs are
expressed on the surface of APCs or, in the case of TLR3,
TLR7/8, and TLR9, on vacuolar membranes, contributing to
proinflammatory and immunogenic signaling after “sensing”
of cargo. Isolated TLRs do not bind ligands directly, but col-
laborate with humoral factors and with other plasma mem-
brane glycoproteins, as in the well-studied case of TLR4, the
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
N
CR
CR Cysteine-rich domain
N
CR Fibronectin type II
CTLD
Potential N-linked glycosylation
C C
ITAM
C C
N N
MR DEC205 DC-SIGN βGR
Murine
Dectin-1
Glycosyl phosphoinositide (GPI)-anchored CD14 receptor
for the plasma lipopolysaccharide-binding protein,68 and
the membrane-associated molecule MD2.
NON–TOLL-LIKE RECEPTORS
NTRs constitute a variety of lectin-like,62,69 scavenger, and
other plasma membrane glycoprotein nonopsonic receptors;
selected examples are illustrated in Figures 8-8 and 8-9. They
include type 1 and 2 transmembrane molecules, with C-type
lectin or lectin-like, collagenous, or immunoglobulin super-
family domains. In addition, macrophages express sialic
acid–binding receptors, members of the SIGLEC family.70
Table 8-4 summarizes known ligands and potential functions
relevant to their role in homeostasis and immunity. Apart
from direct recognition of selected self, modified self, and
foreign structures, they play an important role in cell-cell
and cell-matrix interactions; in phagocytosis and endocy-
tosis; and in less well-understood processes, such as targeted
delivery of antigens to peripheral lymphoid organs.71
Receptors that promote rapid clearance of apoptotic
cells, directly or after opsonization by complement and other
extracellular proteins, are shown in Figure 8-10. More recent
research has shown collaboration of different receptors at the
cell surface,72 regulating cellular responses. Other regulatory
surface receptors include transmembrane molecules with
tyrosine-based activating (ITAM) or inhibitory (ITIM) cyto-
plasmic motifs, for example, C-type lectin-like molecules,
immunoglobulin superfamily members such as Fc receptors,
discussed further subsequently, and receptor-ligand pairs that
143
Figure 8-8  Selected lectin-like rece­
ptors expressed by macrophages and
dendritic cells (DCs). (See Table 8-4
for ligands.) These receptors play an
­important role in nonopsonic adhesion, 
C cell-cell interactions, endocytosis, and 
phagocytosis. The cysteine-rich do-
C
main of the mannose receptor130
has been postulated to play a role in
transport of glycoconjugates to peri­
pheral lymphoid organs, for clearance
or antigen-dependent activation of
B lymphocytes.71 Dec205 has been
used to show efficient targeting of an-
ITAM ITAM tigens to DCs.131 The ITAM-like motif
of Dectin-1, the β-glucan receptor, is
N N essential for myeloid cell responses to
βGR-A βGR-B fungal particles, in collaboration with
TLR69,72,132 and NLR.109 CTLD, C-type
Human Dectin-1 lectin domain.
regulate macrophage activation (CD200/CD200R)73 and
phagocytosis (SIRPα and CD47) (Fig. 8-11).74
COMPLEMENT RECEPTORS
Complement receptors are heterogeneous receptors exp­ressed
by APCs (Fig. 8-12) that mediate direct and lectin and anti-
body-dependent binding of activated complement compo-
nents and play a role in cell migration and phagocytosis and
immune regulation.75,76 The discovery of a novel comple-
ment receptor selectively expressed by Kupffer cells suggests
that mononuclear phagocyte heterogeneity in complement
receptor function merits ongoing study.77 Genetic deficien-
cies in complement receptor expression and their ligands
have provided insights into the pathogenesis of autoimmune
diseases and of rheumatic joint injury.78 ­Complement deposi-
tion on follicular DCs has been implicated in the activation
of B cell responses.50,79 Complement regulatory proteins are
expressed by many cell types and APCs and play an impor-
tant role in limiting cell activation. In addition, comple-
ment receptors collaborate with other receptors to modulate
myeloid cell responses. Their signaling pathways are less well
defined than those of Fc receptors.
Fc RECEPTORS
APCs express diverse receptors for monomeric and com-
plexed immunoglobulins regulating effector responses to
antigens.80-83 Figure 8-13 illustrates the properties of human
Fc receptors with activating or inhibitory motifs in their
 144 GORDON  |  Mononuclear Phagocytes in Rheumatic Diseases

C C
C
SRCR

Collagen-like

C
C C

α-Helical
coiled coil
CTLD

N N N N N N N N N C N C GPI-anchor N
SR-AI SR-AII MARCO CD163 CD36 CD14 LOX-1
Figure 8-9  Selected scavenger receptors. These non-TLRs consist of diverse structures and bind a range of microbial and endogenous ligands (see
Table 8-4). They differ in regulation and expression, providing useful markers of macrophage activation (CD14, MARCO),133-137 heterogeneity (CD163,
the scavenger receptor for clearance of haptoglobin-hemoglobin complexes),119 and lipid homeostasis (SR-A, CD36, LOX-1).138,139 Scavenger receptors
such as MARCO can collaborate with TLRs in APC responses to microbes.140 CTLD, C-type lectin domain.

cytoplasmic tails. Fc receptor polymorphisms have been
implicated in autoimmune human diseases such as systemic
lupus erythematosus. Studies on genetically manipulated
mouse models have led to considerable insights into the role
of individual Fc receptors in immunopathogenesis. Fc recep-
tors are able to cooperate with chemokine84 and other recep-
tors. The nature and role of Fc-independent interactions of
antibody with APCs are under investigation and include lec-
tin-like recognition of sialylated85 and mannosyl residues.
NOD-LIKE RECEPTORS
The identification of microbial peptidoglycan breakdown
and other products as ligands for an intracellular family
of LRR-containing cytosolic sensors that regulate caspase
activation and IL-1β secretion provided a major impetus to
this burgeoning field.86 Other chapters describe examples
of NLRs and their role in hyperinflammatory syndromes.
NLRs also participate in signaling pathways implicated in
TLR-induced and NTR-induced functions.
RESPONSES AND MODULATION
For convenience, I distinguish the cellular pathways that
precede altered gene transcription and subsequent effector
responses. This section summarizes some of the major cell
biologic effects, resulting in antigen processing and presen-
tation by APCs. Other chapters address related topics, such
as recognition of lipids by CD1 and intracellular trafficking
of related molecules.
PHAGOCYTOSIS AND ENDOCYTOSIS:
ANTIGEN PROCESSING
The vacuolar apparatus of APCs is illustrated schematically
in Figure 8-14.87-90 Internalization of the plasma membrane
results in phagosome/endosome formation, with progressive
acidification and digestion, depending on delivery of ves-
icles and their hydrolytic contents. Membrane and recep-
tors are recruited, modified by maturation, and retrieved by
recycling. Further fusion with Golgi-derived vesicles and
primary lysosomes yields phagolysosomes and secondary
lysosomes, reaching a pH of approximately 5.5 to 6.0.
Depending on the bulk of plasma membrane internalized
and the size of the particle, the uptake process involves
cytoskeletal components91 and small GTPases92 and dock-
ing machinery. Guanosine triphosphate (GTP) hydrolysis
provides an important mechanism to control intracellular
membrane traffic and coupling to the cytoskeleton.93 Cyto-
kines such as interferon-γ can have a major effect on activa-
tion and relocation of GTP-binding proteins, contributing
to host cell–pathogen interactions.94,95
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION 145

Table 8-4  Ligands for Selected Nonopsonic, Non–Toll-like Receptors

Class Receptor Microbial Ligands Endogenous Ligands Function
Scavenger   SR-A I/II Gram-positive/  Apoptotic cells Phagocytosis
receptors gram-negative bacteria
Lipoteichoic acid Modified low-density   Endocytosis
lipoproteins
Lipid A AGE-modified proteins Adhesion
Neisserial surface   β Amyloid Foam cell formation
proteins
MARCO Gram-positive/  Marginal zone B   Adhesion
gram-negative bacteria lymphocytes
Neisserial surface proteins Uteroglobin-related protein Phagocytosis
Innate activation
CD36 Diacylated lipopeptide from   Apoptotic cells (with   Uptake, exchange 
gram-positive bacteria thrombospondin and   of lipids
vitronectin receptor)
Plasmodium falciparum–  High-density lipoprotein Adhesion
parasitized erythrocytes (HDL)
Outer rod segments
Lectins Dectin-1 β-Glucan T lymphocytes   Fungal uptake and  
(noncarbohydrate) immunomodulation
DC-SIGN Mannosyl/fucosyl   ICAM 2/3  Adhesion 
glycoconjugates including viruses T lymphocytes  Endocytosis 
(e.g., human immunodeficiency Lysosomal hydrolases Endocytosis
virus-1, dengue)
Mannose receptor: Mannosyl/fucosyl   Thyroglobulin  Adhesion
CRD glycoconjugates on   Ribonuclease B 
bacteria, viruses, fungi, parasites Amylase
Cysteine-rich   Sulfated carbohydrates in Antigen targeting
domain marginal zone (spleen)  
and subcapsular sinus
(lymph node)
Fibronectin type II Collagens Adhesion
domain
Note. This table illustrates the dual recognition properties and diverse cellular functions of selected non–Toll-like receptors. For further details, see references
15, 20, 62, 139, 147-150.

Proteomic analysis of isolated phagolysosomes has
revealed more than 600 constituents, classified according to
the source of membrane and various putative functions.96
Although most membrane constituents are derived from
the plasma membrane, descriptions of contributions from
the endoplasmic reticulum have caused considerable inter-
est, with different estimates of their extent.96,97 Another
controversial aspect relates to the role of TLR engagement
in enhancing the kinetics of the maturation process, a tran-
scription-independent process.98 Variations on this theme
occur depending on the cell type and maturity, whether the
extracellular cargo is microbial or host-derived, and whether
the process arises by autophagy,99 rather than heterophagy.
The role of autophagy in cellular resistance to intracellular
pathogens, such as M. tuberculosis, is receiving increasing
consideration.100,101
Immature DCs are actively endocytic and phagocytic,
but poor APCs, whereas mature DCs downregulate uptake,
but acquire highly efficient APC function.25 Changes asso-
ciated with DC maturation and antigen presentation are
illustrated in Figure 8-15. Pathogenic intracellular organ-
isms vary in their subversion of the aforementioned process,
interfering with different stages, such as cytosolic signaling
mechanisms, fusion, or acidification, and in selected cases,
inducing a novel membrane composition.102,103 Organisms
can replicate in immature or mature compartments, or trans-
locate their genome to the cytosol by acid-induced envelope
fusion or by disruption of the vacuolar membrane.
SIGNALING
The best-characterized signaling responses in macrophages
include the TLR,104 type I interferon,105 and Fc recep-
tor–induced pathways.106,107 The various signaling cascades
are complex, interact with one another, and result in phos-
phorylation/dephosphorylation, formation of cytosolic pro-
tein complexes, and activation of signaling proteins such as
nuclear factor B, which enter the nucleus to regulate tran-
scription. In the case of TLR-dependent sensing, a restricted
number of adapters, such as MyD88, channel the flow of
information into the cell (see Fig. 8-7). There is emerging
evidence that NTR-induced signaling (e.g., by the β-­glucan
receptor [Dectin-1] cytoplasmic ITAM-like motif)69 col-
laborates with TLR and NLR signaling pathways, with dif-
ferential involvement of syk108 and CARD9109 in effector
responses in different mononuclear phagocytes. Distinct,
but interlinked pathways involve interferon regulatory fac-
tors, part of an amplification pathway with broader immu-
noregulatory functions than antiviral responses alone.105 A
more recently defined cytosolic specialized antiviral pathway
 146 GORDON  |  Mononuclear Phagocytes in Rheumatic Diseases

b. Recognition of altered self

Phagocyte Scavenger receptors Mer
β2-GPI receptor
CD36 PtdSer
LOX-1
CD68
β2-GPI ανβ3vitronectin
SRA
Gas6 receptor

a. Innate recognition MFGE8 ABC1

of non-self
ABC1 c. Recognition of
PtdSer non-detaching
exposure self
TSP1 binding
Apoptotic sites Integrins
C3b
β2-Integrin cell (ανβ3, ανβ5)
receptors C1q

TSP1
CD91 CD36

CD91 ACAMP3
MBL Disabled
CD31
ICAM3 CD31

CD14

Figure 8-10  Phagocytic receptors for apoptotic cell phagocytosis. Macrophages (MØ) and immature myeloid dendritic cells (DC) are the main 
immune cells involved in the clearance of apoptotic cells. They express broadly similar multiple receptors, which can bind directly or via opsonic soluble
proteins (e.g., mannose-binding lectins [MBL] to ligands). Phosphatidylserine (PtdSer) becomes exposed on the outer surface of the apoptotic cell, and
a receptor for this ligand has been long sought. A new receptor (TIM4 and related TIM1) has been discovered on resident MØ, with specificity for PtdSer.
Other MØ populations use MFGE8 (a milk fat globulin protein secreted by MØ) as an opsonin. Discrimination of nonself and altered self may involve
combinations of different phagocyte receptors. Apoptotic cell uptake results in an anti-inflammatory response by MØ (e.g., release of TGF-β and PGE2),
but also has been implicated in cross-presentation by DC. (From Savill J, Dransfield I, Gregory C, et al: A blast from the past: Clearance of apoptotic cells regu-
lates immune responses. Nat Rev Immunol 2:965-975, 2002.) MFGE8, milk fat globule-EGF factor 8 protein; PGE, prostaglandin E2; PtdSer, phosphatidy­m­serine;
TIM, T cell immunoglobulin mucin; TGF, transforming growth factor.

involves interaction with mitochondrial components. Cell

surface or intracellular proteins serve as negative regulators,
as exemplified by members of the SOCS family.
Figure 8-15 illustrates the pathway by which effector
response–derived antigens are transported to the cytosol
during synthesis, are processed in proteasomes, and become
associated with MHC class I molecules for presentation at
the cell surface. Effector response chaperones, such as heat
shock protein, may contribute to the surface delivery of
antigens. The inflammasome is another example of a multi-
protein assembly within the cytosol, by which NLRs bring
about IL-1 activation.
Several of the above-described processes involve proteo-
lytic enzymes, including cathepsins and caspases, and lipid-
interacting chaperones. The ubiquitin pathway provides an
important mechanism for cytosolic degradation of proteins.
In addition, vacuolar H+ATPases contribute to acidification
in the different mononuclear phagocytes, especially relevant
to osteoclast function. Comparison and further elucidation
of the coupling between extracellular and intracellular
signaling pathways in macrophages, DCs, and osteoclasts
would be of great interest.
EFFERENT PATHWAYS: GENE EXPRESSION
AND SECRETION
The complex responses of different mononuclear phago-
cytes to intrinsic and extrinsic stimuli have been defined
by microarray studies and, in some cases, by protein and
functional analysis. Although the major differentiation
pathways (macrophages, immature and mature DCs, and
osteoclasts) involve selective, stereotypic changes in gene
expression, the extensive heterogeneity and plasticity of
phenotypes characterizing these cells in situ have begun
to be appreciated only more recently.8,110 Macrophages are
known to express numerous nuclear receptors,111 which
undergo dynamic changes depending on cellular differen-
tiation and the microenvironment. Exposure to particular
cytokines, hormones, and other stimuli, such as microbial
antigens and immune complexes, modulates gene expression
profoundly and selectively and induces post-­transcriptional
and post-translational changes. Through alternative splic-
ing, glycosylation, and poorly characterized protein modi-
fications (e.g., methylation and acetylation of nuclear
and cytoplasmic macromolecules), it provides different
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION 147

CD200 CD47

ITAM
S Immunoglobulin
ITIM S domain S S
S S
+ – Charged
S
amino acids S
S
S
S S
S S

S S S S
S S S S

+ – – + – –

TREM1/DAP12 TREM2/DAP12 CD200R

SIRPα
Figure 8-11  Surface receptors that, singly or as paired receptors, modulate macrophage responses. Illustrated are immunoglobulin superfamily and associ-
ated transmembrane molecules using ITAM or ITIM motifs to generate activating or inhibitory signals. DAP12 can associate with a range of other membrane
molecules as signaling partner in macrophages, dendritic cells, and natural killer cells.142 Note charged amino acid residues in transmembrane domains. CD200
and CD47, which are broadly expressed in tissues, generate inhibitory signals in macrophages via CD200R and SIRPα.74 In CD200 ko mice, macrophages show
spontaneous activation.125 Macrophages themselves are induced by Toll-like receptor stimuli to express CD200 (Mukhopadhyay S: unpublished).

N-terminal repeats with

β propeller structure S
Immunoglobulin
S domain
I-domain

I-like domain
GPI anchor
Short consensus repeats/
Complement control Cysteine rich domain
protein repeats

S
V
S

S S
C2 V
S S

αM β2 αX β2

CR1 CR3 CR4 huCRig(L) huCRig(S) CD55 CD59

Complement regulators
Figure 8-12  Complement receptors and membrane regulators expressed by macrophages. CR1 is broadly expressed by nucleated cells, acting as a
“sink” for activated complement; CR3 (CD11b/CD18), a phagocytic receptor for C3b-coated particles, and CR4 (CD11c/CD18) are β2 integrins, which,
together with LFA-1 (CD11a/CD18), mediate adhesion of myeloid cells to endothelium and extracellular matrix and migration. huCRIg (L and S) are
long and short forms of a newly described complement-binding receptor on Kupffer cells, which mediate uptake of opsonized bacteria.77 CD55143 and
CD59144 are Glycosyl phosphoinositide (GPI)-anchored regulators of complement activation.
 148 GORDON  |  Mononuclear Phagocytes in Rheumatic Diseases

Ig-like domain Mucin-rich region

Lectin-like domain ITAM

ITIM
GPI
ITAM-like

α α α α

β β
YY YY YY YY
ζ ζ ζ Y
FcαR1 Fcα/µR FcγRI FcγRIIa FcγRIIb FcγRIIIa FcγRIIIb FcεRI FcεRII
Figure 8-13  Human Fc receptors. Myeloid cells express a range of classic Fc receptors that initiate a variety of cellular responses, including phago-
cytosis, antibody-dependent cell-mediated toxicity, antigen presentation, respiratory burst, and release of inflammatory mediators. Immunoglobulin
subclasses are bound by extracellular domains; signaling via cytoplasmic ITAM or ITIM is mediated by associated membrane-spanning polypeptides.
Activation and inhibitory receptors usually are coexpressed on the cell surface and function in concert, determining the magnitude of effector cell
responses. A range of Fc receptor–like molecules (Ig-SF extracellular domains), with similar ITAM/ITIM cytoplasmic motifs, are mainly expressed by B or
T/natural killer cells; they may regulate lymphocytic differentiation and responses.145 GPI, Glycosyl phosphoinositide; ITAM, Immunotyrosine activation
motif; ITIM, Immunotyrosine inhibition motif.

Early endosome
Actin polymerization
Pseudopod formation/
Antigen membrane invagination
Figure 8-14  Schematic presentation of presentation Membrane recruitment
phagocytosis and endocytosis. Particu-
Membrane closure
lates are taken up by actin-dependent se- Rab4, Rab5
quential maturation processes, involving and other
membrane fusion and fission, which in- fusion Late endosome
Partial proteins
tersect with the endocytic pathway at sev-
degradation
eral stages. Cytosolic small GTPases (Rabs)
determine organelle-specific interactions. Phagosome maturation by
Membrane is recycled to the plasma interaction with endocytic
membrane, with processed antigen (see pathway
Fig. 8-15). Progressive acidification and Rab7, Rab9,
delivery of lysosomal hydrolases result cathepsin
in terminal degradation. Compartment Phagolysosome
membranes express marker proteins such
as LAMP1; the pan-macrophage CD68 an- Lysosome
tigen is associated with late endosomes
and lysosomes. LAMP1, LAMP2, LAMP3

­ ononuclear phagocytes with extensive diversity in adapta-

m Table 8-6 shows a classification of characteristic effects
tion and function.
Table 8-5 provides a partial list of macrophage secretory
products; these include low-molecular-weight metabolites
(oxygen, nitrogen, and lipid-derived) implicated in inflamma-
tion and its resolution112 and in antimicrobial responses; other
products include proinflammatory and anti-inflammatory
cytokines and neutral proteinases. The cell biology of the
secretory pathway in macrophages has only begun to be
studied.113 The differential effect of T helper type 1 and T
helper type 2 cytokines on nitric oxide production versus its
destruction is thought to be relevant to microbicidal mech-
anisms versus repair.114 The secretory activity of DCs and
osteoclasts is less defined except in regard to their special-
ized functions (lymphocyte activation, bone remodeling).
induced by prototypic activating and inhibitory stimuli. It
is convenient to distinguish the phenotype of mononuclear
phagocytes stimulated by an innate stimulus, including
exposure to microbial products such as lipopolysaccharide;
by a classic immune activating cytokine such as interferon-γ;
and by IL-4/IL-13 induction of an alternative activation
pathway7,115,116 by IL-4/IL-13.57 Other stereotypic responses
are induced by immune complexes that selectively modu-
late IL-12/IL-10 expression.117 Innate and immune cytokine
stimuli are able to potentiate one another.
Conversely, deactivating stimuli such as IL-10 and TGF-
β64 and glucocorticosteroids118 induce their own distinc-
tive patterns of gene expression. Upregulation of CD163,
a hemoglobin-haptoglobin scavenger receptor is a striking
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION 149

CD8
CD4
MHC I-peptide
Plasma Endocytosed
antigens MHC I-peptide
membrane

Cytosolic
antigens C

Cytosolic Endosome
Proteasome B
division of
endocytosed Antigenic
A antigen peptides
Pathogen
MIIC/CIIV

Golgi
TAP

Antigen MHC II-I MHC I-CLP MHC II-peptide

peptides
Endoplasmic
reticulum

MHC I MHC I-peptide MHC I-I

Figure 8-15  Different antigen-processing pathways for the major histocompatibility complex (MHC) class I and class II molecules. A, MHC class I
molecules present peptides that are primarily derived from endogenously synthesized proteins of either self or pathogen origin. These proteins are
degraded into peptides by the proteasome and transported through the transporters of antigen-processing (TAP) molecules into the endoplasmic
reticulum for loading on MHC class I molecules. B, MHC class II molecules present proteins that enter the cell through the endocytic route. During
maturation of MHC class II molecules, they are prevented from binding to endogenous antigens in the endoplasmic reticulum by association with the
invariant chain (li). Invariant chain–MHC class II complexes (MHC II-li) move through the Golgi to the MIIC/CIIV compartment where the invariant chain
is degraded to class II–associated invariant-chain peptide (CLIP). CLIP is removed from the CLIP–MHC class II (MHC-CLIP) complexes and exchanged for
antigenic peptide. C, Dendritic cells can endocytose antigens from other cells and cross-present them to CD8+ cytotoxic T lymphocytes. In most cases,
these antigens also are processed into the MHC class II presentation pathway for recognition by CD4+ helper T cells. CIIV, MHC II vesicles; MIIC, MHC II
loading compartment. (From Heath WR, Carbone FR: Cross-presentation in viral immunity and self-tolerance. Nat Rev Immunol 1:126-134, 2001.)

example of glucocorticosteroid-enhanced endocytic func-

tion.119 These extracellular mediators and poorly defined
interactions with surface expressed molecules make the dif-
ferent APCs reciprocally interactive with other immune
cells (helper and regulatory CD4 T cells, CD8 T cells, natu-
ral killer cells, and Natural Killer T (NKT) cells) and with
nonhematopoietic cells, such as fibroblasts, epithelium, and
neurons. Induced responses on macrophages include altered
MHC class II and costimulatory molecule expression, marker
antigens involved in cellular interactions, phagocytosis and
antigen presentation, and altered secretion. As a result, dif-
ferent mononuclear phagocytes are able to modulate their
interactions with CD4 T lymphocytes, with endothelial and
other cell types, with extracellular matrix, and with bone to
induce trophic and cytostatic/cidal and catabolic effects.
Human inborn errors of defects in macrophage
responses to intracellular pathogens such as mycobac-
teria120 have validated studies on cell activation in the
mouse.121 The ­immunosuppressive effects of macrophages
in immune tolerance to an allogeneic fetus have been
ascribed to induction of indoleamine-2,3-dioxygenase,
which catabolizes the essential amino acid, L-trypto-
phan.122 Macrophage products act mainly locally, but also
can have profound systemic effects, regulating metabolic
and regulatory responses within the host. These effector
programs and products provide targets for pharmacologic
intervention.
RELEVANCE TO SELECTED RHEUMATIC DISEASES
Although the different mononuclear phagocytes have
been addressed as part of a general host homeostatic sys-
tem, there are good examples in human rheumatic dis-
eases and in animal models of arthritis in which these
cells play a role in either the local initiation or effec-
tor phase of the pathology. Primary defects in osteoclast
­differentiation and function contribute to osteopetrosis or,
if overactive, osteoporosis. I have referred to autoinflam-
matory syndromes and the newly discovered NLRs. Gout
also may fall in this category because uric acid provides a
metabolic stimulus for NLR activation. Macrophages play
a role in the pathogenesis and complications of osteoar-
thritis, including the response to foreign implants. DCs
and macrophages play a part in the induction and effec-
tor mechanisms of autoimmune arthritis, in concert with
T lymphocytes and B lymphocytes and their products,
immune complexes and complement. The catabolic role
of TNF-α, IL-1, neutral proteinases, reactive oxygen radi-
cals, and arachidonate metabolites is well known. Less well
appreciated are the trophic interactions of macrophages
with fibroblasts, through production of TGF-β, growth
and angiogenic factors, and modulation by alternative
activation pathways.123
Although animal models may not mimic the natural
diseases in humans, more recent examples suggest that
 150 GORDON  |  Mononuclear Phagocytes in Rheumatic Diseases

Table 8-5  Selected Secretion Products of Macrophages

Proteins Product Comment
Enzymes Lysozyme Bulk product
Urokinase-type plasminogen activator Regulated by inflammation
Collagenase Regulated by inflammation
Elastase Regulated by inflammation
Metalloproteinases Also inhibitors
Complement All components and regulators
Arginase Alternative activation
Angiotensin-converting   Induced glucocorticoids, granulomas
enzyme
Chitotriosidase Gaucher’s disease, lysosomal storage disease
Inhibitors Acid hydrolases All classes (mainly intracellular)
TIMP
Chemokines Many C-C, C-X-C, CX3C (e.g., MCP, RANTES, IL-8) Initiates acute and long-term recruitment of myeloid  
and lymphoid cells
Cytokines IL-1β, TNF-α, IL-6, IL-10, IL-12, IL-18, IL-23 Proinflammatory and anti-inflammatory
Type I interferon Also antagonists (e.g., IL-1Ra)
Autocrine and paracrine amplification
Apolipoproteins Apolipoprotein E Local source, bone marrow origin after  
adoptive transfer
Growth/differentiation TGF-β
factors Also other family members (activins)
M-CSF   Myeloid growth and differentiation
GM-CSF
FGF   Fibrosis
PDGF   Repair
VEGF   Angiogenesis
Opsonins Fibronectin,   Also uncharacterized receptor on  
pentraxin (PTX3) macrophages
Soluble receptors Mannose receptor Soluble mannose receptor
Cationic peptides Defensins Subpopulations and species variation
Lipids Procoagulant Initiation clotting
Arachidonate metabolites Proinflammatory and anti-inflammatory  
mediators
Prostaglandins
Leukotrienes
Thromboxanes
Resolvins
Metabolites Reactive oxygen intermediates
Reactive nitrogen intermediates
Hemoglobin breakdown (bile pigments)
Iron, vitamin B12 binding protein
Vitamin D metabolites
Note. For references, see references 152 and 153. FGF, fibroblast growth factor; GM-CSF, granulocyte macrophage colony stimulating factor; M-CSF,
M-colony stimulating factor; PDGF, platelet derived growth factor; TIMP, tissue inhibitor of mellaproteinase; VEGF, vascular endothelial growth factor.

APC membrane receptors involved in recognition of modi-

fied self and foreign microbial ligands could be relevant
to arthritis—in streptococcal rheumatic disease and in
exacerbation of recurrent arthritis by zymosan in T cell
transgenic mice, acting via Dectin-1, the APC receptor for
β-glucans.124 In addition, inhibitory plasma membrane
receptor interactions (CD200/CD200R) limit macrophage
activation and collagen-induced murine arthritis.73,125
Impaired clearance of apoptotic cells by (uncharacterized)
C1q receptors of mononuclear phagocytes may contribute to
autoimmune diseases such as lupus.78 Finally, macrophages
could contribute to wider connective tissue disorders, such
as scleroderma, because they are able to regulate fibroblast
and matrix synthesis and turnover through metalloprotein-
ases and TGF-β; dysregulation of these pathways has been
neglected as a pathogenetic mechanism in these metabolic
diseases.
ISSUES FOR FURTHER INVESTIGATION
As we take stock of how increasing knowledge of mononu-
clear phagocyte biology has affected pathogenesis and ther-
apy of rheumatic diseases, further questions arise regarding
basic mechanisms and new selective targets. Treating mac-
rophages, DCs, and osteoclasts as specialized forms of a uni-
tary mononuclear phagocyte family helps to bring out their
common and distinctive properties. The selective expres-
sion and signaling pathways of pattern recognition receptors
by macrophages and DCs are still poorly characterized—the
regulation of TLR signaling has already received consider-
able attention as a possible drug target. Discrimination of
microbial and host-derived ligands may account for mim-
icry and autoantigen cross-reactivity, but mechanisms of
regulating homeostatic and tolerogenic responses are still
unclear.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION 151

Table 8-6  Immunomodulation of Macrophage Phenotype

Stimulus Category Markers Function
Microbial (bacterial) Innate Induction of MARCO Enhanced phagocytosis
Activation Costimulatory molecules Antigen presentation
CD200 Inhibition (CD200R)
Interferon-γ Classic activation Induction of MHC II Cell-mediated immunity/ 
delayed-type hypersensitivity
Potentiation of innate markers
TNF-α Proinflammatory
iNOS induction Antimicrobial (NO), signaling
NADPH, respiratory burst Host defense, inflammation
LGP47 induction Association with phagosome/ 
intracellular pathogen killing
Downregulation of MR Unknown
Modulation of FcR expression
Proteosomal composition Antigen presentation
IL-4/IL-13 Alternative activation Enhanced MR Endocytosis
Upregulation Induction of arginase Humoral immunity
Induction of YM1 Th2-responses
FIZZ1 (mouse) Allergy, antiparasitic
Induction of CCL17 (MDC) and CCL22   Immunity
(TARC)
Fusion, giant cell formation Repair/fibrosis
CD23 (FcRε)
IL-10 Deactivation Downregulation of MHC II
TGF-β Downregulation, ­ 
proinflammatory
NO and ROI
Immune complexes Modified activation Selective IL-12 downregulation
IL-10 induction
Glucocorticoids Deactivation CD163 induction Anti-inflammatory
Monocyte recruitment   Homeostatic clearance
downregulated
ACE induction Hemoglobin/haptoglobin  
complexes
Stabilin induction
Note. For references, see references 7, 22, and 154. ACE, angiotensin converting enzyme; LGP47, lysosomal glycoprotein; MDC, macrophage derived
­chemokine; MR, mannose receptor; NADPH, nicotinamide adenine inucleotide phosphate-oxidase; iNOS, inducible nitric oxide synthase; ROI, reactive
­oxygen intermediates; TARC, thymus and activation-regulated chemokine.

On the effector side, success with anti-TNF-α monoclo- Acknowledgments

nal antibodies suggests that targeting of individual effector
molecules can succeed, especially when these are expressed
at the cell surface and are proximal in cascade reactions.
The role of inhibitory/activating pairs of surface molecules
as modulators of APC pathways is unexplored and likely to
move center stage. One neglected aspect of differentiation
and function for osteoclasts and granuloma macrophages
is the mechanism of fusion and the possible functional
­significance of multinuclear giant cell formation.126 Basic
knowledge is still lacking and may provide useful insights
into the pathogenesis of metabolic bone diseases and the
increased perception of links with the immune system, both
innate and adaptive.
Although powerful tools of mRNA and proteomic analy-
sis and for gene silencing by RNAi inhibition have become
available, the heterogeneity and complexity of mononuclear
phagocytes in human tissues, especially at early stages of dis-
ease, need improved characterization in situ and at differ-
ent sites. Much remains to be learned concerning trophic
processes and repair and their relationship to resolution of
inflammation and fibrosis. These involve cell-cell interac-
tions that are still poorly understood, but increasingly ame-
nable to investigation.
The author thanks Christine Holt for preparation of the manuscript and
present and past members of his laboratory for help with illustrations. Work
in the Gordon laboratory has been supported by the Medical Research
Council, United Kingdom; the Wellcome Trust; the Arthritis Research
Campaign; and the British Heart Foundation.
REFERENCES
  1. Feldman M, Nagase H, Saklatvala J, et al (eds): The scientific basis of
rheumatology. Arthritis Res 4:51, 2002.
  2. Stojanov S, Kastner DL: Familial autoinflammatory diseases: Genet-
ics, pathogenesis and treatment. Curr Opin Rheumatol 17:586-599,
2005.
  3. Ezekowitz RA, Hoffmann JA (eds): Innate Immunity. Boston,
Humana Press, 2003.
  4. Kaufmann SHE, Medzhitov R, Gordon S (eds): Innate Immunity.
Norwalk, Conn, ASM Press, 2004.
  5. Takayanagi H: Osteoimmunology: Shared mechanisms and cross-
talk between the immune and bone systems. Nat Rev Immunol 7:
292-304, 2007.
  6. van Furth E: Mononuclear Phagocytes: Biology of Monocytes and
Macrophages. Leiden, Kluwer Publications 1992.
  7. Gordon S: Alternative activation of macrophages. Nat Rev Immu-
nol 3:23-35, 2003.
  8. Gordon S: Mononuclear phagocytes in immune defence. In Roitt I,
Brostoff B, Male D (eds): Immunology, ch 9, 7th ed. Edinburgh,
Mosby, 2005.
 152 GORDON  |  Mononuclear Phagocytes in Rheumatic Diseases
  9. Gordon S: Macrophages and the immune response. In Paul W (ed):  
Fundamental Immunology, 5th ed. Philadelphia, Lippincott Raven,
2003, pp 481-495.
  10. Gordon S: Macrophages and phagocytosis. In Paul W (ed): Fundamen-  
tal Immunology, 6th ed, ch17. Philadelphia, Lippincott Williams &
Wilkins, 2008.
  11. Banchereau J, et al: Immunobiology of dendritic cells. Annu Rev  
Immunol 18:767-811, 2000.
  12. Gordon S, Hughes DA: Macrophages and their origins: Heterogene-
ity in relation to tissue microenvironment. In Lipscomb M, Russell S
(eds): Lung macrophages and dendritic cells in health and disease.  
New York, Marcel Dekker, pp 3-31, 1997.
  13. Steinman RM, Cohn ZA: Pillars Article: Identification of a novel  
cell type in peripheral lymphoid organs of mice, I: Morphology,
quantitation, tissue distribution. J Exp Med 137:1142-1162, 1973.
  14. Moser M: In Paul W (ed): Fundamental Immunology, 6th ed.  
Philadelphia,­ Lippincott Williams & Wilkins, 2008.
  15. Shortman K, Naik SH: Steady-state and inflammatory dendritic-cell
development. Nat Rev Immunol 7:19-30, 2007.  
  16. McKenzie EJ, Taylor PR, Stillion RJ, et al: Mannose receptor expres-
sion and function define a new population of murine dendritic cells.
J Immunol 178:4975-4983, 2007.  
  17. Facchetti F, Vermi W, Mason D, et al: The plasmacytoid monocyte/
interferon producing cells. Virchows Arch 443:703-717, 2003.
  18. Bruzzaniti A, Baron R: Molecular regulation of osteoclast activity.  
Rev Endocr Metab Disord 7:123-139, 2006.
  19. Gordon S, Taylor PR: Monocyte and macrophage heterogeneity.  
Nat Rev Immunol 5:953-964, 2005.
  20. Pluddemann A, Mukhopadhyay S, Gordon S: The interaction of
macrophage receptors with bacterial ligands. Expert Rev Mol Med  
8:1-25, 2006.
  21. Fadok VA, Bratton DL, Henson PM: Phagocyte receptors for apop-
totic cells: Recognition, uptake, and consequences. J Clin Invest
108:957-962, 2001.  
  22. Mantovani A, Sica A, Locati M: New vistas on macrophage differen-
tiation and activation. Eur J Immunol 37:14-16, 2007.
  23. Meylan E, Tschopp J, Karin M: Intracellular pattern recognition
receptors in the host response. Nature 442:39-44, 2006.  
  24. Albert ML, Sauter B, Bhardwaj N: Dendritic cells acquire antigen
from apoptotic cells and induce class I-restricted CTLs. Nature  
392:86-89, 1998.
  25. Trombetta ES, Mellman I: Cell biology of antigen processing in vitro  
and in vivo. Annu Rev Immunol 23:975-1028, 2005.
  26. Crocker PR, Morris L, Gordon S: Novel cell surface adhesion recep-  
tors involved in interactions between stromal macrophages and hae-
matopoietic cells. J Cell Sci Suppl 9:185-206, 1988.  
  27. Elliott J, Johnston JA: SOCS: Role in inflammation, allergy and
homeostasis. Trends Immunol 25:434-440, 2004.
  28. Chitu V, Stanley ER: Colony-stimulating factor-1 in immunity and  
inflammation. Curr Opin Immunol 18:39-48, 2006.
  29. Witmer-Pack MD, Hughes DA, Schuler G, et al: Identification of  
macrophages and dendritic cells in the osteopetrotic (op/op) mouse.
J Cell Sci 104(Pt 4):1021-1029, 1993.
  30. Wiktor-Jedrzejczak W, Gordon S: Cytokine regulation of the mac-  
rophage (M phi) system studied using the colony stimulating factor-
1-deficient op/op mouse. Physiol Rev 76:927-947, 1996.  
  31. Anderson KL, Smith KA, Conners K, et al: Myeloid development is
selectively disrupted in PU.1 null mice. Blood 91:3702-3710, 1998.  
  32. Dahl R, Walsh JC, Lancki D, et al: Regulation of macrophage and
neutrophil cell fates by the PU.1:C/EBPalpha ratio and granulocyte  
colony-stimulating factor. Nat Immunol 4:1029-1036, 2003.
  33. Aziz A, Vanhille L, Mohideen P, et al: Development of macrophages
with altered actin organization in the absence of MafB. Mol Cell  
Biol 26:6808-6818, 31(3)584-592, 2006.
  34. Ziegler-Heitbrock L: The CD14+ CD16+ blood monocytes: Their
role in infection and inflammation. J Leukoc Biol 81(3):584-592,  
2007.
  35. Fogg DK, Sibon C, Miled C, et al: A clonogenic bone marrow pro-
genitor specific for macrophages and dendritic cells. Science 311:83-  
87, 2006.
  36. Geissmann F, Jung S, Littman DR: Blood monocytes consist of two  
principal subsets with distinct migratory properties. Immunity 19:
71-82, 2003.
  37. Tacke F, Randolph GJ: Migratory fate and differentiation of blood  
monocyte subsets. Immunobiology 211:609-618, 2006.
38. Zlotnik A, Yoshie O, Nomiyama H: The chemokine and chemokine
receptor superfamilies and their molecular evolution. Genome Biol
7:243, 2006.
39. Stacey M, Lin HH, Gordon S, et al: LNB-TM7, a group of seven-
transmembrane proteins related to family-B G-protein-coupled
receptors. Trends Biochem Sci 25:284-289, 2000.
40. Stacey M, Chang GW, Davies JQ, et al: The epidermal growth factor-
like domains of the human EMR2 receptor mediate cell attachment
through chondroitin sulfate glycosaminoglycans. Blood 102:2916-2924,
2003.
41. Gordon S: Macrophage heterogeneity and tissue lipids. J Clin Invest
117:89-93, 2007.
42. Tsou CL, Peters W,  Si Y,  et al: Critical roles for CCR2 and MCP-3
in monocyte mobilization from bone marrow and recruitment to
inflammatory sites. J Clin Invest 117:902-909, 2007.
43. Luster AD, Alon R, von Andrian UH: Immune cell migration in
inflammation: Present and future therapeutic targets. Nat Immunol
6:1182-1190, 2005.
44. Schenkel AR, Mamdouh Z, Chen X, et al: CD99 plays a major role
in the migration of monocytes through endothelial junctions. Nat
Immunol 3:143-150, 2002.
45. Carman CV, Springer TA: A transmigratory cup in leukocyte diape-
desis both through individual vascular endothelial cells and between
them. J Cell Biol 167:377-388, 2004.
46. Harris ES, McIntyre TM, Prescott SM, et al: The leukocyte integrins.
J Biol Chem 275:23409-23412, 2000.
47. Anderson DC, Springer TA: Leukocyte adhesion deficiency: An
inherited defect in the Mac-1, LFA-1, and p150,95 glycoproteins.
Annu Rev Med 38:175-194, 1987.
48. Rosen H, Gordon S: Monoclonal antibody to the murine type 3
complement receptor inhibits adhesion of myelomonocytic cells in
vitro and inflammatory cell recruitment in vivo. J Exp Med 166:
1685-1701, 1987.
49. Gordon S, Lawson L, Rabinowitz S, et al: Antigen markers of macro-
phage differentiation in murine tissues. In Russell S, Gordon S (eds):
Macrophage biology and activation. Curr Top Microbiol Immunol
181:1-37, 1992.
50. Kosco-Vilbois MH: Are follicular dendritic cells really good for noth-
ing? Nat Rev Immunol 3:764-769, 2003.
51. Kraal G, Mebius R: New insights into the cell biology of the marginal
zone of the spleen. Int Rev Cytol 250:175-215, 2006.
52. Colonna M: TREMs in the immune system and beyond. Nat Rev
Immunol 3:445-453, 2003.
53. Adams DO: The granulomatous inflammatory response: A review.
Am J Pathol 84:164-191, 1976.
54. Keshav S, Chung P, Milon G, et al: Lysozyme is an inducible marker
of macrophage activation in murine tissues as demonstrated by in situ
hybridization. J Exp Med 174:1049-1058, 1991.
55. Chen EH, Grote E, Mohler W, et al: Cell-cell fusion. FEBS Lett
581:2181-2193, 2007.
56. McInnes A, Rennick DM: Interleukin 4 induces cultured monocytes/
macrophages to form giant multinucleated cells. J Exp Med 167:
598-611, 1988.
57. Brombacher F: The role of interleukin-13 in infectious diseases and
allergy. Bioessays 22:646-656, 2000.
58. Ginhoux F, Tacke F, Angeli V, et al: Langerhans cells arise from
monocytes in vivo. Nat Immunol 7:265-273, 2006.
59. Janeway CA Jr, Medzhitov R: Innate immune recognition. Annu
Rev Immunol 20:197-216, 2002.
60. Medzhitov R, Preston-Hurlburt P, Janeway CA Jr: A human homo-
logue of the Drosophila Toll protein signals activation of adaptive
immunity. Nature 388:394-397, 1997.
61. Poltorak A, He X, Smirnova I, et al: Defective LPS signaling in
C3H/HeJ and C57BL/10ScCr mice: Mutations in Tlr4 gene. Science
282:2085-2088, 1998.
62. Taylor PR, Martinez-Pomares L, Stacey M, et al: Macrophage
receptors and immune recognition. Annu Rev Immunol 23:901-
944, 2005.
63. Gordon S: Pattern recognition receptors: Doubling up for the innate
immune response. Cell 111:927-930, 2002.
64. Freire-de-Lima CG, Nascimento DO, Soares MB, et al: Uptake of
apoptotic cells drives the growth of a pathogenic trypanosome in mac-
rophages. Nature 403:199-203, 2000.
65. Akira S, Takeda K, Kaisho T: Toll-like receptors: Critical proteins link-
ing innate and acquired immunity. Nat Immunol 2:675-680, 2001.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
  66. Beutler B, Hoebe K, Georgel P, et al: Genetic analysis of innate
immunity: Identification and function of the TIR adapter proteins.
Adv Exp Med Biol 560:29-39, 2005.
  67. Doyle SL, O’Neill LA: Toll-like receptors: From the discovery
of NFkappaB to new insights into transcriptional regulations in
innate immunity. Biochem Pharmacol 72:1102-1113, 2006.
  68. Haziot A, Ferrero E, Köntgen F, et al: Resistance to endotoxin shock
and reduced dissemination of gram-negative bacteria in CD14-defi-
cient mice. Immunity 4:407-414, 1996.
  69. Brown GD: Dectin-1: A signalling non-TLR pattern-recognition
receptor. Nat Rev Immunol 6:33-43, 2006.
  70. Crocker PR, Paulson JC, Varki A: Siglecs and their roles in the
immune system. Nat Rev Immunol 7:255-266, 2007.
  71. Taylor PR, Gordon S, Martinez-Pomares L: The mannose recep-
tor: Linking homeostasis and immunity through sugar recognition.
Trends Immunol 26:104-110, 2005.
  72. Dostert C, Tschopp J: DEteCTINg fungal pathogens. Nat Immunol
8:17-18, 2007.
  73. Barclay AN, Wright GJ, Brooke G, et al: CD200 and membrane pro-
tein interactions in the control of myeloid cells. Trends Immunol
23:285-290, 2002.
  74. Barclay AN, Brown MH: The SIRP family of receptors and
immune regulation. Nat Rev Immunol 6:457-464, 2006.
  75. Barrington R, Zhang M, Fischer M, et al: The role of complement in
inflammation and adaptive immunity. Immunol Rev 180:5-15, 2001.
  76. Roozendaal R, Carroll MC: Emerging patterns in complement-
­mediated pathogen recognition. Cell 125:29-32, 2006.
  77. Helmy KY, Katschke KJ Jr, Gorgani NN, et al: CRIg: A macro-
phage complement receptor required for phagocytosis of circulating
pathogens. Cell 124:915-927, 2006.
  78. Taylor PR, Carugati A, Fadok VA, et al: A hierarchical role for classi-
cal pathway complement proteins in the clearance of apoptotic cells
in vivo. J Exp Med 192:359-366, 2000.
  79. Taylor PR, Pickering MC, Kosco-Vilbois MH, et al: The follicular­
dendritic cell restricted epitope, FDC-M2, is complement
C4: Localization of immune complexes in mouse tissues. Eur J Immu-
nol 32:1888-1896, 2002.
  80. Daeron M: Fc receptor biology. Annu Rev Immunol 15:203-234,
1997.
  81. Daeron M, Lesourne R: Negative signaling in Fc receptor complexes.
Adv Immunol 89:39-86, 2006.
  82. Ravetch JV, Bolland S: IgG Fc receptors. Annu Rev Immunol
19:275-290, 2001.
  83. Hogarth PM: Fc receptors are major mediators of antibody based inflam-
mation in autoimmunity. Curr Opin Immunol 14:798-802, 2002.
  84. Florey OJ, Johns M, Esho OO, et al: Antiendothelial cell antibod-
ies mediate enhanced leukocyte adhesion to cytokine-activated
endothelial cells through a novel mechanism requiring cooperation
between FcγRIIa and CXCR1/2. Blood 109:3881-3889, 2007.
  85. Kaneko Y, Nimmerjahn F, Ravetch JV: Anti-inflammatory activ-
ity of immunoglobulin G resulting from Fc sialylation. Science 313:
670-673, 2006.
  86. Fritz JH, Ferrero RL, Philpott DJ, et al: Nod-like proteins in immu-
nity, inflammation and disease. Nat Immunol 7:1250-1257, 2006.
  87. Aderem A, Underhill DM: Mechanisms of phagocytosis in macro-
phages. Annu Rev Immunol 17:593-623, 1999.
  88. Gordon S (ed): Phagocytosis—The Host. Stamford, Conn, JAI Press,
1999.
  89. Heinsbroek SEM, Gordon S: Macrophages. In Brown GD, Netea
M (eds): Immunology of Fungal Infections, ch2. Dordrecht, The
Netherlands,­ Springer, 2007, pp 3-25.
  90. Stuart LM, Ezekowitz RA: Phagocytosis: Elegant complexity.
Immunity 22:539-550, 2005.
  91. May RC, Machesky LM: Phagocytosis and the actin cytoskeleton.
J Cell Sci 114:1061-1077, 2001.
  92. Caron E, Hall A: Identification of two distinct mechanisms of phagocyto-
sis controlled by different Rho GTPases. Science 282:1717-1721, 1998.
  93. Martens S, Sabel K, Lange R, et al: Mechanisms regulating the posi-
tioning of mouse p47 resistance GTPases LRG-47 and IIGP1 on
cellular membranes: Retargeting to plasma membrane induced by
phagocytosis. J Immunol 173:2594-2606, 2004.
  94. MacMicking JD: Immune control of phagosomal bacteria by p47
GTPases. Curr Opin Microbiol 8:74-82, 2005.
  95. MacMicking JD, Taylor GA, McKinney JD: Immune control
of tuberculosis by IFN-gamma-inducible LRG-47. Science 302:
654-659, 2003.
153
  96. Desjardins M: ER-mediated phagocytosis: A new membrane for new
functions. Nat Rev Immunol 3:280-291, 2003.
  97. Touret N, Paroutis P, Terebiznik M, et al: Quantitative and dynamic
assessment of the contribution of the ER to phagosome formation.
Cell 123:157-170, 2005.
  98. Blander JM, Medzhitov R: Regulation of phagosome maturation by
signals from toll-like receptors. Science 304:1014-1018, 2004.
  99. Swanson MS: Autophagy: Eating for good health. J Immunol
177:4945-4951, 2006.
100. Deretic V: Autophagy in innate and adaptive immunity. Trends
Immunol 26:523-528, 2005.
101. Gutierrez MG, Master SS, Singh SB, et al: Autophagy is a defense
mechanism inhibiting BCG and Mycobacterium tuberculosis survival
in infected macrophages. Cell 119:753-766, 2004.
102. Gorvel J-P (ed): Intracellular Pathogens in Membrane Interactions
and Vacuole Biogenesis. Amsterdam, Kluwer Academic/Plenum,
2003.
103. Roy CR, Salcedo SP, Gorvel JP: Pathogen-endoplasmic-reticulum
interactions: In through the out door. Nat Rev Immunol 6:136-147,
2006.
104. Akira S, Takeda K: Toll-like receptor signalling. Nat Rev Immunol
4:499-511, 2004.
105. Honda K, Taniguchi T: IRFs: Master regulators of signalling by Toll-
like receptors and cytosolic pattern-recognition receptors. Nat Rev
Immunol 6:644-658, 2006.
106. Greenberg S: Diversity in phagocytic signalling. J Cell Sci 114:
1039-1040, 2001.
107. Nimmerjahn F, Ravetch JV: Fcgamma receptors: Old friends and
new family members. Immunity 24:19-28, 2006.
108. Crowley MT, Costello PS, Fitzer-Attas CJ, et al: A critical role for
Syk in signal transduction and phagocytosis mediated by Fcgamma
receptors on macrophages. J Exp Med 186:1027-1039, 1997.
109. Gross O, Gewies A, Finger K, et al: Card9 controls a non-TLR signal-
ling pathway for innate anti-fungal immunity. Nature 442:651-656,
2006.
110. Stout RD, Suttles J: Functional plasticity of macrophages: Revers-
ible adaptation to changing microenvironments. J Leukoc Biol 76:
509-513, 2004.
111. Barish GD, Downes M, Alaynick WA, et al: A nuclear receptor atlas:
Macrophage activation. Mol Endocrinol 19:2466-2477, 2005.
112. Serhan CN, Brain SD, Buckley CD, et al: Resolution of inflamma-
tion: State of the art, definitions and terms. FASEB J 21:325-332,
2007.
113. Murray RZ, Kay JG, Sangermani DG, et al: A role for the phagosome
in cytokine secretion. Science 310:1492-1495, 2005.
114. Munder M, Eichmann K, Modolell M: Alternative metabolic states
in murine macrophages reflected by the nitric oxide synthase/argi-
nase balance: Competitive regulation by CD4+ T cells correlates
with Th1/Th2 phenotype. J Immunol 160:5347-5354, 1998.
115. Stein M, Keshav S, Harris N, et al: Interleukin 4 potently enhances
murine macrophage mannose receptor activity: A marker of alternative
immunologic macrophage activation. J Exp Med 176:287-292, 1992.
116. Goerdt S, Orfanos CE: Other functions, other genes: Alternative
activation of antigen-presenting cells. Immunity 10:137-142, 1999.
117. Anderson CF, Mosser DM: A novel phenotype for an activated
macrophage: The type 2 activated macrophage. J Leukoc Biol 72:
101-106, 2002.
118. Yona S, Gordon S: Inflammation: Glucocorticoids turn the mono-
cyte switch. Immunol Cell Biol 85:81-82, 2007.
119. Kristiansen M, Graverson JH, Jacobsen C, et al: Identification of the
haemoglobin scavenger receptor. Nature 409:198-201, 2001.
120. Casanova JL, Abel L: Genetic dissection of immunity to mycobac-
teria: The human model. Annu Rev Immunol 20:581-620, 2002.
121. Dalton DK, Pitts-Meek S, Keshav S, et al: Multiple defects of immune
cell function in mice with disrupted interferon-gamma genes.
Science 259:1739-1742, 1993.
122. Mellor AL, Munn DH: Tryptophan catabolism and regulation of
adaptive immunity. J Immunol 170:5809-5813, 2003.
123. Wynn TA: Fibrotic disease and the T(H)1/T(H)2 paradigm. Nat
Rev Immunol 4:583-594, 2004.
124. Yoshitomi H, Sakaguchi N, Kobayashi K, et al: A role for fungal
β-glucans and their receptor Dectin-1 in the induction of autoimmune
arthritis in genetically susceptible mice. J Exp Med 201:949-960, 2005.
125. Hoek RM, Ruuls SR, Murphy CA, et al: Down-regulation of the
macrophage lineage through interaction with OX2 (CD200).
Science 290:1768-1771, 2000.
 154 GORDON  |  Mononuclear Phagocytes in Rheumatic Diseases
126. Helming L, Gordon S: Macrophage fusion induced by IL-4 alterna-
tive activation is a multistage process involving multiple target mol-
ecules. Eur J Immunol 37:33-42, 2007.
127. Lin HH, Faunce DE, Stacey M, et al: The macrophage F4/80 receptor
is required for the induction of antigen-specific efferent regulatory
T cells in peripheral tolerance. J Exp Med 201:1615-1625, 2005.
128. Hamann J, Vogel B, van Schijndel GM, et al: The seven-span trans-
membrane receptor CD97 has a cellular ligand (CD55, DAF). J Exp
Med 184:1185-1189, 1996.
129. O’Neill LA, Bowie AG: The family of five: TIR-domain-contain-
ing adaptors in Toll-like receptor signalling. Nat Rev Immunol 7:
353-364, 2007.
130. East L, Isacke CM: The mannose receptor family. Biochim Biophys
Acta 1572:364-386, 2002.
131. Bonifaz L, Donnyay D, Mahnke K, et al: Efficient targeting of protein
antigen to the dendritic cell receptor DEC-205 in the steady state
leads to antigen presentation on major histocompatibility complex
class I products and peripheral CD8+ T cell tolerance. J Exp Med
196:1627-1638, 2002.
132. Taylor PR, Tsoni SV, Willment JA, et al: Dectin-1 is required for
beta-glucan recognition and control of fungal infection. Nat Immu-
nol 8:31-38, 2007.
133. Elomaa O, Kangas M, Sahlberg C, et al: Cloning of a novel bacte-
ria-binding receptor structurally related to scavenger receptors and
expressed in a subset of macrophages. Cell 80:603-609, 1995.
134. Mukhopadhyay S, Bowdish D, Gordon S: The scavenger receptor
MARCO. Mod Asp Immunobiol 20:15, 2006.
135. Mukhopadhyay S, Chen Y, Sankala M, et al: MARCO, an innate
activation marker of macrophages, is a class A scavenger receptor for
Neisseria meningitidis. Eur J Immunol 36:940-949, 2006.
136. Mukhopadhyay S, Gordon S: The role of scavenger receptors in
pathogen recognition and innate immunity. Immunobiology 209:
39-49, 2004.
137. Mukhopadhyay S, Peiser L, Gordon S: Activation of murine mac-
rophages by Neisseria meningitidis and IFN-gamma in vitro: Distinct
roles of class A scavenger and Toll-like pattern recognition recep-
tors in selective modulation of surface phenotype. J Leukoc Biol 76:
577-584, 2004.
138. Febbraio M, Hajjar DP, Silverstein RL: CD36: A class B scavenger
receptor involved in angiogenesis, atherosclerosis, inflammation, and
lipid metabolism. J Clin Invest 108:785-791, 2001.
139. Peiser L, Mukhopadhyay S, Gordon S: Scavenger receptors in innate
immunity. Curr Opin Immunol 14:123-128, 2002.
140. Mukhopadhyay S, Herre J, Brown GD, et al: The potential for Toll-
like receptors to collaborate with other innate immune receptors.
Immunology 112:521-530, 2004.
141. Savill J, Dransfield I, Gregory C, et al: A blast from the past: Clear-
ance of apoptotic cells regulates immune responses. Nat Rev Immu-
nol 2:965-975, 2002.
142. Turnbull IR, Colonna M: Activating and inhibitory functions of
DAP12. Nat Rev Immunol 7:155-161, 2007.
143. White J, Lukacik P, Esser D, et al: Biological activity, membrane-
targeting modification, and crystallization of soluble human decay
accelerating factor expressed in E. coli. Protein Sci 13:2406-2415,
2004.
144. Farkas I, Baranyi L, Ishikawa Y, et al: CD59 blocks not only the inser-
tion of C9 into MAC but inhibits ion channel formation by homolo-
gous C5b-8 as well as C5b-9. J Physiol 539:537-545, 2002.
145. Maltais LJ, Lovering RC, Taranin AV, et al: New nomenclature for Fc
receptor-like molecules. Nat Immunol 7:431-432, 2006.
146. Heath WR, Carbone FR: Cross-presentation in viral immunity and
self-tolerance. Nat Rev Immunol 1:126-134, 2001.
147. Geijtenbeek TB, Engering A, Van Kooyk Y: DC-SIGN, a C-type
lectin on dendritic cells that unveils many aspects of dendritic cell
biology. J Leukoc Biol 71:921-931, 2002.
148. Kraal G, van der Laan LJ, Elomaa O, et al: The macrophage receptor
MARCO. Microbes Infect 2:313-316, 2000.
149. Platt N, Gordon S: Is the class A macrophage scavenger recep-
tor (SR-A) multifunctional? The mouse’s tale. J Clin Invest 108:
649-654, 2001.
150. Taylor PR, Zamze S, Stillion RJ, et al: Development of a specific sys-
tem for targeting protein to metallophilic macrophages. Proc Natl
Acad Sci U S A 101:1963-1968, 2004.
151. van der Laan LJ, Döpp EA, Haworth R, et al: Regulation and func-
tional involvement of macrophage scavenger receptor MARCO in
clearance of bacteria in vivo. J Immunol 162:939-947, 1999.
152. MacMicking J, Xie QW, Nathan C: Nitric oxide and macrophage
function. Annu Rev Immunol 15:323-350, 1997.
153. Nathan CF: Secretory products of macrophages. J Clin Invest
79:319-326, 1987.
154. Herbert DR, Hölscher C, Mohrs M, et al: Alternative macrophage
activation is essential for survival during schistosomiasis and down-
modulates T helper 1 responses and immunopathology. Immunity
20:623-635, 2004.
 9 T Lymphocytes
KEY POINTS
T cells develop primarily in the thymus.
Thymic selection consists of a positive phase in which T cells
must recognize self-MHC molecules, and a negative phase
in which thymocytes bearing high-affinity TCRs for self-MHC
peptide are deleted through apoptosis.
Developing T cells first express the TCR β chain during the late
CD4–CD8– stage. Expression of one TCR β chain suppresses
further rearrangement and expression of another TCR β chain
(allelic exclusion). The TCR β chain pairs briefly with the
invariant pre-Tα chain until the actual TCR α chain rearranges
at the CD4+CD8+ stage. The α chain is not subject to allelic
exclusion, so further rearrangements are possible.
T cells emerge from the thymus as naive T cells that are
quiescent and, when activated, express low to negligible
levels of most cytokines. Once they acquire a memory
phenotype (CD44 high, CD45RO+), they can produce high
levels of cytokines.
Naive T cells can spontaneously undergo homeostatic
proliferation to self-MHC peptides in peripheral lymphoid
tissues to generate a critical number of T cells. This requires
IL-7 and IL-15.
The importance of the thymus is underscored by the complete
absence of T cells in patients in whom a thymus has failed to
develop (e.g., complete DiGeorge syndrome).
The immune system is foremost an organ of defense against
infection. The evolutionary pressures that have molded the
particular structures of the immune response and promoted
a highly diverse repertoire clearly derive from infectious
agents. Two very different strategies exist. The more primi-
tive innate immune response (see Chapter 16) uses a lim-
ited repertoire of nonpolymorphic receptors that recognize
structural motifs that are common to many microorganisms.
These include small glycolipids and lipopeptides. The evolu-
tionarily newer adaptive immune response (see Chapter 17)
relies on the generation of myriad different receptors that
might recognize a wide array of foreign compounds from
infectious agents. Whereas the innate immune response is
rapid, the adaptive one permits a wider array of responses as
well as immune memory.
T lymphocyte development constantly confronts the
dilemma of combating infection without provoking a
response in the host. The trade-off of generating an increas-
ingly varied population of antigen receptors that can rec-
ognize a wide spectrum of pathogens is the progressive risk
of producing self-reactive lymphocytes that can provoke
an autoimmune diathesis. To minimize the possibility of
creating self-reactive cells, T lymphocytes are subjected to
RALPH C. BUDD  •  KAREN A. FORTNER
a rigorous selection process during development in the thy-
mus. In addition, premature activation of mature T cells is
prevented by requiring two signals for activation. Finally,
the tremendous expansion of T cells that occurs during the
response to an infection is resolved by the active induction
of cell death. In perhaps no other organ are the processes
of cell proliferation and death more dynamically displayed
than by lymphocytes during an immune response. The con-
sequences of inefficient lymphocyte removal at any one
of these junctures can be devastating to the health of the
organism. This is vividly displayed in both humans and mice,
where naturally arising mutations in death receptors such as
Fas result in the massive accumulation of lymphocytes and
autoimmune sequelae. These are discussed in more detail in
Chapter 24.
The activation of T lymphocytes yields a variety of effec-
tor functions that are pivotal to arresting infectious pro-
cesses. Cytolytic T cells can kill infected cells through the
expression of perforin, which produces holes in cell mem-
branes, or ligands for death receptors such as Fas or tumor
necrosis factor-α (TNF-α) receptor. The production of
T cell cytokines such as interferon-γ (IFN-γ) can inhibit
viral replication, whereas other cytokines such as interleu-
kin (IL)-4 and IL-5 are critical for optimal B cell growth and
immunoglobulin production.1,2 However, this same arma-
mentarium can also precipitate damage to host tissues and
provoke autoimmune responses. This is most dramatically
apparent when T cell infiltration can be observed histologi-
cally, such as in the synovium of inflammatory arthritides,
pancreatic islets in type 1 diabetes, and the central nervous
system in multiple sclerosis. Damage in these cases need not
be the direct result of recognition of target tissues by T cells.
T cells may be activated elsewhere and then migrate to the
tissue and damage innocent bystander cells. T cells may also
promote an autoimmune diathesis through the augmenta-
tion of B cell responses.
In describing the development and function of T lym-
phocytes in this chapter, emphasis is placed on the vari-
ous junctures where autoreactive T cells may arise through
their inefficient elimination, accidental clonal expansion
by means of cross-reactivity between infectious and self-
antigens, or nonspecific activation and resulting bystander
injury.
T CELL DEVELOPMENT
T cells must overcome two stringent hurdles during their
development. First, a T cell must successfully rearrange the
genes encoding the two chains of the T cell antigen receptor
(TCR). Second, a T cell must survive thymic selection, dur-
ing which self-reactive T cells are eliminated. The ­survival
155
 156 BUDD  |  T Lymphocytes
rate of these two independent selection processes is less
than 3%, which is in part necessary to minimize the chance
of autoreactive T cells escaping to the periphery.
The TCR is an 80- to 90-kD disulfide-linked heterodi-
mer composed of a 48- to 54-kD α chain and a 37- to 42-kD
β chain. An alternative TCR composed of γ and δ chains
is expressed on 2% to 3% of peripheral blood T cells and is
discussed later. The TCR has an extracellular ligand bind-
ing pocket and a short cytoplasmic tail that, by itself, cannot
signal. Consequently, it is noncovalently associated with as
many as five invariant chains of the CD3 complex. Not sur-
prisingly, the structure of the TCR gene is very similar to
what was first described for immunoglobulin genes in B cells
(see details in Chapter 10). Each overcame the problem of
how to encode approximately 10 million different T or B cell
specificities within the human genome, which contains only
about 30,000 genes. To economically package this diversity,
the process of gene rearrangement and splicing evolved
using machinery similar to that which already existed to
promote gene translocations. The β and δ chain genes of
the TCR contain four segments known as the V (variable),
D (diversity), J (joining), and C (constant) regions. The α
and γ chains are similar but lack the J component. Each of
the segments has several family members (approximately 50
to 100 V, 15 D, 6 to 60 J, and 1 to 2 C members). An orderly
process occurs during TCR gene rearrangement in which a
D segment is spliced adjacent to a J segment, which is sub-
sequently spliced to a V segment. Following transcription,
the VDJ sequence is spliced to a C segment to produce a
mature TCR messenger RNA. Arithmetically, this random
rearrangement of a single chain of the TCR locus can give
rise to a minimum of 50V × 15D × 6J × 2C, or about 9000
possible combinations. At the site of each splice, which
must occur in-frame to be functional, additional nucleotides
not encoded by the genome (so-called N-region nucleo-
tides) can be incorporated, adding further diversity to the
rearranging gene. Theoretically, the combinations from the
two TCR chains, plus N-region diversity, yield at least 108
possible combinations. The cutting, rearranging, and splic-
ing are directed by specific enzymes. Mutations in the genes
for these processes can result in an arrest in lymphocyte
development. For example, mutation in the gene encoding
a DNA-dependent protein kinase required for receptor gene
recombination results in a severe combined immunodefi-
ciency (SCID).
TCR gene recombination thus represents the first of the
two major hurdles for developing lymphocytes. Because the
developing T cell has two copies of each chromosome, there
are two chances to successfully rearrange each of the two
TCR chains. As soon as successful rearrangement occurs,
further β-chain rearrangements on either the same or the
other chromosome are suppressed, a process known as allelic
exclusion. This limits the chance of dual TCR expression by
an individual T cell. The high percentage of T cells that
contain rearrangements of both β-chain genes attests to the
inefficiency of this complex event. Rearrangement of the α
chain occurs later in thymocyte development in a similar
fashion, although without apparent allelic exclusion. This
can lead to dual TCR expression by a single T cell.
Development of T cells occurs within a microenviron-
ment provided by the thymic epithelial stroma. The thymic
anlage is formed from embryonic ectoderm and endoderm
and is then colonized by hematopoietic cells, which give rise
to dendritic cells, macrophages, and developing T cells.2 The
hematopoietic and epithelial components combine to form
two histologically defined compartments: the cortex, which
contains immature thymocytes, and the medulla, which
contains mature thymocytes (Fig. 9-1A). As few as 50 to 100
bone marrow–derived stem cells enter the thymus daily.3
The stages of thymocyte development can be defined
by the status of rearrangement and expression of the two
genes that encode the α and β chains of the TCR and the
expression of CD4 and CD8, proceeding in an orderly fash-
ion: CD4–CD8– → CD4+CD8+ → CD4+CD8– or CD4–CD8+
(Fig. 9-1B). CD4 and CD8 define, respectively, the helper
and cytolytic subsets of mature T cells.
CD4–CD8– thymocytes can be further subdivided based
on their expression of CD25 (the high-affinity IL-2 receptor
α chain) and CD44 (the hyaluronate receptor).4 Develop-
ment proceeds in this order: CD25–CD44+ → CD25+CD44+
→ CD25+CD44– → CD25–CD44–. These subpopulations
correspond to discrete stages of thymocyte differentia-
tion. CD25–CD44+ cells express low levels of CD4, and
their TCR genes are in germline configuration. These cells
downregulate CD4 and upregulate CD25 to give rise to
CD25+CD44+ thymocytes, which now express surface CD2
and low levels of CD3ε. At the next stage (CD25+CD44–),
there is a brief burst of proliferation, followed by upregula-
tion of the recombination-activating enzymes RAG-1 and
RAG-2, and the concomitant rearrangement of the genes
of the TCR β chain. A small subpopulation of T cells rear-
ranges and expresses a second pair of TCR genes known as
γ and δ. Productive TCR β-chain rearrangement results in
downregulation of RAG and a second proliferative burst.
Loss of CD25 then yields CD25–CD44– thymocytes.
The TCR β chain cannot be stably expressed without an
α chain. Because the TCR α chain has not yet rearranged,
a surrogate invariant TCR pre-α chain is disulfide linked
to the β chain.5 When associated with components of the
CD3 complex, this allows a low-level surface expression of a
pre-TCR and progression to the next developmental stage.
Failure to successfully rearrange the TCR β chain results in
developmental arrest at the transition from CD25+CD44– to
CD25–CD44–. This occurs in RAG-deficient mice as well as
in mice and humans with SCID.6,7
A number of signaling molecules are required for early T
cell development (Fig. 9-2).8 The IKAROS gene encodes a
family of transcription factors required for the development
of cells of lymphoid origin. Notch-1, a molecule known to
regulate cell fate decisions, is also required at the earliest
stage of T cell lineage development.9 Cytokines, including
IL-7, promote the survival and expansion of the earliest thy-
mocytes.10 In mice deficient for IL-7, its receptor components
IL-7Rα or γc, or the cytokine receptor-associated signaling
molecule JAK-3, thymocyte development is inhibited at the
CD25–CD44+ stage. In humans, mutations in γc or JAK-3
result in the most frequent form of SCID.11 Pre-TCR sig-
naling is required for the transition from CD25+CD44– to
CD25–CD44–. Thus, loss of signaling components, includ-
ing Lck, SLP-76, and LAT-1, results in a block at this stage
of T cell development.12 TCR signals are also required for
differentiation of CD4+ or CD8+ cells. Humans deficient in
ZAP-70 have CD4+ but not CD8+ T cells in the thymus and
periphery.13
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION 157

TCR-β TCR-β
A + pre-Tα B
scid
Rag1&2

CD44+CD25- CD44+CD25+ CD44-CD25+ CD44-CD25- CD4-CD8+ CD4+CD8+

CD8
TCR-α

CD4-CD8- CD4+CD8+

CD4-CD8- CD4+CD8-
CD4+
or CD8+ CD4

Medulla

Cortex

Thymus
Figure 9-1  Sequence of thymocyte development. A, The earliest thymocyte precursors lack expression of CD4 and CD8 (CD4–CD8–). These can be
divided into four subpopulations based on the sequential expression of CD44 and CD25. It is at the CD44–CD25+ stage that the T cell antigen recep-
tor (TCR) β chain rearranges. The scid mutation or deficiencies in the rearrangement enzymes RAG-1 and RAG-2 result in an inability to rearrange the
β chain, and maturational arrest occurs at this stage. Those thymocytes that successfully rearrange the β chain express it associated with a surrogate
α chain known as pre-Tα. Concomitant with a proliferative burst, development can then progress to the CD4+CD8+ stage in the cortex, where the TCR
α chain rearranges and pairs with the β chain to express a mature TCR complex. These cells then undergo thymic positive and negative selection (see
Fig. 9-3B). Successful completion of this rigorous selection process results in mature CD4+ or CD8+ T cells in the medulla, which eventually migrate to
peripheral lymphoid sites. B, Schematic flow cytometry showing subpopulations of thymocytes defined by CD4 and CD8 expression in their relative
proportions.

Corticosubcapsular region Cortex C-M Medulla Figure 9-2  Sequence of αβ T cell

junction (10-15%) development in the thymus. The
CD4-CD8- (5-8%) Selection earliest thymocyte precursors lack
CD4+ expression of CD4 and CD8 (CD4–
- CD8- CD8–). These can be divided into
Lymph. CD25- CD25+ CD25+ CD25- CD4+ + four subpopulations based on the
precursor CD44+ CD44+ CD44- CD44- CD8+ - sequential expression of CD25 and
CD4- CD44. At the CD25+CD44– stage,
(65-85%)
CD8+ the T cell antigen receptor (TCR) β
chain rearranges and associates a
Ikaros, Notch-1 TCR β-chain TCR α-chain surrogate α chain known as pre-Tα.
(5-10%)
rearrangement rearrangement Concomitant with a proliferative
burst, thymocytes progress to the
IL-7Ra, IL-7, c-kit/gc CD4+CD8+ stage, rearrange the TCR
α chain, and express a mature TCR
RAG, Ku-80, Lck/Fyn, ZAP-70/Syk complex. These cells then undergo
thymic positive and negative selec-
Jak 3, SLP-76, LAT-1, p38 tion. Those thymocytes that survive
this rigorous selection process dif-
Lck ferentiate into mature CD4+ or CD8+
T cells. Also shown are the various
CD45, ZAP-70, Vav signaling molecules involved at spe-
cific stages of thymic development.

CD25–CD44– cells upregulate expression of CD4 and
CD8 to become CD4+CD8+. It is as a CD4+CD8+ thymo-
cyte that the α chain of the TCR rearranges. Unlike the β
chain, allelic exclusion of the α chain is not apparent. Rear-
rangement of the α chain can occur simultaneously on both
chromosomes, and if one attempt is unsuccessful, repeat
rearrangements to other Vα segments are possible. Dual
TCR expression has been reported in as many as 30% of
mature T cells in which the same T cell expresses different
α chains paired with the same β chain.14 However, in most
 158 BUDD  |  T Lymphocytes
cases of dual TCR α chains, one is downregulated during
positive selection by Lck and Cbl through ubiquitination,
endocytosis, and degradation.15
Although the structure of immunoglobulin and TCR
are quite similar, they recognize fundamentally differ-
ent antigens. Immunoglobulins recognize intact antigens
in isolation, either soluble or membrane bound, and are
often sensitive to the tertiary structure. The TCRαβ rec-
ognizes linear stretches of antigen peptide fragments bound
within the grooves of either major histocompatibility
complex (MHC) class I or class II molecules (Fig. 9-3A).
Thymic selection molds the repertoire of emerging TCRs
so that they recognize peptides within the groove of self-
MHC molecules, ensuring the self-MHC restriction of
T cell responses. The MHC structure is described in detail in
Chapter 17. Pockets within the MHC groove bind particu-
lar residues along the peptide sequence of 7 to 9 amino acids
for MHC class I molecules and 9 to 15 amino acids for MHC
class II molecules. As a result, depending on the particular
MHC molecule, certain amino acids make strong contact
with the MHC groove, while others contact the TCR.
The contact between the TCR and antigen-MHC has
been revealed by crystal structure to be remarkably flat,
rather than the deep lock-and-key structure one might
imagine.16,17 The TCR axis is tipped about 30 degrees to
the long axis of the MHC class I molecule and is slightly
more skewed to MHC class II. The affinity of the TCR for
antigen-MHC is in the micromolar range.18 This is less than
many antibody-antigen affinities and is several logs less than
many enzyme-substrate affinities. This has led to the notion
that TCR interactions with antigen-MHC are brief and that
successful activation of the T cell requires multiple interac-
tions, resulting in a cumulative signal.
Once the T cell has successfully rearranged and expressed
a TCR in association with the CD3 complex, it encounters
the second major hurdle in T cell development: thymic
selection. Selection has two phases, positive and negative,
and the outcome is based largely on the intensity of TCR
signaling in response to interactions with self-MHC pep-
tides expressed on thymic epithelium and dendritic cells.
TCR signals that are either too weak (death by neglect)
or too intense (negative selection) result in elimination
by apoptosis, whereas those with intermediate signaling
intensity survive positive selection (Fig. 9-3B). Successful
positive selection at the CD4+CD8+ stage is coincident with
upregulation of surface TCR, the activation markers CD5
and CD69, and the survival factor Bcl-2.19-21 T cells bearing
a TCR that recognizes MHC class I maintain CD8 expres-
sion, downregulate CD4, and become CD4–CD8+. T cells
expressing a TCR that recognizes MHC class II become
CD4+CD8–.
Not surprisingly, a variety of signaling molecules acti-
vated by TCR engagement are important to thymic selec-
tion. Lck, the Ras∏Raf-1∏MEK1∏ERK kinase cascade, the
kinase ZAP-70, and the phosphatases CD45 and calcineurin
are involved with positive selection.12 Among these, the Ras
∏ERK pathway is particularly important, because dominant
negative versions of these molecules can disrupt positive
selection. Conversely, an activator of Ras known as GRP1
assists the positive selection of thymocytes expressing weakly
selecting signals.22 These molecules are discussed in more
detail in the section on TCR signaling. By contrast, although
A
T cell
α β
Superantigen
Peptide
MHC
APC
B
Negtive selection
(apoptosis)
TCR density
Positive
selection
Null selection
(apoptosis)
TCR signal intensity
Figure 9-3  T cell antigen receptor (TCR) interaction with the major
histocompatibility complex (MHC)–peptide complex. A, Polymorphic
residues within the variable region of the α and β chains of the TCR make
contact with determinants of the MHC molecule on an antigen present-
ing cell (APC), as well as with the peptide fragment that sits in the MHC
binding groove. B, Diagram illustrating that during thymocyte devel-
opment, those TCRs conferring either a very low signal intensity (null 
selection) or a high signal intensity (negative selection) lead to apoptosis.
Only those thymocytes whose TCRs can engage MHC peptides and con-
fer moderate signal intensity survive by positive selection.
a number of molecules may promote negative selection (e.g.,
the MAP kinases JNK and p38), there appears to be suf-
ficient redundancy so that elimination of any one of these
molecules rarely affects the deletion of thymocytes. The few
exceptions include CD40, CD40L, and CD30; preservation
of at least some thymocytes bearing self-reactive TCR can be
observed in mice deficient in these molecules.23-25
The survivors of these two stringent processes of TCR
gene rearrangement and thymic selection represent less than
3% of total immature thymocytes. This is reflected by the
presence of a high rate of cell death in developing thymo-
cytes. This can be visualized by the measurement of DNA
degradation, a hallmark of apoptosis, as shown in ­Figure 9-4.
The survivors become either CD4+ helper or CD8+ cytolytic
T cells and reside in the thymic medulla for 12 to 14 days
before emigrating to the periphery. The decision to become
a CD4+ versus a CD8+ T cell involves further developmen-
tal signals, including (once again) Notch-1.26 Notch-1 sig-
naling is required for progression to CD8+ but not CD4+
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
Figure 9-4  TUNEL assay for nicked DNA in thymocytes undergoing
apoptosis. Thymus sections were made from wild-type mice, fixed, and
stained by the TUNEL assay. The DNA repair enzyme TdT inserts biotinyl-
ated d-uridine triphosphate residues at sites of double-stranded breaks,
which is then revealed by avidin horeseradish peroxidase. Sections are
shown at magnifications of 100× (top) and 400× (bottom). Apoptotic
thymocytes reflect those undergoing negative selection from a T cell an-
tigen receptor (TCR) signal that is either too intense or too weak.
thymocytes. This parallels the observation that long TCR
interactions are required for CD4 progression, whereas
shorter TCR engagement is required for CD8 progression.27
EXTRATHYMIC T CELL DEVELOPMENT
In the absence of a thymus, as in nude (nu/nu) mice, T cell
populations in the lymph nodes and spleen are severely
reduced and oligoclonal, but they are not absent. Whereas
some of this extrathymic T cell development occurs within
the liver, a significant amount also occurs in contact with
the intestinal epithelium.
Intestinal intraepithelial lymphocytes (IELs) have a
very different phenotype from thymus-derived T cells. In
contrast to the predominance of CD4+ T cells over CD8+
T cells in normal lymph nodes, IELs contain large propor-
tions of CD8+ and CD4– T cells.28 Many CD8+ IELs express
only the CD8 α chain as an αα homodimer. γδ T cells also
­constitute a significant portion of IELs. The antigens to
which IELs respond are not known, although in humans,
some of the γδ IELs recognize the MHC class Ib molecule
MICA.29 IELs have a memory CD44+ T cell phenotype (see
later) and are cytolytic when freshly isolated.
ABNORMALITIES OF HUMAN T CELL
DEVELOPMENT
Given the vast number of developmental events in T cell
development, it is not surprising that a multiplicity of
causes can underlie human T cell immunodeficiencies.30,31
159
The influence of the thymic stroma on thymocyte ontogeny
is underscored in the DiGeorge anomaly, in which develop-
ment of the pharyngeal pouches is disrupted and the thy-
mic rudiment fails to form.32 This results in the failure of
normal T cell development. Less severe T cell deficiencies
are associated with a failure to express MHC class I or class
II (the “bare lymphocyte syndrome”); these deficiencies are
directly involved with interactions required to induce the
positive selection of, respectively, CD8+ and CD4+ mature
T cells.
Metabolic disorders can affect thymocytes more directly.
The absence of functional adenosine deaminase and purine
nucleoside phosphorylase results in the buildup of metabolic
by-products that are toxic to developing T and B lympho-
cytes. This ultimately produces forms of SCID.
The inability to express a number of surface molecules
important in TCR and cytokine signaling also has the
potential to perturb development. The failure to express
TCR-CD3 components (specifically CD3γ and CD3ε),
CD18, and IL-2Rγ has been noted among patients who
exhibit varying degrees of T cell deficiency or dysfunction.33
All these molecules are involved in the signaling of thy-
mocyte development and survival, and their absence clearly
has the potential to alter developmental fate.
PERIPHERAL MIGRATION OF T CELLS
The migration of naive T cells to peripheral lymphoid struc-
tures or their infiltration into other tissues requires the coor-
dinated regulation of an array of cell adhesion molecules.
T cell recirculation is essential for host surveillance and is
carefully regulated by a specific array of homing receptors.
Entry from the circulation to tissues occurs via two main
anatomic sites: the flat endothelium of the blood vessels,
or specialized postcapillary venules known as high endo-
thelial venules.34 A three-step model has been proposed for
lymphocyte migration: rolling, adhesion, and migration.35
L-selectin expressed by naive T cells binds via lectin domains
to carbohydrate moieties of GlyCAM-1 and CD34 (col-
lectively known as peripheral node addressin), which are
expressed on endothelial cells, particularly high endothelial
venules. The weak binding of CD62L to its ligand mediates
a weak adhesion to the vessel wall; this, combined with the
force of blood flow, results in rolling of the T cell along the
endothelium. The increased cell contact facilitates the inter-
action of a second adhesion molecule on lymphocytes, the
integrin leukocyte function–associated antigen 1 (LFA-1;
CD11a/CD18), with its ligands, intercellular adhesion mol-
ecule 1 (ICAM-1; CD54) and ICAM-2 (CD102). This
results in the arrest of rolling and firm attachment. Migra-
tion into the extracellular matrix of tissues may involve
additional lymphocyte cell surface molecules such as the
hyaluronate receptor (CD44) or integrin α4β7 (CD49d/β7),
which binds the mucosal addressin cell adhesion molecule
1 on endothelium of Peyer’s patches and other endothelial
cells.
Other cytokines known as chemokines may contribute
to lymphocyte homing. Chemokines are structurally and
functionally related to proteins bearing an affinity for hepa-
ran sulfate proteoglycan, and they promote the migration of
various cell types.36,37 The chemokines RANTES, MIP-1α,
MIP-1β, MCP-1, and IL-8 are produced by a number of cell
 160 BUDD  |  T Lymphocytes

types, including endothelium, activated T cells, and mono-

cytes, and are present at inflammatory sites such as rheuma-
toid synovium (see also Chapter 65).
A more recently recognized capacity of peripheral T
cells is homeostatic proliferation.38,39 This reflects the ability
of mature T cells to undergo proliferation in lymphopenic
environments. Experimentally, this is studied by the adop-
tive transfer of T cells into RAG-deficient or irradiated
mice. However, a similar phenomenon occurs in normal
newborn mice.40 Newborn mice do not acquire adult lev-
els of T cells until day 7 in lymph nodes and day 15 in the
spleen. Because this expansion of T cells requires the pres-
ence of self-MHC peptides, as well as IL-7 and IL-15, this
area may be of considerable interest in future studies of auto-
immune mechanisms. In this regard, it is notable that one of
the standard models of autoimmunity is day 3 thymectomy,
which results in lymphopenia.41,42 It will be useful to deter-
mine the possible contribution of homeostatic proliferation
to this syndrome.
ACTIVATION OF T CELLS
T cell activation initiates intracellular signaling cascades,
which activate transcription factors and induce new gene
transcription. This ultimately results in proliferation, effector
function, or death, depending on the developmental stage of
the cell. To guard against premature or excessive activation,
T cells require two independent signals for full activation.
Signal 1 is an antigen-specific signal provided by the bind-
ing of the TCR to antigenic peptide complexed with MHC.
Signal 2 is mediated by either cytokines or the engagement
of costimulatory molecules such as B7-1 (CD80) and B7-2
(CD86) on the antigen presenting cell (APC).
T CELL RECEPTORS AND TYROSINE KINASES
TCRs αβ and γδ have very short cytoplasmic domains and,
by themselves, are unable to transduce signals.43,44 The
molecules of the noncovalently associated CD3 complex
couple the TCR to intracellular signaling machinery (Fig.
9-5). The CD3 complex contains nonpolymorphic members
known as CD3ε, CD3γ, CD3δ, as well as ζ and η chains that
are alternatively spliced forms of the same gene and are not
genetically linked to the CD3 complex. Although the func-
tional stoichiometry of the TCR complex is not completely
defined, current data indicate that each TCR heterodimer is
associated with three dimers: CD3εγ, CD3εδ, and ζζ or ζ.45
CD3ε, γ, and δ have an immunoglobulin-like extracellular
domain, a transmembrane region, and a modest cytoplasmic
domain, whereas ζ contains a longer cytoplasmic tail. The
transmembrane domains of ζ and the CD3 chains contain
a negatively charged residue that interacts with positively
charged amino acids in the transmembrane domain of the
TCR.
None of the proteins in the TCR complex has intrinsic
enzymatic activity. Instead, the cytoplasmic domains of the
invariant CD3 chains contain conserved activation domains
that are required for coupling the TCR to intracellular sig-
naling molecules. These immunoreceptor tyrosine-based
activation motifs (ITAMS) contain a minimal functional
consensus sequence of paired tyrosines (Y) and leucines
(L): (D/E)XXYXXL(X)6-8YXXL.46 ITAMs are substrates
for cytoplasmic protein tyrosine kinases (PTKs) and, upon
phosphorylation, recruit additional molecules to the TCR
complex.47 Each ζ chain contains three ITAMs, whereas
there is one in each of the CD3ε, γ, and δ chains. Thus,
each TCR complex can contain 10 ITAMs.

TCR

CD3
CD4

γ1 PLC PIP2
Ras PLC γ1
LAT
LAT

SOS Itk Itk

Grb2
ZAP-70
SL

DAG
76

Gads Gads
P

P-

Vav Lck
-7

IP3
SL
6

c-FLIP
Nck PKCθ

RIP1 Casp8
Rac CARMA1
Raf-1 Actin TRAF2 Ca2+
Rho Bcl-10
cytoskeleton MALT1
Cyclosporin
FK-506
MKKK IKKαβγ
MEK1 Calcineurin
MKK4 MKK7 IκB α/β
NF-κB p50/p65
ERK NFAT
JNK

c-Jun
Fos

NFAT AP-1 NF-κB Oct

IL-2
Figure 9-5  T cell antigen receptor (TCR) signal pathways. Diagram showing the principal signal pathways resulting from TCR activation and how
they impinge on the regulatory region of the interleukin-2 (IL-2) gene. See text for details.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
Activation of PTKs is one of the earliest signaling
events following TCR stimulation. Four families of PTKs
are known to be involved in TCR signaling: Src, Csk, Tec,
and Syk.48 The Src family members Lck and Fyn(T) have
a central role in TCR signaling and are expressed exclu-
sively in lymphoid cells. Src PTKs contain multiple struc-
tural domains, including (1) N-terminal myristylation
and palmitylation sites, which allow association with the
plasma membrane; (2) a Src homology (SH) 3 domain,
which associates with proline-rich sequences; (3) an SH2
domain that binds phosphotyrosine-containing proteins;
and (4) a carboxy-terminal negative regulatory site. Their
catalytic activity is regulated by the balance between the
actions of kinases and phosphatases. Activity is repressed by
phosphorylation of a conserved carboxy-terminal tyrosine,
and dephosphorylation by the phosphatase CD45 is criti-
cal for the initiation of TCR-mediated signal transduction.
In addition, autophosphorylation of other tyrosines within
the kinase domain enhances catalytic activity. Lck is physi-
cally and functionally associated with CD4 and CD8. Fifty
percent to 90% of total Lck molecules are associated with
CD4, and 10% to 25% with CD8. CD4 and CD8 physi-
cally associate with the TCR-CD3 complex during antigen
stimulation as a result of their interaction with MHC class
II and class I molecules and thus enhance TCR-mediated
signals by recruiting Lck to the TCR complex. Lck phos-
phorylates the CD3 chains, TCRζ, ZAP-70, phospholipase
C-γ1 (PLCγ1), Vav, and Shc. Fyn binds TCRζ and CD3ε,
and although their substrates are less well defined, T cells
lacking Fyn have diminished response to TCR signals.49,50
In addition, the SH2 and SH3 domains of Src PTKs can
mediate their association with phosphotyrosine- and pro-
line-containing molecules, respectively.
Somewhat less is known about the Csk and Tec PTKs.
Csk negatively regulates TCR signaling by phosphorylat-
ing the carboxy-terminal tyrosine of Lck and Fyn. Dephos-
phorylation of this negative regulatory tyrosine is mediated
by the transmembrane tyrosine phosphatase CD45. CD45
activity is essential for TCR signaling, and CD45-deficient
T cells fail to activate by TCR stimulation.51 The Tec
family member Itk is preferentially expressed in T cells. T
cells from Itk-deficient mice have a diminished response to
TCR stimulation.52 The mechanism by which Itk regulates
TCR signaling has not been determined, although recent
studies have shown that Itk is an important component
of the pathway leading to increased intracellular calcium
ions (Ca2+).
Phosphorylation of the ITAMs on the CD3 complex
recruits the Syk kinase family member ZAP-70 by its tan-
dem SH2 domains. ZAP-70 is expressed exclusively in T
cells and is required for TCR signaling. Like the Src family
PTKs, ZAP-70 is positively and negatively regulated by its
phosphorylation. Phosphorylation of tyrosine 493 by Lck
activates ZAP-70 kinase activity. In murine thymocytes and
ex vivo T cells, inactive nonphosphorylated ZAP-70 is con-
stitutively associated with the basally phosphorylated TCRζ
chain via the SH2 domain of ZAP-70.53 TCR stimulation
is required for ZAP-70 phosphorylation and activation. The
recruitment of ZAP-70 to the TCR complex facilitates the
tyrosine phosphorylation and activation of ZAP-70 by Lck.
These data suggest that the receptor signaling complex in
these cells is primed, but an initiating event is still required
161
to activate ZAP-70. ZAP-70 can also act as a scaffold, bind-
ing to other proteins via its SH2 domain and phosphory-
lated tyrosines. ZAP-70 interacts with Lck, Ras-GAP, Vav,
the phosphatase SHP-1, and Cbl, and these proteins may
regulate ZAP-70 activity or serve as substrates. ZAP-70
phosphorylates tyrosine residues in a number of molecules,
including PLCγ1 and the adapter proteins SLP-76 and LAT
(see the next section).
ADAPTER PROTEINS
Phosphorylation of tyrosine residues in ITAMs and PTKs
following TCR stimulation creates docking sites for
adapter proteins. Adapter proteins contain no known
enzymatic or transcriptional activities but mediate pro-
tein-protein interactions or protein-lipid interactions.54
They function to bring proteins in proximity to their
substrates and regulators, as well as to sequester signal-
ing molecules to specific subcellular locations. The pro-
tein complexes formed can function as either positive or
negative regulators of TCR signaling, depending on the
molecules they contain.
Two critical adapter proteins for linking proximal and
distal TCR signaling events are SH2-domain-containing
leukocyte protein of 76 kD (SLP-76) and Linker for activa-
tion of T cells (LAT) (see Fig. 9-5). Loss of these adapter
proteins has profound consequences for T cell develop-
ment. Mice deficient for LAT or SLP-76 manifest a block in
T cell development at the CD4–CD8– CD25+CD44+ stage.
LAT is constitutively localized to lipid rafts and, following
TCR stimulation, is phosphorylated on tyrosine residues by
ZAP-70. Phosphorylated LAT then recruits SH2-domain-
containing proteins, including PLCγ1, the p85 subunit of
phosphoinositide-3 kinase, IL-2 inducible kinase (Itk), and
the adapters Grb2 and Gads. Because the SH3 domain of
Gads is constitutively associated with SLP-76, this brings
SLP-76 to the complex, where it is phosphorylated by
ZAP-70. SLP-76 contains three protein binding motifs:
tyrosine phosphorylation sites, a proline-rich region,and an
SH2 domain. The N-terminus of SLP-76 contains tyrosine
residues that associate with the SH2 domains of Vav, the
adapter Nck, and Itk. Vav is a 95-kD protein that acts as a
guanine nucleotide exchange factor for the Rho/Rac/cdc42
family of small G proteins. Through its association with Vav
and Nck, SLP-76 links TCR signals to Rac/Rho GTPases
and the actin cytoskeleton. The association of phosphory-
lated LAT with PLCγ1 and Grb2 couples the TCR signal-
ing to both the Ras and the phosphatidylinositol pathways
(see later). Grb2 contains a central SH2 domain flanked
by two SH3 domains and associates with the proline-rich
regions of Sos, a guanine nucleotide exchange factor for
Ras. Recruitment of Grb2 to the receptor brings Sos to the
plasma membrane, where it activates Ras. The complex of
LAT, SLP-76/Gads, PLCγ1, and associated molecules results
in the full activation of PLCγ1 and activation of Ras.
The use of statins as immunomodulators has generated
interest. Although statins have been prescribed extensively
as cholesterol-lowering agents, recent studies suggest that
they may have a role in the treatment of autoimmune dis-
eases. Modulation of post-translational protein prenylation
appears to be a key mechanism by which statins confer this
function.55
 162 BUDD  |  T Lymphocytes
In addition to acting as positive regulators for TCR
signaling, adapters can mediate negative regulation.
As described previously, the activity of the Src family of
kinases is regulated by the interaction of kinases (Csk) and
phosphatases (CD45) specific for inhibitory C-terminal
phosphotyrosine, which is determined by the subcellular
localization of these regulatory molecules. The interac-
tions that control the localization of CD45 are currently
being analyzed. The localization of Csk is dependent on an
adapter known as phosphoprotein, associated with glyco-
sphingolipid-enriched microdomains (PAG or Csk binding
protein [CBP]). Collectively, the data suggest the following
model: PAG is constitutively associated with membrane
lipid rafts. In resting cells, PAG or CBP is tyrosine-phos-
phorylated, which mediates the association with the SH2
domains of Csk. This colocalizes Csk with Src family
kinases in lipid rafts and controls the basal activation of
Src family PTK through the phosphorylation of inhibitory
C-terminal tyrosine residues. Upon TCR stimulation, PAG
becomes dephosphorylated, resulting in the dissociation of
Csk. Loss of Csk from the rafts reverses the inhibition of
Src PTK activity and permits the initiation of downstream
signaling.
A second mechanism by which adapter proteins can
negatively regulate TCR stability is through the regulation
of protein stability. c-Cbl and Cbl-b are members of a con-
served family of proteins that contains a highly conserved
N-terminal region containing a tyrosine kinase–binding and
RING-finger domains. The c-Cbl RING finger domain binds
the E2 ubiquitin-conjugating enzymes. Active E2 enzymes
are brought into proximity with tyrosine kinase–binding
proteins, resulting in their ubiquitination and degradation
by the proteasome complex. Syk and ZAP-70 associate with
c-Cbl, whereas Vav, ZAP-70, Lck, PLCγ1, and the p85 sub-
unit of PI3K associate with Cbl-b.
DOWNSTREAM TRANSCRIPTION FACTORS
The previously mentioned signaling events couple TCR
stimulation to downstream pathways, culminating in the
changes in gene transcription that are required for prolifera-
tion and effector function (see Fig. 9-5). One of the best-
characterized genes induced following T cell activation is
the T cell growth factor IL-2. Transcription of the IL-2 gene
is regulated in part by the transcription factors activator pro-
tein-1 (AP-1), nuclear factor of activated T cells (NFAT),
and nuclear factor κB (NFκB), all of which are activated
following TCR stimulation. Proximal signaling events lead
to the activation of Ras and PLCγ.56,57 Ras initiates a cas-
cade of kinases, including Raf-1, MEK, and the MAP kinase
ERK, which leads to the production of the transcription
factor Fos. Ligation of the costimulatory molecule CD28
results in the activation of another member of the MAP
kinase family, c-Jun N-terminal kinase (JNK), and phos-
phorylation of the transcription factor c-Jun. c-Jun and Fos
associate to form AP-1. PLCγ hydrolyzes membrane inositol
phospholipids to generate phosphoinositide second messen-
gers, including inositol 1,4,5 triphosphate (IP3) and diac-
ylglycerol. IP3 stimulates the mobilization of calcium from
intracellular stores. Diacylglycerol activates protein kinase
C (especially PKCθ in T cells) and, along with CARMA,
connects with the NFκB pathway.57
Increased intracellular calcium is central to many forms
of cellular activation. Calcium activates the ­ calcium-
­calmodulin–dependent serine phosphatase calcineurin,
which dephosphorylates NFAT. 58 Dephosphorylated NFAT
translocates to the nucleus and, together with AP-1, forms
a trimolecular transcription factor for the IL-2 gene. The
immunosuppressive agents cyclosporine-A and FK-506 spe-
cifically inhibit the calcium-dependent activation of calci-
neurin, thereby blocking the activation of NFAT and the
transcription of NFAT-dependent cytokines such as IL-2,
IL-3, IL-4, and granulocyte-macrophage colony-stimulating
factor. Recently, it has been appreciated that differences in
the amplitude and duration of calcium signals mediate dif-
ferent functional outcomes. Although high spikes of cal-
cium are easily measured in lymphocytes during the first
10 minutes following antigen stimulation, sustained low-
level calcium spikes over a few hours are necessary for full
activation.59 These more subtle calcium fluxes appear to be
controlled by cyclic ADP-ribose and ryanodine receptors.60
Selective inhibitors exist for these molecules, leading to the
potential for new, specific blockers of T cell activation.
A surprising discovery in the field of T cell activation was
the observation that caspase activity, particularly caspase-8,
is required to initiate T cell proliferation and activation of
NFκB.61-63 Previously, the role of caspases had been confined
to apoptosis. However, it is now appreciated that following
TCR ligation, caspase-8 is activated and forms a complex
that includes the NFκB adapter proteins CARMA1, Bcl-10,
and MALT1.64 How T cells manage to activate a sufficient
level of caspase to promote proliferation, but not so much as
to precipitate cell death, remains a mystery. However, this
means that inhibition of caspase activity might be therapeu-
tic in preventing T cell activation.
COSTIMULATION
Signal 2 is mediated either by growth factor cytokines or
through a costimulatory molecule; the prototype of the lat-
ter is CD28 interacting with B7-1 (CD80) or B7-2 (CD86).
CD28 is a disulfide-linked homodimer constitutively
expressed on the surface of T cells.65 Virtually all murine
T cells express CD28, whereas in human T cells, nearly all
CD4+ and 50% of CD8+ cells express CD28. The CD28–
subset of T cells appears to represent a population that has
undergone chronic activation and can manifest suppres-
sive activity.66 Increased levels of CD28– T cells have been
reported in several inflammatory and infectious conditions,
including Wegener’s granulomatosis, cytomegalovirus, and
mononucleosis.67-69 The cytoplasmic domain of CD28 has no
known enzymatic activity, but it does contain one SH2 and
two SH3 binding sites. CD28 interacts with PI3-kinase and
GRB2 and promotes JNK activation, as noted earlier. CD28
ligation alone does not transmit a proliferative response to
T cells; however, in conjunction with TCR engagement, it
augments IL-2 production at the level of both transcription
and translation. It also increases the production of other
cytokines, including IL-4, IL-5, IL-13, IFN-γ, and TNF-α, as
well as the chemokines IL-8 and RANTES.70
The ligands for CD28, CD80 (B7-1), and CD86 (B7-2)
are expressed in a restricted distribution on B cells, dendritic
cells, monocytes, and activated T cells. CD80 and CD86
have similar structures but share only 25% amino acid
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION 163

homology. They each contain rather short cytoplasmic tails microdomains called rafts.74,75 Rafts are composed primarily
that may signal directly, and they bind to CD28 with differ- of glycosphingolipids and cholesterol and are enriched in
ent avidities. signaling molecules, actin, and actin-binding proteins.76,77
Src family kinases, Ras-like G proteins, LAT, and phospha-
tidylinositol-anchored membrane proteins have all been
IMMUNOLOGIC SYNAPSE
shown to localize to raft domains.
Antigen-specific interaction between the T cell and the During T cell stimulation, activation of many tyrosine
APC results in the formation of a specialized contact region kinases precedes formation of the immunologic synapse.78,79
called the immunologic synapse (Fig. 9-6) or supramolecu- After TCR ligation, rafts become enriched for Vav, Shc,
lar activation cluster.71 Synapse formation is an active, phosphorylated ζ, PLCγ1, and ZAP-70.75,80 TCR engage-
dynamic process that requires a specific antigen to drive ment induces rapid reorganization of the T cell cytoskel-
synapse formation; TCR-MHC interaction alone is not suf- eton toward the T cell–APC junction.81 This results in the
ficient. The synapse also overcomes the obstacles to close redistribution of surface molecules and the release of cyto-
T cell–APC contact involving short molecules (e.g., TCR, kines and cytotoxic mediators toward the site of the T cell
MHC, CD4, CD8) caused by interactions recruiting long stimulus.82 Among the known molecules connecting signal-
molecules (ICAM-1, LFA-1, CD45). Two stages of assembly ing and structural proteins is the Wiskott-Aldrich syndrome
have been described. During the nascent stage, cell adhe- protein (WASp). WASp contains a GTPase binding domain
sion molecules—such as ICAM-1 on APCs and LFA-1 on that mediates binding to activated cdc42.83 This promotes
T cells—make contact in a central zone, surrounded by an the coupling of TCR engagement to actin polymerization.
annulus of close contact between MHC and TCR.71 Within Full T cell activation requires engagement of a minimum
minutes, the engaged TCR migrates to the central area, of about 100 to 200 MHC-peptide molecules on an APC,
resulting in a mature synapse in which the initial relation- which can serially stimulate 2000 to 8000 TCRs. It has been
ships are reversed—the central area now contains TCR, estimated that naive T cells also require a sustained signal
CD2, CD28, and CD4 and is enriched for Lck, Fyn, and for 15 to 20 hours to commit to proliferation.84 T cells face
PKCθ.72,73 Surrounding the central domain is a peripheral a number of obstacles to achieving full activation, includ-
ring that contains CD45, LFA-1, and associated talin. T ing the small size of the TCR and MHC molecules com-
cell activation leads to compartmentalization of activated pared with other cell surface molecules, the low affinity of
TCR and TCR signaling molecules to plasma membrane TCR for the MHC-peptide complex, and the low number

Time (minutes)
0.5 1.5 3 5 10 30 60

B
10 µm

250 5000 500

Density MHC-pep. (µm–2)

Total MHC-pep. (molec.)

Density ICAM-1 (µm–2)

200 4000 400

150 3000 300

100 2000 200

50 1000 100

0 0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60 0 10 20 30 40 50 60
C Time (min) D Time (min) E Time (min)
Figure 9-6  Formation of the immunologic synapse. A, Contact areas of T cells over time, indicated as dark gray against a light background. B, Im-
ages containing Oregon green Ek-antigen (mouse cytochrome peptide 88-103) and Cy5 intercellular adhesion molecule 1 (ICAM-1). C, Density of ac-
cumulated Ek-MCC88-103. D, Total accumulated Ek-MCC88-103. E, Density of accumulated ICAM-1. (From Grakoui A, et al: The immunological synapse: A
molecular machine controlling T cell activation. Science 285:221-227, 1999.)
 164 BUDD  |  T Lymphocytes
of MHC molecules present on the APC that contain anti-
genic peptide.85 The immunologic synapse may provide the
mechanism for overcoming these barriers and achieving the
duration of TCR stimulation necessary to commit the cell
to proliferation.71 The spatial organization of the synapse
juxtaposes the membranes of the APC and T cell, facili-
tating the interaction of the TCR and MHC-peptide com-
plex. The available MHC-peptide complexes and TCRs are
concentrated at the site of contact via actin cytoskeleton-
mediated transport. The multistep process of mature syn-
apse formation may also enable the T cell to discriminate
between the potential antigen-containing MHC-peptide
complexes it encounters on the APC surface. Recently, it
has been shown that costimulatory signals may contribute to
synapse formation by initiating the transport of membrane
rafts containing the kinases and adapter molecules required
for TCR signaling to the site of contact.86,87
Although it has been appreciated for some time that
chronic infection can lead to an unresponsive state, or
“exhausted” T cells, the molecular explanation was un­­
known. More recently, it was observed that chronically
activated CD8+ T cells contain more mRNA encoding the
inhibitory receptor PD1 (programmed death 1) that acutely
activates CD8+ T cells.88 In parallel, one of the ligands for
PD1, PDL1, was highly expressed by chronically infected
splenocytes. Treatment of mice with a blocking antibody
to PDL1 caused virus-specific CD8+ T cells to undergo
marked expansion. The fact that many tumors also express
PDL1 enhances the interest in reversing the suppression of
immune responses during infection and tumorigenesis. The
fact that PD1-deficient mice develop spontaneous auto-
immunity leads to the possibility of manipulating PD1 for
therapeutic purposes.89
TOLERANCE AND CONTROL OF AUTOREACTIVE
T CELLS
The immune system is constantly confronted by the prob-
lem of how to ensure that T cells are activated only when
there is a true need to respond to a foreign pathogen and not
merely a self-component. This is not a trivial point because,
like all biologic filters, the thymus is not 100% efficient, and
not all self-reactive T cells are eliminated. Hence, a variety
of failsafe mechanisms are engaged to suppress the prema-
ture clonal expansion of these errant T cells. Part of nature’s
ingenious solution was to require two distinct signals from
separate molecules to be coordinately triggered in order for
T cell activation and proliferation to proceed. If only one
signal is received, the T cell will not proliferate and will
actually enter a nonresponsive state known as tolerance or
anergy.
The anergy that results from the absence of a CD28
costimulatory signal manifests at a signal level by failure to
fully couple the TCR signal to the Ras-MAP kinase path-
way and consequent AP-1 transcriptional activity. An addi-
tional method of provoking an incomplete TCR signal and
unresponsiveness is to make amino acid substitutions in the
recognized peptide antigen. These so-called altered peptide
ligands cause a suboptimal phosphorylation of TCRζ and
consequent inefficient recruitment of ZAP-70.90
Following the discovery of CD28 as a costimulatory mol-
ecule, a related structure known as CTLA-4 was found to
bind to CD80 and CD86 with a 20-fold higher affinity than
CD28. Unlike CD28, CTLA-4 is expressed only transiently
following T cell activation and confers an inhibitory sig-
nal for T cell proliferation.91 The mechanism by which this
occurs is not clearly defined, although two reports suggested
that the phosphatase SHP-2 may associate with the cyto-
plasmic tail of CTLA-4.86,92 A more recent report showed
that CTLA-4 signaling keeps CD3ζ out of the immunologic
synapse and thus diminishes the density of surface TCR and
its consequent signaling.93 In this capacity, CTLA-4 func-
tions to limit T cell clonal expansion induced by CD28.
The consequences of the loss of this negative regulation
are striking. The genetic deletion of the ctla-4 gene in mice
results in enormous uncontrolled T cell expansion and an
autoimmune diathesis.94,95
Chronic exposure to certain inflammatory cytokines,
most notably TNF-α, can also induce anergy. It has been
known for some time that T cells from rheumatoid synovium
manifest profound deficiencies of proliferation and cyto-
kine production.96,97 Because TNF-α is one of the major
cytokines detectable in rheumatoid synovial fluid, it was
soon recognized that chronic exposure of T cell clones to
TNF-α for 10 to 12 days suppresses proliferative and cyto-
kine responses to antigen by as much as 70%.98 Further, a
single administration of anti–TNF-α receptor monoclo-
nal antibody to patients with rheumatoid arthritis rapidly
restores the response of peripheral T cells to mitogens and
recall antigens.98 Similar observations have been made in
TCR transgenic mice following TNF-α exposure.99 The
observation that chronic TNF-α exposure inhibits calcium
responses following TCR ligation99 supports the view that
TNF-α may uncouple TCR signaling. It is conceivable that
other members of the TNF-α family may invoke similar
T cell anergy.
An additional negative regulator for T cells is B
lymphocyte­–induced maturation protein 1 (Blimp-1), pre-
viously thought to be expressed only in B lymphocytes.
Blimp-1–deficient mice manifest augmented levels of
peripheral effector T cells and develop severe colitis as early
as 6 weeks of age.100 Blimp-1 mRNA expression increases
with TCR stimulation, and Blimp-1–deficient T cells pro-
liferate more and produce more IL-2 and IFN-γ following
activation.100
Another layer of regulation involves the long-held
theoretical concept that a subset of T cells might exist
that can actively suppress the immune response. In the
past, clear demonstration of these cells was elusive, as was
their acceptance by the immunology community. More
recently, however, a phenotypically defined subpopulation
of CD4+CD25+ regulatory T cells has been identified with
an ability to inhibit antigen-induced proliferation.101 This
subset is expressed in the periphery at a low frequency and
appears to be thymic dependent. The latter point may be
of interest, because it suggests that the absence of regula-
tory T cells following day 3 thymectomy may be involved
with the subsequent development of autoimmune disease in
these animals.102 Indeed, diminished levels of CD4+CD25+
regulatory T cells have been observed in other autoimmune
syndromes, and the transfer of regulatory T cells to autoim-
mune mice has resulted in some alleviation of symptoms.
At present, the lineage of CD4+CD25+ cells is unclear, as
is their exact mechanism of suppression. The production of
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
transforming growth factor-β (TGF-β) and IL-10 appears
to be critical to the suppressive activity of these cells.103
CD4+CD25+ cells express Foxp3, a regulatory gene that,
upon transfection into naive T cells, confers the functional
characteristics of regulatory T cells.104 More recently, an
alternative marker appears to be low levels of surface IL-7
receptor α chain (CD127).105 This is an active area of
research because of the potential therapeutic implications
for autoimmune diseases and the possible role in generating
IL-17–producing CD4+ T cells (Th17; see later).106
SUBSETS AND FUNCTIONS
OF PERIPHERAL T CELLS
CD4 HELPER AND CD8 CYTOLYTIC T CELLS
αβ T cells can be divided into two main subsets based on
their recognition of peptides presented by MHC class I or
class II molecules and their respective expression of CD8 or
CD4. CD4+ and CD8+ T cells have different functions and
recognize antigens derived from different cellular compart-
ments. The peptides presented by MHC class I molecules
are produced by the proteasomes107,108 and can be derived
from either self-proteins or intracellular foreign proteins,
as might occur during viral infection. MHC class II–bound
peptides are derived largely from extracellular infectious
agents or self–cell surface proteins that have been engulfed
and degraded in the lysosomal complex.
CD4+ T cells express a variety of cytokines and cell sur-
face molecules that are important to B cell proliferation,
immunoglobulin production, and CD8+ T cell function.
After antigen stimulation, CD4+ T cells differentiate into
two major classes of effector T cells based on their cytokine
profiles: T helper 1 (Th1) and T helper 2 (Th2).109 The CD4
molecule is structurally related to immunoglobulins and has
an affinity for nonpolymorphic residues on the MHC class
II molecule. In this capacity, CD4 presumably increases the
efficiency with which CD4+ T cells recognize antigen in the
context of class II molecules, which are restricted in their
expression to B cells, macrophages, dendritic cells, and a few
other tissues during states of inflammation. In addition, the
cytoplasmic tail of CD4 binds to Lck and promotes signaling
by the TCR, as described earlier. However, ligation of CD4
before engagement of the TCR renders the T cell suscep-
tible to apoptosis on subsequent engagement of the TCR.110
This is clinically important in human immunodeficiency
virus (HIV) infections in which the gp120 molecule of HIV
binds to CD4 and primes the T cell to undergo cell death
when later triggered by the TCR.111 Accelerated ­apoptosis
of CD4+ T cells has been demonstrated in patients with
acquired immunodeficiency syndrome (AIDS).112
CD8+ T cells are very efficient killers of pathogen-infected
cells. Given the ubiquitous expression of class I molecules,
mature cytolytic T cells can recognize viral infections in a
wide array of cells, in contrast to the more restricted distri-
bution of class II molecules and their recognition by CD4+
T cells. Cytolytic T cells induce lysis of target cells through
the production of perforin, which induces holes in cell
membranes, and through the expression of FasL and TNF-α,
which induce apoptosis. In this capacity, cytolytic T cells
kill virally infected target cells in an attempt to restrict
the spread of infection. Similar to CD4, CD8 manifests an
165
a­ ffinity for MHC class I molecules, enhances the signaling
of cytolytic T cells, and also binds Lck by its cytoplasmic
tail.
T CELLS IN THE INNATE IMMUNE RESPONSE
In addition to the broad array of antigens recognized by
αβ T cells, there is growing appreciation that the immune
system contains small subpopulations of specialized T cells
that may recognize conserved structures that are uniquely
expressed either by prokaryotic pathogens or on stressed
host cells. These are discussed in detail in Chapter 8. Such
common antigenic motifs include bacterial lipoproteins
recognized by Toll-like receptor (TLR)-2 and TLR-4, dou-
ble-stranded RNA from RNA viruses that binds TLR-3,
methylated cytosine residues in bacterial CpG sequences,
or anti-DNA–DNA complexes that bind to TLR-9.113 TLR
expressed by APCs can trigger the release of cytokines and
costimulatory molecules for T cells. Another family of mol-
ecules that likely binds bacterial components is CD1. CD1
structurally resembles MHC class I but contains a deeper
and more hydrophobic binding pocket that can accommo-
date certain lipopeptides and glycolipids.114 By using such
molecular strategies to focus on common and nonpolymor-
phic molecules, the immune system may be able to respond
quickly during the early phase of infection. This response is
part of innate immunity. Even though it may represent the
remnants of an evolutionarily primitive immune response,
it provides a vital early-defense system. Among T cells, this
function is provided by γδ and natural killer (NK) T cells.
γδ T Cells
Among immunology’s oddities, the γδ T cell is one of the
oddest. Study of these cells was precipitated by the serendip-
itous discovery of rearranged genes while searching for the
TCR α-chain gene, rather than by a preexisting knowledge
of their presence and biologic function.115 Structurally, the
γ-chain locus contains at least 14 Vγ region genes, of which
6 are pseudogenes, each capable of rearranging to any of 5
Jγ regions and 2 Cγ regions. The δ-chain genes are nested
within the α-chain gene locus between Vα and Jα. There
are about six Vδ regions, two Dδ and two Jδ regions, and a
single Cδ gene. Transcription of rearranged γ and δ genes
begins before that of αβ genes and is apparent on days 15 to
17 of mouse thymus development, after which it declines in
the adult thymus. In addition to the ordered appearance of
TCR γδ before TCR αβ, there is a highly ordered expression
of γ and δ V-region genes during early thymic development.
This results in successive waves of oligoclonal γδ T cells
migrating to the periphery. The reason for this remarkable
regimentation remains unclear.
γδ T cells manifest a number of differences from αβ T
cells. For instance, γδ T cells are often anatomically seques-
tered to epithelial barriers or sites of inflammation,116 and
they frequently manifest cytotoxicity toward a broad array
of targets.117 In contrast to αβ T cells, γδ T cells can respond
to antigen directly, without evidence of MHC restriction,118
or, conversely, they can react to MHC molecules without
peptide.119
Human γδ T cell clones, particularly those expressing
the Vδ2 gene and derived from peripheral blood of normal
 166 BUDD  |  T Lymphocytes
individuals or synovial fluid of rheumatoid arthritis patients,
frequently react to mycobacterial extracts.120 Although a
few of these Vδ2 clones respond to a heat shock protein, the
major stimulatory components were recently identified as
phosphate-containing nonpeptide molecules such as nucleo-
tide triphosphates,121 prenyl pyrophosphate,122,123 and alkyl-
amines.124 These molecules are, respectively, subunits in
DNA and RNA, substrates in lipid metabolism for the syn-
thesis of farnesyl pyrophosphate, and products of pathogenic
organisms. In mammalian cells, farnesyl addition is a criti-
cal modification for targeting certain signaling molecules to
the cell membrane, such as Ras. This process appears to be
critical to cell transformation. These phosphate-containing
nonpeptides can be found in both microbial and mammalian
cells. This suggests that γδ cells may recognize a class of anti-
gens shared by a number of pathogens, as well as by damaged
or transformed mammalian cells, and it may provide insight
into the role of γδ cells in infection and their accumula-
tion at sites of inflammation. Recently, a subpopulation of
γδ T cells typically found in the intestine and expressing the
Vδ1 gene (which is also found in inflamed synovial fluid)
was shown to react to newly discovered MHC class I–like
molecules known as MICA and MICB.29 Unlike classic
MHC class I molecules, which are expressed ubiquitously
and continuously, MICA and MICB expression appears to
be restricted to gut epithelium and occurs only during times
of stress, similar to a heat shock response.
The contribution of γδ T cells to defense against infec-
tion has been examined in mice using a number of patho-
gens, including Listeria,125 Leishmania,126 Mycobacterium,127
Plasmodium,128 and Salmonella.129 All these studies showed
a moderately protective role for γδ T cells. In some cases of
rapid bacterial growth, γδ T cells appear to provide protec-
tion early during infection by reducing bacterial growth,125
whereas in less virulent infections such as influenza or Sen-
dai virus, γδ cells appear later in inflammatory lesions.130
In only a few instances, however, have γδ clones derived
from an infected animal been shown to react to the caus-
ative organism. In human infections, γδ T cells from cuta-
neous lesions of leprosy patients respond to Mycobacterium
leprae,131 and γδ cells from Lyme arthritis synovial fluid
respond to the causative spirochete, Borrelia burgdorferi.132
This response by Lyme synovial γδ cells requires lipidated
hexapeptides of the outer surface proteins of B. burgdorferi,
and the tripalmitate lipid moiety is as important for activa-
tion of γδ T cells as the hexapeptide portion is.
γδ T cells accumulate at inflammatory sites in autoim-
mune disorders such as rheumatoid arthritis,133 celiac dis-
ease,134 and sarcoidosis.135 The reason for this accumulation
remains an enigma. However, there is evidence that γδ cells
can be highly cytolytic toward a variety of tissues, including
CD4+ T cells,136 in part owing to their high and sustained
expression of surface Fas-ligand.137 Their presence can
strongly bias the cytokine profiles of the infiltrating CD4+
cells—in some instances, toward Th1 profiles,138 and in oth-
ers, toward Th2.139
Natural Killer T Cells
A minor subpopulation of T cells bearing the NK determi-
nant manifests perhaps the most restricted of TCR reper-
toires and determinants of recognition. NK T cells are found
within the CD4+ and CD4–CD8– subsets of T cells and, in
both mouse and human, express a very limited number of
TCR Vβ chains and an invariant α chain (Vα 14 in mice
and Vα 24 in humans).140 Further, most NK T cells are
restricted in their response to a monomorphic MHC class
I–like molecule, CD1d. Recent crystallographic analysis
of CD1d has shown that it contains a deeper groove than
traditional MHC molecules and is highly hydrophobic, sug-
gesting that it may bind lipid moieties.114 Until recently, the
sea sponge sphingolipid α-galactosylceramide was the only
known CD1d ligand. Now both endogenous and bacterial
(sphingomonas and B. burgdorferi) sources of CD1d-binding
sphingolipids have been identified.141,142 This may represent
another type of innate T cell response whereby bacterial lip-
ids or lipopeptides may be presented to NK T cells to pro-
voke a rapid early immune response.
The potential importance of NK T cells in autoimmune
disease stems from their production of high levels of certain
cytokines, particularly IL-4 and IFN-γ.140 In this capacity,
the IL-4 response may be important for modulating inflam-
matory responses dominated by Th1 infiltrates. This has
been noted in the nonobese diabetic (NOD) mouse model
of diabetes, which has reduced levels of NK T cells.143 Adop-
tive transfer of NK T cells into NOD mice blocks the onset
of diabetes.144 A recent study extended this observation to
human type 1 diabetes. The NK T cells of diabetic individu-
als produced more IFN-γ and less IL-4 than did the cells of
their unaffected siblings.145 More recently, NK T cells were
found to be the predominant CD4+ T cells in the airways of
asthma patients.146 Thus, this minor population of T cells
may play a pivotal role in early innate responses to certain
infections and in the regulation of inflammatory lesions.
Naive versus Memory T Cells
CD4+ and CD8+ T cells emigrate from the thymus bear-
ing a naive phenotype. Naive T cells produce IL-2 but only
low levels of other cytokines; as a result, they manifest little
B cell helper activity. They express high levels of Bcl-2
and can survive for extended periods without antigen but
require the presence of MHC molecules. Naive T cells cir-
culate from the blood to lymphoid tissues of the spleen and
lymph nodes, which concentrates antigen, APCs, T cells,
and B cells. Particularly important APCs in this environ-
ment are dendritic cells, which are derived from both lym-
phoid and myeloid progenitors and are particularly adept at
concentrating and presenting antigen. Dendritic cells can
migrate from other areas of the body, such as the skin, and
thus transport antigen to lymphoid tissues (see Chapter 8).
These specialized cells express high and constitutive levels
of MHC class II and costimulatory molecules B7-1 (CD80)
and B7-2 (CD86), which are critical to promoting the pro-
liferation of naive T cells. In this capacity, dendritic cells are
particularly adept at promoting clonal expansion of antigen-
specific T cells, which may be present at a frequency as low
as 1 in 106 before immunization but can increase to 1 in 100
or more within 1 week. The recent development of anti-
gen peptide–MHC tetramer technology has led to the more
direct quantitation of these values using flow cytometry and
suggests that their frequency may be considerably higher.147
During the process of clonal expansion of naive
T cells and their differentiation into effector and ­eventually
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
Table 9-1  Surface Markers on Naive and Memory T Cells
Molecule Other Designation Molecular Weight (kD)
CD58 LFA-3 45-66
CD2 T11 50
CD11a/CD18 LFA-1 180-195
CD29 130
CD45RO 220
CD45RA 80-95
CD44 Pgp-1 90
CD54 ICAM-1 120
CD26 40
CD7 Multichain complex
CD3
CD, cluster of differentiation; ICAM, intercellular adhesion molecule; LFA, leukocyte function–associated antigen; TCR, T cell antigen receptor; VLA, very late
activation antigen.
memory T cells, as many as 100 genes are induced. These
manifest primarily as increased expression of certain sur-
face molecules involved with cell adhesion and migration
(CD44, ICAM-1, LFA-1, α4β1 and α4β7 integrins, the
chemokine receptor CXCR3), activation (change of CD45
from high-molecular-weight CD45RA to lower-molecular-
weight CD45RO isotype), cytokine production (increased
production of IFN-γ, IL-3, IL-4, and IL-5), and death recep-
tors (e.g., Fas/CD95) (Table 9-1). More transiently induced
are CD69, the survival factor Bcl-xL, and the high-­affinity
IL-2 receptor α chain (CD25), the last being necessary
for T cell proliferation. Survival of effector T cells to the
memory stage is partly dependent on the cytokines IL-7 and
IL-15.148,149
The concept of immune memory has existed since
­Jenner’s first successful vaccinations for smallpox. For years,
memory T cells could be identified at a functional level only
by demonstrating the presence of an enhanced proliferative
response of PBL from an individual previously vaccinated
against an antigen, such as tetanus toxoid. In the late 1980s,
a series of cell surface memory T cell markers were identi-
fied. The first of these was CD44, the hyaluronate recep-
tor.150 Surface CD44 is low on mature, single, positive T
cells as they emerge from the thymus, but its expression is
upregulated upon the first encounter with antigen stimu-
lation in the periphery. Several other markers have been
shown to change upon primary antigenic stimulation. Most
notable for human T cells is CD45; an isoform known as
CD45RA is expressed on naive T cells, whereas CD45RO
expression characterizes memory T cells (see Table 9-1).
By using these markers, it has been possible to identify a
variety of differences between naive and memory T cells.
Activation of memory T cells appears to be more efficient
than that of naive T cells and is not absolutely dependent
on costimulation. Memory T cells are also able to migrate to
nonlymphoid tissues such as lungs, skin, liver, and joints.151
Th1 versus Th2 T Cells
Th1 cells participate in cell-mediated inflammatory reac-
tions, activate macrophages, and produce IL-2, TNF-β,
and IFN-γ. Th2 cells activate B cells and produce IL-4,
167
Expression
Characteristic Memory Naive
Ligand for CD2 ++ +
Alternative activation pathway +++ ++
Receptor for ICAM-1, ICAM-2, ICAM-3 +++ ++
β chain of β1 (VLA) integrins ++++ +
Isoform of CD45 ++++ −
Isoform of CD45 − ++++
Receptor for hyaluronic acid +++ ++
Counterreceptor for LFA-1 + −
Dipeptidyl peptidase IV + −
T cell lineage marker +/− ++
Part of TCR complex + +
IL-5, IL-6, and IL-10. IL-4 and IL-5 are important B cell
growth factors, as are surface CD40-ligand and the recently
described BAFF.152 In addition, IL-4 promotes B cell secre-
tion of immunoglobulin (Ig) G1 and IgE, whereas IFN-γ
drives IgG2a production. Because Th1 and Th2 cells medi-
ate different functions, the type of response generated can
influence susceptibility to disease.
In response to repetitive antigenic stimulation, respond-
ing CD4+ cells can differentiate into effector cells express-
ing polarized patterns of cytokine production. Th1 cells are
characterized by the production of IL-2, IFN-γ, TNF-α, and
TNF-β, whereas Th2 cells produce IL-4, IL-5, IL-6, IL-10,
and IL-13.109 A list of cytokines and their properties is pro-
vided in Table 9-2. These patterns have been best character-
ized during chronic infections. In general, a Th1 response
helps eradicate intracellular microorganisms, such as Leish-
mania major and Brucella abortus,109 whereas a Th2 cell
response can better control extracellular pathogens, such
as the helminth Nippostrongylus brasiliensis.153 The cytokine
profiles of Th1 and Th2 cells are mutually inhibitory; thus,
the Th1 cytokine IFN-γ, or IL-12 from APCs, suppresses
Th2 responses and augments Th1 cytokine gene expression,
whereas the Th2 cytokine IL-4, or IL-6 from APCs, pro-
motes the opposite pattern.154 Polarization of the cytokine
environment also occurs at the sites of inflammation in many
autoimmune syndromes. Th2 skewing has been observed in
models of systemic lupus erythematosus, where increased
levels of immunoglobulins and autoantibodies are typical,
as well as in chronic allergic conditions such as asthma.155
Frequently, however, the infiltrating lymphocytes exhibit a
bias toward Th1 cytokines. This occurs with brain-infiltrating
lymphocytes in multiple sclerosis and its animal model,
experimental allergic encephalomyelitis,156 β islet lympho-
cytes in diabetes,157 and synovial lymphocytes in inflamma-
tory arthritides.158,159 Unlike the beneficial effects of Th1
responses during infections, these same cytokines can be
quite deleterious in autoimmune disorders. Thus, therapies
based on the inhibition of certain Th1 cytokines have gen-
erated considerable interest and are often ameliorative, such
as anti–TNF-α treatment of experimental allergic encepha-
lomyelitis156 and rheumatoid arthritis.160 Several cytokines
can be pleiotropic, and predicting the effects of modulating
 168

Table 9-2  T Cell–Derived Cytokines

Mr of Natural Amino Acids of Chromo-
Cytokine Abbreviation Other Names Protein (kD) Mature Protein some Receptor Activity
Interleukin-1α IL-1α Lymphocyte-activating factor (LAF), 17.5 159 2 Single chain; CDw121a; type I receptor;   Costimulates T and B cell activation and
BUDD 

endogenous pyrogen (EP),   80 kD or p80; binds IL-1α, IL-1β, and the secretion of cytokines (IL-2, IFN-γ)
| 

catabolin, osteoclast-activating   IL-1R antagonist and antibody; increases killing by NK

factor (OAF), epidermal cell-derived cells; myriad effects on nonlymphoid
thymocyte-activating factor (ETAF) cells
Interleukin-2 IL-2 T cell growth factor (TCGF) 15-20 133 4 Low affinity: CD25, IL-2Rα, p55; CD122, Promotes growth and differentiation of
IL-2Rβ, p75; non-IL-2 binding p64   activated T and B cells; activates NK
IL-2γ cells, macrophages
T Lymphocytes

Intermediate affinity: IL-2R,  

IL-2Rα/γ, or IL-2Rβ/γ
High affinity:  
IL-2Rα/β/γ; γ chain associated with  
IL-4R, IL-7R, IL-15R (? IL-13R and IL-9R)
Interleukin-3 IL-3 Multipotential colony-stimulating   14-30 133 5 α Chain, p70; β chain, p120; IL-3R   Stimulates proliferation and  
factor (multi-CSF), mast cell growth   associated with tyrosine kinase   differentiation of precursors of all hema-
factor (MCGF), erythroid colony- activity topoietic cell lineages
­stimulating factor (ECSF), mega­
karyocyte colony-stimulating factor
(Meg-CSF), eosinophil  
colony-­stimulating factor (Eo-CSF)
Interleukin-4 IL-4 B cell stimulatory factor 1 (BSF-1),   15-20 129 5 High affinity: IL-4Rα + IL-2R γ chain or Promotes growth and differentiation of  
B cell differentiation factor-γ   “common γ chain,” γc; CD124 is   T cells, B cells; enhances tumoricidal
(BCDF-γ), T cell growth factor-2   IL-4R α chain, p140 activity of macrophages, but inhibits
(TCGF-2), mast cell growth factor-2 Low affinity:   IL-1 and TNF-α production
(MCGF-2) IL-4R reported; soluble IL-4Rα is a
potent IL-4 antagonist
Interleukin-5 IL-5 B cell growth factor II (BCGF-II), T cell 45 115 5 Low affinity: CD125, IL-5α, p60; IL-5R β Promotes growth of cytotoxic cells and
replacing factor, eosinophil   chain is nonbinding and shared with differentiation of B cells
differentiation factor (EDF),   IL-3R and GM-CSFR
IgA-enhancing factor (IgA-EF)
Interleukin-6 IL-6 Interferon-β2 (IFN-β2), B cell   26 183 7 High affinity: IL-6R α chain (p80) +   Enhances IL-2 production from T cells and
stimulatory factor-2 (BSF-2),   gp130; gp130 is nonbinding and   Ig production by B cells; myriad effects
hepatocyte stimulatory factor II   forms homodimer when complexed on nonlymphoid cells
(HSF-II), hybridoma plasmacytoma with IL-6Rα; acts to transduce signal
growth factor (HPGF, IL-HP1),  
myeloma cell growth factor (MCGF)
Interleukin-8 IL-8 Neutrophil-activating protein (NAP-1), 6-8 77 4 High affinity: IL-8R, CDw128, p58-67 Produced by many cell types; acts mainly
granulocyte chemotactic protein   Low affinity: IL-8R also binds   as neutrophil and lymphocyte  
(GCP) GRO/MGSA, NAP-2 chemoattractant and activation factor
Interleukin-9 IL-9 P40, mast cell growth-enhancing   32-39 126 5 IL-9R is single chain, p64 Produced by activated Th2 cells; enhances
activity; T cell growth factor-3 T cell proliferation, mast cell lines, and
erythroid precursors
Interleukin-10 IL-10 Cytokine synthesis inhibition factor   35-40 160 1 IL-10R is single chain, p90-110 Stimulates Th2 cell and thymocyte growth;
(CSIF) inhibits Th1 cell proliferation, IL-2 and
B cell-derived T cell growth factor   IFN-γ production; inhibits  
(B-TCGF) macrophage cytokine production
 Interleukin-12 IL-12 Natural killer cell stimulatory factor   30-33 196/306 ? High affinity: IL-12R, type I receptor,   Induces Th1 cell differentiation; enhances
(NKSF) 35-44 p180 IFN-γ production by T cells and NK cells;
Cytotoxic lymphocyte maturation   stimulates proliferation of T cells
factor (CLMF)
Interleukin-13 IL-13 Murine P600 10-17 112 5 Unknown; may share nonbinding chain Produced by activated T cells; inhibits
with IL-4 production of inflammatory cytokines
(IL-1β, IL-6, TNF-α, IL-8); induces CD23
on B cells, promotes human B cell prolif-
eration and Ig secretion
Interleukin-14 IL-14 High-molecular-weight B cell growth   60 483 ? Unknown; also binds Bb component of Produced by activated T cells; promotes B
factor (HMW-BCGF) complement cell proliferation, inhibits Ig secretion;
shares homology with complement
factor Bb
Interleukin-15 IL-15 None 14-15 114 ? IL-15R binding chain unknown; shares Produced by many cells; enhances T cell
IL-2Rβ and γ chains (not IL-2Rα) proliferation, cytotoxic activity, and LAK
activity
Granulocyte- GM-CSF Colony-stimulating factor-α,   22 127 5 Low affinity: CDw116, α chain, p80; β Activates macrophages
macrophage pluripoietin, colony-stimulating   chain, p130, shared with IL-3R and  
colony- factor 2 IL-5R
stimulating High affinity: α + β chains
factor
Interferon-γ IFN-γ Macrophage-activating factor (MAF) 20-25 143 12 High affinity: CDw119, binding chain, Enhances differentiation of T and B cells;
PART 2 

second chain transduces signal counteracts effects of IL-4; enhances kill-

| 

ing by NK cells; activates macrophages;

induces class II MHC molecules on many
nonimmune cells
Lymphotoxin LT Tumor necrosis factor-β (TNF-β) 25 171 6 Type I receptor, CD120a, p55; second LT and TNF-α have the same activities; en-
receptor is type II receptor, CD120b, hance T and B cell proliferation; enhance
p75; both members of NGFR/TNFR B cell differentiation; increase killing by
superfamily NK cells
Tumor necro- TNF-α Cachectin 52 157 6 Same as LT Same as LT
sis factor-α
Transforming TGF-β None 25 2 × 112 19/14 Three receptors; high-affinity type I and Inhibits T cell growth and cytokine  
growth   II, p55 and p80; low-affinity type III, secretion; inhibits B cell growth and  
factor-β p250-300 differentiation; counteracts effects of
IFN-γ on nonimmune cells
Ig, immunoglobulin LAK, lymphokine-activated killer; NGFR, nerve growth factor receptor; TNFR, tumor necrosis factor receptor.
CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
169
 170 BUDD  |  T Lymphocytes
their levels can be complex. For example, despite the ten-
dency of IL-6 to promote a Th2 cytokine profile, blocking
IL-6 can be extremely beneficial in rheumatoid arthritis.161
Costimulatory molecules and other cytokines ­ produced
by APCs can also influence cytokine polarization. B7-1
(CD80) and IL-12 promote a Th1 emergence,162,163 whereas
B7-2 (CD86), IL-4, and IL-6 can evoke a Th2 predomi-
nance.154,163,164 Th1 cells are also generally thought to be
more readily tolerogenic and more susceptible to activa-
tion-induced cell death (AICD),165,166 perhaps because they
express more Fas-ligand than Th2 cells do.167,168 Increased
expression of Fas-associating phosphatase-1 by Th2 cells
may also contribute to their resistance to AICD by inhib-
iting Fas signaling.165 However, other investigators have
observed that Th1 and Th2 cells are equally sensitive to
apoptosis upon Fas ligation.167
Th17 Cells
A newly recognized subset of IL-17–producing CD4+ T cells
(Th17) is critical for promoting a variety of autoimmune
disorders. TGF-β (possibly originating from regulatory T
cells), accompanied by IL-6 (probably from dendritic cells),
appears to be pivotal for the appearance of Th17 cells.103,106
IL-23 may also be important for the survival, but perhaps
not the appearance, of Th17 cells. Injections of IL-23 into
skin produce increased IL-17 in the epidermis and inflam-
matory lesions that resemble psoriasis.169,170 Th17 cells are
increased in human psoriatic plaques,170 in rheumatoid
arthritis synovial fluid, and in multiple sclerosis.171
MOLECULAR MIMICRY
Perhaps the oldest concept of autoimmunity is that of molec-
ular mimicry, the notion that the immune system’s response
to a foreign substance may provoke cross-reactivity to a self-
protein. This is best established in rheumatic heart disease,
where a B cell antibody response to a group A streptococ-
cal cell wall component can precipitate cross-reactivity to
cardiac myosin. Similarly for T cells, investigators used the
peptide sequence of myelin basic protein (MBP) recognized
by specific T cell clones from patients with multiple sclerosis
to search a database of infectious agents. Some of the can-
didate sequences obtained were able to stimulate the MBP-
reactive T cell clones.172 This suggested for the first time
that T cells responding to an infectious agent might mani-
fest cross-reactivity to self-peptides. More recently, it was
observed that one of the outer surface proteins of B. burgdor-
feri, known as OspA, may trigger a cross-reactive response
in Lyme arthritis.173 Further, a T cell immunodominant pep-
tide of OspA has been identified in a subset of HLA-DR4
patients who are more resistant to antibiotic treatment and
manifest an antibody as well as a T cell response to OspA.174
By using a sequence algorithm to identify homologous pep-
tides that bind the DR4 pocket, a sequence in LFA-1 was
identified that bound DR4 and stimulated a T cell response
from these OspA-reactive patients.173 The technology of
peptide-MHC tetramers discussed earlier will enable inves-
tigators to determine whether a given subpopulation of T
cells within an inflammatory synovium might manifest dual
specificity for both a foreign pathogen and a cross-reacting
self-protein.
DEATH OF T CELLS
The rapid removal of effector T cells following clearance
of the infection is as important as the initial clonal expan-
sion of responding T cells for the health of the organism.
Failure to clear activated lymphocytes increases the risk
of cross-reactivity with self-antigens and a sustained auto-
immune reaction. To ensure the rapid resolution of the
immune response, a number of processes promote active
cell death of clonally expanded T cells. One means to con-
trol T cell proliferation is through the limited availability
of growth factors. Upon activation, T cells express recep-
tors for various growth cytokines for approximately 7 to
10 days, but they produce cytokines only during the first
48 hours. This results in an unstable situation in which
T cells tend to outgrow the availability of cytokines. For
example, T cells expressing IL-2R in the absence of IL-2
rapidly undergo programmed cell death. Another method
is the restimulation of TCRs on actively dividing T cells,
which triggers AICD.
The discovery of a family of death receptors expressed by
T cells elucidated an additional regulatory process. These
molecules are described more extensively in Chapter 24 on
apoptosis and are discussed here only as they relate to T cell
function. The best described of these is Fas (CD95). Both
Fas-deficient mice175 and humans bearing Fas mutations
(Canele-Smith syndrome)176 manifest a profound lymph-
adenopathy accompanied by an autoimmune diathesis. This
underscores the importance of efficiently removing T cells
after their activation. Nearly all cells have some level of sur-
face Fas, whereas expression of its ligand (FasL) is restricted
primarily to activated T cells and B cells. Consequently, reg-
ulation of Fas-mediated apoptosis is, to a large extent, under
the governance of the immune system. FasL expression
has also been reported in certain components of the eye,
the Sertoli cells of the testis, and perhaps some tumors.177
Expression of FasL by these nonlymphoid cells is thought to
prevent immune responses at sites where such inflammation
might cause tissue damage. For years, immunologists have
been aware of these so-called immune-privileged sites where
immune responses are difficult to initiate.
During T cell activation, expression of FasL is rapidly
induced at the level of RNA, and the ability to kill Fas-
­sensitive target cells is easily demonstrated. Expression of
surface FasL protein has been difficult to demonstrate, how-
ever. This may be due to the sensitivity of surface FasL to
certain proteinases, which results in its rapid cleavage and
release from the cell, similar to the release of TNF-α, another
member of the Fas family. Resting T cells are not sensitive
to Fas-induced death but must first enter the cell cycle for
approximately 3 days. During this period, the cellular level
of an endogenous Fas inhibitor known as c-FLIP is down-
regulated, and this presumably allows Fas signaling to prog-
ress.178 Thus, c-FLIP may function to protect resting T cells
from unnecessary death and restrict apoptosis to activated T
cells to limit their expansion. The importance of c-FLIP as
an inhibitor of cell death is reinforced by the discovery that
certain herpesviruses express a homologue of mammalian
FLIP called E8 that can prevent the death of host cells.179
E8 expression may alter the tumorigenic potential of herpes-
viruses.180 Although it is generally well accepted that Fas is
a major regulator of AICD in vitro, it is less clear whether
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
Fas plays much of a role in the death and removal of T cells
following activation in vivo.
The sequence of T cell activation followed by cell death
is graphically displayed after the administration to mice of
bacterially or virally derived compounds called superanti-
gens. Superantigens activate T cells by directly cross-linking
MHC class II molecules with particular β-chain V families
of the TCR (see Fig. 9-3). The superantigen staphylococ-
cal enterotoxin B strongly activates Vβ8+ T cells.181 This
initiates a rapid expansion of Vβ8+ T cells over 2 to 3 days,
followed by an equally rapid loss of these cells, such that by
day 7, very few Vβ8+ T cells remain. A similar process of
T cell activation occurs in the human disease toxic shock
syndrome, in which a related staphylococcal toxin stimu-
lates the expansion of Vβ2+ T cells.182 The devastating
illness that results from this profound activation of a large
proportion of T cells underscores the need to rapidly elimi-
nate such cells. At least some of the damage in toxic shock
syndrome likely results from the extensive T cell expression
of FasL and TNF-α, particularly in certain tissues such as
the liver. Hepatocytes are exquisitely sensitive to damage by
these ligands.183 Activation of T lymphocytes leads to hom-
ing to the liver, and the administration of antigen to TCR
transgenic mice can yield a syndrome resembling autoim-
mune hepatitis.184 Thus, certain autoimmune disorders may
result from the death or damage of “innocent bystander”
cells as a consequence of the migration of activated T cells
to an organ and nonspecific damage due to the expression of
FasL family members.
It was initially assumed that Fas would largely regulate
this process; however, Fas-deficient mice eliminate T cells
activated by either traditional antigens or superantigens
nearly as efficiently as wild-type mice do. It now appears
that a more recently identified family of compounds known
as Bim, Bad, and Bax regulate AICD in vivo.185 These mol-
ecules are related to the cell survival molecule Bcl-2, but
they are more truncated, containing only the BH3 domain
of Bcl-2—hence their designation as the “BH3-only” fam-
ily. They function as sentinels within the cell, in that they
are attached to various cytoskeletal proteins and organelles
and can sense cellular damage. If damage occurs, they are
released from these sequestered areas and migrate to the
mitochondria to inhibit the survival function of Bcl-2.185
T CELLS AT SITES OF INFLAMMATION
The observation that T cells infiltrate target organs in
­autoimmune diseases such as rheumatoid arthritis, type 1 dia-
betes mellitus, and multiple sclerosis quickly led to an analy-
sis of T cell subsets and, more recently, to a more detailed
study of the T cell repertoire based on TCR expression. In
many of these disorders, the known HLA class II association
is paralleled by a predominant, though by no means exclu-
sive, infiltration of CD4+ T cells.186 Many of these CD4+ T
cells also manifest a Th1-like cytokine profile, as discussed
earlier.156,157,159 Evidence of the importance of these CD4+
cells derives from numerous studies showing the efficacy of
CD4 depletion in animal models of these disorders.187 A
parallel situation often occurs in humans with these autoim-
mune diseases who concurrently become infected with HIV.
The CD4 depletion that occurs during AIDS can actually
ameliorate rheumatoid arthritis.188 ­ However, the resulting
171
CD8 predominance during HIV infection can exacerbate
psoriatic arthritis and Sjögren’s syndrome, suggesting that
the CD8+ subset of T cells may be important in these dis-
orders.188
The ability to assess the TCR repertoire of infiltrating
T cells has provided additional insight into the degree of
clonality present within inflamed tissues. Many studies have
examined the TCR β chain because of its association with
superantigen responses, as well as the TCR α chain.189 Based
on dozens of publications, it is clear that a very broad array
of TCR types is observed in most T cell–mediated diseases.
In the few studies that have analyzed the earliest lesions, T
cell oligoclonality has been observed.190 However, it is pos-
sible that the oligoclonality in these situations may simply
reflect the limited number of T cells in early lesions. These
are nonetheless provocative findings that warrant further
analysis.
REFERENCES
  1. Pawson T, Scott JD: Signaling through scaffold, anchoring, and
adaptor proteins. Science 278:2075-2080, 1997.
  2. Rudd CE: Adaptors and molecular scaffolds in immune cell signaling.
Cell 96:5-8, 1999.
  3. Scollay R, Smith J, Stauffer V: Dynamics of early T cells: Prothymo-
cyte migration and proliferation in the adult mouse thymus. Immu-
nol Rev 91:129-157, 1986.
  4. Godfrey DI, Kennedy J, Suda T, Zlotnik A: A developmental path-
way involving four phenotypically and functionally distinct subsets of
CD3-CD4-CD8- triple-negative adult mouse thymocytes defined by
CD44 and CD25 expression. J Immunol 150:4244-4252, 1993.
  5. von Boehmer H, Fehling HJ: Structure and function of the pre-T cell
receptor. Annu Rev Immunol 15:433-452, 1997.
  6. Bosma GC, Custer RP, Bosma MJ: A severe combined immunodefi-
ciency mutation in the mouse. Nature 301:527-530, 1983.
  7. Mombaerts P, et al: RAG-1-deficient mice have no mature B and
T lymphocytes. Cell 68:869-877, 1992.
  8. Rodewald HR, Fehling HJ: Molecular and cellular events in early
thymocyte development. Adv Immunol 69:1-112, 1998.
  9. Radtke F, et al: Deficient T cell fate specification in mice with an
induced inactivation of Notch1. Immunity 10:547-558, 1999.
10. Peschon JJ, et al: Early lymphocyte expansion is severely impaired
in interleukin 7 receptor-deficient mice. J Exp Med 180:1955-1960,
1994.
11. Uribe L, Weinberg KI: X-linked SCID and other defects of cytokine
pathways. Semin Hematol 35:299-309, 1998.
12. Farrar MA, Doerfler P, Sauer K: Signal transduction pathways regu-
lating the development of alpha beta T cells. Biochim Biophys Acta
1377:F35-F78, 1998.
13. Elder ME, et al: Human severe combined immunodeficiency due to
a defect in ZAP-70, a T cell tyrosine kinase. Science 264:1596-1599,
1994.
14. Padovan E, et al: Expression of two T cell receptor α chains: Dual
receptor T cells. Science 262:422-424, 1993.
15. Niederberger N, et al: Allelic exclusion of the TCR α chain is an
active process requiring TCR-mediated signaling and c-Cbl. J Immu-
nol 170:4557-4563, 2003.
16. Garboczi DN, et al: Structure of the complex between human T-cell
receptor, viral peptide and HLA-A2. Nature 384:134-141, 1996.
17. Garcia KC, et al: An ab T cell receptor structure at 2.5 A and its ori-
entation in the TCR-MHC complex. Science 274:209-219, 1996.
18. Matsui K, et al: Low affinity interaction of peptide-MHC complexes
with T cell receptors. Science 254:1788-1791, 1991.
19. Yamashita I, Nagata T, Tada T, Nakayama T: CD69 cell surface
expression identifies developing thymocytes which audition for
T cell antigen receptor-mediated positive selection. Int Immunol
5:1139-1150, 1993.
20. Punt JA, Osborne BA, Takahama Y, et al: Negative selection of
CD4+CD8+ thymocytes by T cell receptor-induced apoptosis
requires a costimulatory signal that can be provided by CD28. J Exp
Med 179:709-713, 1994.
 172 BUDD  |  T Lymphocytes
21. Linette GP, et al: Bcl-2 is upregulated at the CD4+ CD8+ stage dur-
ing positive selection and promotes thymocyte differentiation at sev-
eral control points. Immunity 1:197-205, 1994.
22. Priatel JJ, Teh SJ, Dower NA, et al: RasGRP1 transduces low-grade
TCR signals which are critical for T cell development, homeostasis,
and differentiation. Immunity 17:617-627, 2002.
23. Amakawa R, et al: Impaired negative selection of T cells in
Hodgkin’s disease antigen CD30-deficient mice. Cell 84:551-562,
1996.
24. Kawabe T, et al: The immune responses in CD40-deficient mice:
Impaired immunoglobulin class switching and germinal center for-
mation. Immunity 1:167-178, 1994.
25. Xu J, et al: Mice deficient for the CD40 ligand. Immunity 1:423-431,
1994.
26. Deftos ML, He YW, Ojala EW, Bevan MJ: Correlating notch signal-
ing with thymocyte maturation. Immunity 9:777-786, 1998.
27. Yasutomo K, Doyle C, Miele L, et al: The duration of antigen recep-
tor signalling determines CD4+ versus CD8+ T-cell lineage fate.
Nature 404:506-510, 2000.
28. Lefrancois L, Puddington L: Extrathymic intestinal T-cell develop-
ment: Virtual reality? Immunol Today 16:16-21, 1995.
29. Groh V, Steinle A, Bauer S, Spies T: Recognition of stress-induced
MHC molecules by intestinal epithelial gd T cells. Science 279:
1737-1740, 1998.
30. Arnaiz-Villena A, et al: Human T-cell activation deficiencies. Immu-
nol Today 13:259-265, 1992.
31. Buckley RH: Primary cellular immunodeficiencies. J Allergy Clin
Immunol 109:747-757, 2002.
32. Hong R: The DiGeorge anomaly. Clin Rev Allergy Immunol 20:
43-60, 2001.
33. Noguchi M, et al: Interleukin-2 receptor gamma chain mutation
results in X-linked severe combined immunodeficiency in humans.
Cell 73:147-157, 1993.
34. van Dinther-Janssen AC, Pals ST, Scheper R, et al: Dendritic cells
and high endothelial venules in the rheumatoid synovial membrane.
J Rheumatol 17:11-17, 1990.
35. Butcher EC, Williams M, Youngman K, et al: Lymphocyte trafficking
and regional immunity. Adv Immunol 72:209-253, 1999.
36. Bodolay E, Koch AE, Kim J, et al: Angiogenesis and chemokines in
rheumatoid arthritis and other systemic inflammatory rheumatic dis-
eases. J Cell Mol Med 6:357-376, 2002.
37. Szekanecz Z, Kim J, Koch AE: Chemokines and chemokine receptors
in rheumatoid arthritis. Semin Immunol 15:15-21, 2003.
38. Troy AE, Shen H: Cutting edge: Homeostatic proliferation of periph-
eral T lymphocytes is regulated by clonal competition. J Immunol
170:672-676, 2003.
39. Goldrath AW, Bogatzki LY, Bevan M:. Naive T cells transiently
acquire a memory-like phenotype during homeostasis-driven prolif-
eration. J Exp Med 192:557-564, 2000.
40. Min B, et al: Neonates support lymphopenia-induced proliferation.
Immunity 18:131-140, 2003.
41. Miller JF, Osoba D: Current concepts of the immunological function
of the thymus. Physiol Rev 47:437-520, 1967.
42. Yunis EJ, et al: Postthymectomy wasting associated with autoimmune
phenomena. I. Antiglobulin-positive anemia in A and C57BL-6 Ks
mice. J Exp Med 125:947-966, 1967.
43. Weiss A, Littman DR: Signal transduction by lymphocyte antigen
receptors. Cell 76:263-274, 1994.
44. Howe LR, Weiss A: Multiple kinases mediate T-cell-receptor signal-
ing. Trends Biochem Sci 20:59-64, 1995.
45. Weissman AM: The T-cell antigen receptor: A multisubunit signal-
ing complex. Chem Immunol 59:1-18, 1994.
46. Cambier JC: Antigen and Fc receptor signaling: The awesome power
of the immunoreceptor tyrosine-based activation motif (ITAM).
J Immunol 155:3281-3285, 1995.
47. Wange RL, Samelson LE: Complex complexes: Signaling at the
TCR. Immunity 5:197-205, 1996.
48. Mustelin T, Tasken K: Positive and negative regulation of T-cell
activation through kinases and phosphatases. Biochem J 371:15-27,
2003.
49. Appleby MW, et al: Defective T cell receptor signaling in mice lack-
ing the thymic isoform of p59fyn. Cell 70:751-763, 1992.
50. Stein PL, Lee HM, Rich S, Soriano P: pp59fyn Mutant mice dis-
play differential signaling in thymocytes and peripheral T cells. Cell
70:741-750, 1992.
51. Koretzky GA, Picus J, Thomas ML, Weiss A: Tyrosine phosphatase
CD45 is essential for coupling T-cell antigen receptor to the phos-
phatidyl inositol pathway. Nature 346:66-68, 1990.
52. Liao XC, Littman DR: Altered T cell receptor signaling and disrupted
T cell development in mice lacking Itk. Immunity 3:757-769, 1995.
53. van Oers NS, Killeen N, Weiss A: ZAP-70 is constitutively associ-
ated with tyrosine-phosphorylated TCR z in murine thymocytes and
lymph node T cells. Immunity 1:675-685, 1994.
54. Jordan MS, Singer AL, Koretzky GA: Adaptors as central mediators of
signal transduction in immune cells. Nat Immunol 4:110-116, 2003.
55. Greenwood J, Steinman L, Zamvil SS: Statin therapy and autoim-
mune disease: From protein prenylation to immunomodulation. Nat
Rev Immunol 6:358-370, 2006.
56. Cantrell D: T cell antigen receptor signal transduction pathways.
Annu Rev Immunol 14:259-274, 1996.
57. Sun Z, et al: PKC-theta is required for TCR-induced NF-kappaB acti-
vation in mature but not immature T lymphocytes. Nature 404:402-
407, 2000.
58. Crabtree GR, Olson EN: NFAT signaling: Choreographing the social
lives of cells. Cell 109(Suppl):S67-S79, 2002.
59. Dolmetsch RE, Lewis RS, Goodnow CC, Healy JI: Differential acti-
vation of transcription factors induced by Ca2+ response amplitude
and duration. Nature 386:855-858, 1997.
60. Guse AH, et al: Regulation of calcium signalling in T lymphocytes by
the second messenger cyclic ADP-ribose. Nature 398:70-73, 1999.
61. Kennedy NJ, Kataoka T, Tschopp J, Budd RC: Caspase activation is
required for T cell proliferation. J Exp Med 190:1891-1896, 1999.
62. Salmena L, et al: Essential role for caspase 8 in T-cell homeostasis and
T-cell-mediated immunity. Genes Dev 17:883-895, 2003.
63. Chun HJ, et al: Pleiotropic defects in lymphocyte activation caused
by caspase-8 mutations lead to human immunodeficiency. Nature
419:395-399, 2002.
64. Su H, et al: Requirement for caspase-8 in NF-kappaB activation by
antigen receptor. Science 307:1465-1468, 2005.
65. Lenschow DJ, Walunas TL, Bluestone JA: CD28/B7 system of T cell
costimulation. Annu Rev Immunol 14:233-258, 1996.
66. Speiser DE, et al: CD28-negative cytolytic effector T cells frequently
express NK receptors and are present at variable proportions in circu-
lating lymphocytes from healthy donors and melanoma patients. Eur
J Immunol 29:1990-1999, 1999.
67. Lamprecht P, et al: CD28 negative T cells are enriched in granulo-
matous lesions of the respiratory tract in Wegener’s granulomatosis.
Thorax 56:751-757, 2001.
68. Fletcher JM, et al: Cytomegalovirus-specific CD4+ T cells in healthy
carriers are continuously driven to replicative exhaustion. J Immunol
175:8218-8225, 2005.
69. Uda H, et al: Expansion of a CD28-intermediate subset among CD8
T cells in patients with infectious mononucleosis. J Virol 76:6602-
6608, 2002.
70. Loetscher P, Seitz M, Baggiolini M, Moser B: Interleukin-2 regulates
CC chemokine receptor expression and chemotactic responsiveness
in T lymphocytes. J Exp Med 184:569-577, 1996.
71. Grakoui A, et al: The immunological synapse: A molecular machine
controlling T cell activation. Science 285:221-227, 1999.
72. Monks CR, Freiberg BA, Kupfer H, et al: Three-dimensional segrega-
tion of supramolecular activation clusters in T cells. Nature 395:82-
86, 1998.
73. Dustin ML, et al: A novel adaptor protein orchestrates receptor pat-
terning and cytoskeletal polarity in T-cell contacts. Cell 94:667-677,
1998.
74. Montixi C, et al: Engagement of T cell receptor triggers its recruit-
ment to low-density detergent-insoluble membrane domains. EMBO
J 17:5334-5348, 1998.
75. Xavier R, Brennan T, Li Q, et al: Membrane compartmentation is
required for efficient T cell activation. Immunity 8:723-732, 1998.
76. Simons K, Ikonen E: Functional rafts in cell membranes. Nature
387:569-572, 1997.
77. Harder T, Simons K: Caveolae, DIGs, and the dynamics of sphin-
golipid-cholesterol microdomains. Curr Opin Cell Biol 9:534-542,
1997.
78. Lee KH, et al: T cell receptor signaling precedes immunological syn-
apse formation. Science 295:1539-1542, 2002.
79. Stoll S, Delon J, Brotz TM, Germain RN: Dynamic imaging of T cell-
dendritic cell interactions in lymph nodes. Science 296:1873-1876,
2002.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
80. Rozdzial MM, Malissen B, Finkel TH: Tyrosine-phosphorylated T
cell receptor z chain associates with the actin cytoskeleton upon acti-
vation of mature T lymphocytes. Immunity 3:623-633, 1995.
81. Kupfer A, Swain SL, Singer SJ: The specific direct interaction of
helper T cells and antigen-presenting B cells. II. Reorientation of the
microtubule organizing center and reorganization of the membrane-
associated cytoskeleton inside the bound helper T cells. J Exp Med
165:1565-1580, 1987.
82. Kupfer A, Mosmann TR, Kupfer H: Polarized expression of cyto-
kines in cell conjugates of helper T cells and splenic B cells. Proc
Natl Acad Sci U S A 88:775-779, 1991.
83. Badour K, et al: The Wiskott-Aldrich syndrome protein acts down-
stream of CD2 and the CD2AP and PSTPIP1 adaptors to promote
formation of the immunological synapse. Immunity 18:141-154,
2003.
84. Iezzi G, Karjalainen K, Lanzavecchia A: The duration of antigenic
stimulation determines the fate of naive and effector T cells. Immu-
nity 8:89-95, 1998.
85. Shaw AS, Dustin ML: Making the T cell receptor go the distance: A
topological view of T cell activation. Immunity 6:361-369, 1997.
86. Lee K-M, et al: Molecular basis of T cell inactivation by CTLA-4.
Science 282:2263-2266, 1998.
87. Viola A, Schroeder S, Sakakibara Y, Lanzavecchia A: T lymphocyte
costimulation mediated by reorganization of membrane microdo-
mains. Science 283:680-682, 1999.
88. Barber DL, et al: Restoring function in exhausted CD8 T cells during
chronic viral infection. Nature 439:682-687, 2006.
89. Nishimura H, Nose M, Hiai H, et al: Development of lupus-like
autoimmune diseases by disruption of the PD-1 gene encoding
an ITIM motif-carrying immunoreceptor. Immunity 11:141-151,
1999.
90. Sloan-Lancaster J, Shaw AS, Rothbard JB, Allen PM: Partial T cell
signaling: Altered phospho-z and lack of Zap70 recruitment in APL-
induced T cell anergy. Cell 79:913-922, 1994.
91. Krummel MF, Allison JP: CD28 and CTLA-4 have opposing effects
on the response of T cells to stimulation. J Exp Med 182:459-465,
1995.
92. Marengere LE, et al: Regulation of T cell receptor signaling by tyro-
sine phosphatase SYP association with CTLA-4. Science 272:1170-
1173, 1996.
93. Chikuma S, Imboden JB, Bluestone JA: Negative regulation of T cell
receptor-lipid raft interaction by cytotoxic T lymphocyte-associated
antigen 4. J Exp Med 197:129-135, 2003.
94. Tivol EA, et al: Loss of CTLA-4 leads to massive lymphoproliferation
and fatal multiorgan tissue destruction, revealing a critical negative
regulatory role of CTLA-4. Immunity 3:541-547, 1995.
95. Waterhouse P, et al: Lymphoproliferative disorders with early lethal-
ity in mice deficient in Ctla-4. Science 270:985-988, 1995.
96. Firestein GS, Zvaifler NJ: Peripheral blood and synovial fluid mono-
cyte activation in inflammatory arthritis. I. A cytofluorographic
study of monocyte differentiation antigens and class II antigens and
their regulation by gamma-interferon. Arthritis Rheum 30:857-863,
1987.
97. Verwilghen J, Vertessen S, Stevens EA, et al: Depressed T-cell reac-
tivity to recall antigens in rheumatoid arthritis. J Clin Immunol
10:90-98, 1990.
98. Cope AP, et al: Chronic exposure to tumor necrosis factor (TNF) in
vitro impairs the activation of T cells through the T cell receptor/
CD3 complex; reversal in vivo by anti-TNF antibodies in patients
with rheumatoid arthritis. J Clin Invest 94:749-760, 1994.
99. Cope AP, et al: Chronic tumor necrosis factor alters T cell responses
by attenuating T cell receptor signaling. J Exp Med 185:1573-1584,
1997.
100. Martins GA, et al: Transcriptional repressor Blimp-1 regulates T cell
homeostasis and function. Nat Immunol 7:457-465, 2006.
101. Sakaguchi S, et al: Immunologic tolerance maintained by CD25+
CD4+ regulatory T cells: Their common role in controlling autoim-
munity, tumor immunity, and transplantation tolerance. Immunol
Rev 182:18-32, 2001.
102. Shevach EM, McHugh RS, Piccirillo CA, Thornton AM: Control of
T-cell activation by CD4+ CD25+ suppressor T cells. Immunol Rev
182:58-67, 2001.
103. Veldhoen M, Hocking RJ, Atkins CJ, et al: TGFbeta in the context
of an inflammatory cytokine milieu supports de novo differentiation
of IL-17-producing T cells. Immunity 24:179-189, 2006.
173
104. Hori S, Nomura T, Sakaguchi S: Control of regulatory T cell
development by the transcription factor Foxp3. Science 299:1057-
1061, 2003.
105. Liu W, et al: CD127 expression inversely correlates with FoxP3
and suppressive function of human CD4+ T reg cells. J Exp Med
203:1701-1711, 2006.
106. Dong C: Diversification of T-helper-cell lineages: Finding the family
root of IL-17-producing cells. Nat Rev Immunol 6:329-333, 2006.
107. Pamer E, Cresswell P: Mechanisms of MHC class I-restricted antigen
processing. Annu Rev Immunol 16:323-358, 1998.
108. Rock KL, Goldberg AL: Degradation of cell proteins and the gen-
eration of MHC class I-presenting peptides. Annu Rev Immunol
17:739-779, 1999.
109. Street NE, et al: Heterogeneity of mouse helper T cells: Evidence
from bulk cultures and limiting dilution cloning for precursors of Th1
and Th2 cells. J Immunol 144:1629-1639, 1990.
110. Newell MK, Haughn LJ, Maroun CR, Julius MH: Death of mature T
cells by separate ligation of CD4 and the T-cell receptor for antigen.
Nature 347:286-289, 1990.
111. Fauci AS: Host factors and the pathogenesis of HIV-induced disease.
Nature 384:529-534, 1996.
112. Casella CR, Finkel TH: Mechanisms of lymphocyte killing by HIV.
Curr Opin Hematol 4:24-31, 1997.
113. Janeway CA Jr, Medzhitov R: Innate immune recognition. Annu
Rev Immunol 20:197-216, 2002.
114. Zeng Z, et al: Crystal structure of mouse CD1: An MHC-like fold
with a large hydrophobic binding groove. Science 277:339-345,
1997.
115. Saito H, et al: Complete primary structure of a heterodimeric T-cell
receptor deduced from cDNA sequences. Nature 309:757-762, 1984.
116. Itohara S, et al: Homing of a gamma delta thymocyte subset with
homogeneous T-cell receptors to mucosal epithelia. Nature 343:
754-757, 1990.
117. Wright A, et al: Cytotoxic T lymphocytes specific for self tumor
immunoglobulin express T cell receptor delta chain. J Exp Med
169:1557-1564, 1989.
118. Sciammas R, et al: Unique antigen recognition by a herpesvirus-
­specific TCR-gamma delta cell. J Immunol 152:5392-5397, 1994.
119. Schild H, et al: The nature of major histocompatibility complex rec-
ognition by gamma delta T cells. Cell 76:29-37, 1994.
120. Holoshitz J, Koning F, Coligan JE, et al: Isolation of CD4– CD8–
mycobacteria-reactive T lymphocyte clones from rheumatoid arthri-
tis synovial fluid. Nature 339:226-229, 1989.
121. Kabelitz D, Bender A, Schondelmaier S, et al: A large fraction of
human peripheral blood gd+ T cells is activated by Mycobacterium
tuberculosis but not by its 65-kD heat shock protein. J Exp Med
171:667-679, 1990.
122. Constant P, et al: Stimulation of human gd T cells by nonpeptidic
mycobacterial ligands. Science 264:267-270, 1994.
123. Tanaka Y, et al: Nonpeptide ligands for human gd T cells. Proc Natl
Acad Sci U S A 91:8175-8179, 1994.
124. Bukowski JF, Morita CT, Brenner MB: Human gd T cells recognize
alkylamines derived from microbes, edible plants, and tea: Implica-
tions for innate immunity. Immunity 11:57-65, 1999.
125. Hiromatsu K, et al: A protective role of gd T cells in primary infection
with Listeria monocytogenes in mice. J Exp Med 175:49-56, 1992.
126. Rosat JP, MacDonald HR, Louis JA: A role for gd+ T cells during
experimental infection of mice with Leishmania major. J Immunol
150:550-555, 1993.
127. Kaufmann SH, Ladel CH: Role of T cell subsets in immunity against
intracellular bacteria: Experimental infections of knock-out mice
with Listeria monocytogenes and Mycobacterium bovis BCG. Immuno-
biology 191:L509-L519, 1994.
128. Tsuji M, et al: gd T cells contribute to immunity against the liver
stages of malaria in ab T-cell-deficient mice. Proc Natl Acad Sci U S A
91:345-349, 1994.
129. Mixter PF, Camerini V, Stone BJ, et al: Mouse T lymphocytes that
express a gamma delta T-cell antigen receptor contribute to resis-
tance to Salmonella infection in vivo. Infect Immun 62:4618-4621,
1994.
130. Carding SR, et al: Late dominance of the inflammatory process in
murine influenza by gd+ T cells. J Exp Med 172:1225-1231, 1990.
131. Modlin RL, et al: Lymphocytes bearing antigen-specific gd T-cell
receptors accumulate in human infectious disease lesions. Nature
339:544-548, 1989.
 174 BUDD  |  T Lymphocytes
132. Vincent MS, et al: Lyme arthritis synovial gamma delta T cells
respond to Borrelia burgdorferi lipoproteins and lipidated hexapep-
tides. J Immunol 161:5762-5771, 1998.
133.  Brennan FM, et al: T cells expressing chain receptors in rheuma-
toid arthritis. J Autoimmun 1:319-326, 1988.
134. Rust C, et al: Phenotypical and functional characterization of small
intestinal TcR T cells in coeliac disease. Scand J Immunol 35:459-
468, 1992.
135. Balbi B, Moller DR, Kirby M, et al: Increased numbers of T lympho-
cytes with gd+ antigen receptors in a subgroup of individuals with
pulmonary sarcoidosis. J Clin Invest 85:1353-1361, 1990.
136. Vincent MS, et al: Apoptosis of Fas high CD4+ synovial T cells
by Borrelia-reactive Fas-ligand(high) gamma delta T cells in Lyme
arthritis. J Exp Med 184:2109-2117, 1996.
137. Roessner K, et al: High expression of Fas ligand by synovial fluid-
derived gamma delta T cells in Lyme arthritis. J Immunol 170:
2702-2710, 2003.
138. Huber S, Shi C, Budd RC: Gamma delta T cells promote a Th1
response during coxsackievirus B3 infection in vivo: Role of Fas and
Fas ligand. J Virol 76:L6487-L6494, 2002.
139. Schramm CM, et al: Proinflammatory roles of T-cell receptor (TCR)
gamma delta and TCR alpha beta lymphocytes in a murine model of
asthma. Am J Respir Cell Mol Biol 22:218-225, 2000.
140. Bendelac A, Rivera MN, Park SH, Roark JH: Mouse CD1-specific
NK1 T cells: Development, specificity, and function. Annu Rev
Immunol 15:535-562, 1997.
141. Kinjo Y, et al: Recognition of bacterial glycosphingolipids by natural
killer T cells. Nature 434:520-525, 2005.
142. Kinjo Y, et al: Natural killer T cells recognize diacylglycerol antigens
from pathogenic bacteria. Nat Immunol 7:978-986, 2006.
143. Lehuen A, et al: Overexpression of natural killer T cells protects
Va14-Ja281 transgenic nonobese diabetic mice against diabetes.
J Exp Med 188:1831-1839, 1998.
144. Baxter AG, Kinder SJ, Hammond KJ, et al: Association between
abTCR+CD4–CD8– T-cell deficiency and IDDM in NOD/Lt mice.
Diabetes 46:572-582, 1997.
145. Wilson SB, et al: Extreme Th1 bias of invariant Va24JaQ T cells in
type 1 diabetes. Nature 391:177-181, 1998.
146. Akbari O, et al: Essential role of NKT cells producing IL-4 and IL-13
in the development of allergen-induced airway hyperreactivity. Nat
Med 9:582-588, 2003.
147. Doherty PC: The new numerology of immunity mediated by virus-
specific CD8+ T cells. Curr Opin Microbiol 1:419-422, 1998.
148. Purton JF, et al: Antiviral CD4+ memory T cells are IL-15 depen-
dent. J Exp Med 204:951-961, 2007.
149. Shin H, Blackburn SD, Blattman JN, Wherry EJ: Viral antigen and
extensive division maintain virus-specific CD8 T cells during chronic
infection. J Exp Med 204:941-949, 2007.
150. Budd RC, Cerottini JC, MacDonald HR: Phenotypic identifica-
tion of memory cytolytic T lymphocytes in a subset of Lyt-2+ cells.
J Immunol 138:1009-1013, 1987.
151. Masopust D, Vezys V, Marzo AL, Lefrancois L: Preferential local-
ization of effector memory cells in nonlymphoid tissue. Science
291:2413-2417, 2001.
152. Schneider P, et al: BAFF, a novel ligand of the tumor necrosis factor
family, stimulates B cell growth. J Exp Med 189:1747-1756, 1999.
153. Coffman RL, Seymour BW, Hudak S, et al: Antibody to interleukin-5
inhibits helminth-induced eosinophilia in mice. Science 245:
308-310, 1989.
154. Rincon M, Anguita J, Nakamura T, et al: Interleukin (IL)-6 directs
the differentiation of IL-4-producing CD4+ T cells. J Exp Med
185:461-469, 1997.
155. Fuss IJ, et al: Characteristic T helper 2 T cell cytokine abnormalities
in autoimmune lymphoproliferative syndrome, a syndrome marked
by defective apoptosis and humoral autoimmunity. J Immunol
158:1912-1918, 1997.
156. Ruddle NH, et al: An antibody to lymphotoxin and tumor necrosis
factor prevents transfer of experimental allergic encephalomyelitis.
J Exp Med 172:1193-1200, 1990.
157. Heath WR, et al: Autoimmune diabetes as a consequence of locally
produced interleukin-2. Nature 359:547-549, 1992.
158. Saxne T, Palladino MA Jr, Heinegard D, et al: Detection of tumor
necrosis factor a but not tumor necrosis factor b in rheumatoid
arthritis synovial fluid and serum. Arthritis Rheum 31:1041-1045,
1988.
159. Yssel H, et al: Borrelia burgdorferi activates a T helper type 1-like T
cell subset in Lyme arthritis. J Exp Med 174:593-601, 1991.
160. Elliott MJ, et al: Randomised double-blind comparison of chime-
ric monoclonal antibody to tumour necrosis factor alpha (cA2)
versus placebo in rheumatoid arthritis. Lancet 344:1105-1110,
1994.
161. Choy EH, et al: Therapeutic benefit of blocking interleukin-6 activ-
ity with an anti-interleukin-6 receptor monoclonal antibody in rheu-
matoid arthritis: A randomized, double-blind, placebo-controlled,
dose-escalation trial. Arthritis Rheum 46:3143-3150, 2002.
162. Hsieh CS, et al: Development of TH1 CD4+ T cells through IL-12
produced by Listeria-induced macrophages. Science 260:547-549,
1993.
163. Kuchroo VK, et al: B7-1 and B7-2 costimulatory molecules activate
differentially the Th1/Th2 developmental pathways: Application to
autoimmune disease therapy. Cell 80:707-718, 1995.
164. Le Gros G, Ben-Sasson SZ, Seder R, et al: Generation of interleu-
kin 4 (IL-4)-producing cells in vivo and in vitro: IL-2 and IL-4 are
required for in vitro generation of IL-4-producing cells. J Exp Med
172:921-929, 1990.
165. Zhang X, et al: Unequal death in T helper cell (Th)1 and Th2 effec-
tors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated
apoptosis. J Exp Med 185:1837-1849, 1997.
166. Gilbert KM, Hoang KD, Weigle WO: Th1 and Th2 clones differ in
their response to a tolerogenic signal. J Immunol 144:2063-2071,
1990.
167. Ramsdell F, et al: Differential ability of Th1 and Th2 T cells to
express Fas ligand and to undergo activation-induced cell death. Int
Immunol 6:1545-1553, 1994.
168. Suda T, et al: Expression of the Fas ligand in cells of T cell lineage.
J Immunol 154:3806-3813, 1995.
169. Zheng Y, et al: Interleukin-22, a T(H)17 cytokine, mediates IL-
23-induced dermal inflammation and acanthosis. Nature 445:648-
651, 2007.
170. Chan JR, et al: IL-23 stimulates epidermal hyperplasia via TNF
and IL-20R2-dependent mechanisms with implications for psoriasis
pathogenesis. J Exp Med 203:2577-2587, 2006.
171.  Steinman L: A brief history of T(H)17, the first major revision
in the T(H)1/T(H)2 hypothesis of T cell-mediated tissue damage.
Nat Med 13:139-145, 2007.
172. Wucherpfennig KW, Strominger JL: Molecular mimicry in T cell-
mediated autoimmunity: Viral peptides activate human T cell clones
specific for myelin basic protein. Cell 80:695-705, 1995.
173.  Gross DM, et al: Identification of LFA-1 as a candidate autoantigen
in treatment-resistant Lyme arthritis. Science 281:703-706, 1998.
174. Kalish RA, Leong JM, Steere AC: Association of treatment-resistant
chronic Lyme arthritis with HLA-DR4 and antibody reactivity to
OspA and OspB of Borrelia burgdorferi. Infect Immun 61:2774-2779,
1993.
175. Budd RC, Van Houten N, Clements J, Mixter PF: Parallels in T lym-
phocyte development between lpr and normal mice. Semin Immunol
6:43-48, 1994.
176. Vaishnaw AK, et al: The molecular basis for apoptotic defects in
patients with CD95 (Fas/Apo-1) mutations. J Clin Invest 103:
355-363, 1999.
177. Griffith TS, Brunner T, Fletcher SM, et al: Fas ligand-induced apop-
tosis as a mechanism of immune privilege. Science 17:1189-1192,
1995.
178. Irmler M, et al: Inhibition of death receptor signals by cellular FLIP.
Nature 388:190-195, 1997.
179. Thome M, et al: Viral FLICE-inhibitory proteins (FLIPs) prevent
apoptosis induced by death receptors. Nature 386:517-521, 1997.
180. Tschopp J, Thome M, Hofmann K, Meinl E: The fight of viruses
against apoptosis. Curr Opin Genet Dev 8:82-87, 1998.
181. Kawabe Y, Ochi A: Selective anergy of V beta 8+,CD4+ T cells
in Staphylococcus enterotoxin B-primed mice. J Exp Med 172:
1065-1070, 1990.
182. Choi Y, et al: Selective expansion of T cells expressing V beta 2 in
toxic shock syndrome. J Exp Med 172:981-984, 1990.
183. Kennedy NJ, Russell JQ, Michail N, Budd RC: Liver damage by infil-
trating CD8+ T cells is Fas dependent. J Immunol 167:6654-6662,
2001.
184. Russell JQ, et al: Liver damage preferentially results from CD8+
T cells triggered by high affinity peptide antigens. J Exp Med 188:
1147-1157, 1998.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
185. Hildeman DA, et al: Activated T cell death in vivo mediated by pro-
apoptotic bcl-2 family member bim. Immunity 16:759-767, 2002.
186. Steere AC, Duray PH, Butcher EC: Spirochetal antigens and lym-
phoid cell surface markers in Lyme synovitis: Comparison with rheu-
matoid synovium and tonsillar lymphoid tissue. Arthritis Rheum
31:487-495, 1988.
187. Ranges GE, Sriram S, Cooper SM: Prevention of type II collagen-
induced arthritis by in vivo treatment with anti-L3T4. J Exp Med
162:1105-1110, 1985.
175
188. Winchester RJ: HIV infection and rheumatic disease. Bull Rheum
Dis 43:5-8, 1994.
189. Wucherpfennig KW, et al: T cell receptor Va-Vβ repertoire and
cytokine gene expression in active multiple sclerosis lesions. J Exp
Med 175:993-1002, 1992.
190. Yang Y, Charlton B, Shimada A, et al: Monoclonal T cells identified
in early NOD islet infiltrates. Immunity 4:189-194, 1996.
 10
KEY POINTS
The immunoglobulin Fc’ region defines the immunoglobulin
isotype (IgM, IgG, IgA, IgE, IgD) and mediates different
effector functions. The variable region of the immunoglobulin
binds to specific antigenic epitopes. The random combination
of multiple V, D, and J gene segments generates a diverse
array of immunoglobulin molecules with myriad antigenic
specificities.
Secreted immunoglobulin mediates distinct effector
functions, including neutralization of antigen, antigen uptake
by phagocytic cells, complement activation, and antigen-
dependent cell-mediated cytotoxicity.
Surface immunoglobulin acts as a signal transduction
molecule and is a major component of the B cell receptor,
which plays a critical role in regulating B cell selection,
survival, and activation.
B cells are derived from hematopoietic precursors in the
bone marrow and undergo several stages of development
and selection before becoming immunocompetent, mature,
naive B cells that reside in peripheral lymphoid organs. The
final stages of B cell development, which occur in peripheral
lymphoid organs following antigen activation, include affinity
maturation and generation of long-lived memory B cells and
immunoglobulin-secreting plasma cells.
Distinct B cell subsets participate in different types of immune
responses to pathogens. Follicular B cells respond to T cell–
dependent antigenic responses; B1 and marginal zone B cells
respond to distinct classes of T cell–independent antigens.
Autoreactive B cells are generated in all individuals. There
are multiple checkpoints that extinguish autoreactive B cells
during early and later stages of B cell development. One or
more of these checkpoints are breached in autoimmune-
prone individuals, leading to the maturation and activation of
autoreactive B cells.
IMMUNOGLOBULIN STRUCTURE
AND FUNCTION
The principal role of B lymphocytes (Fig. 10-1) in the
immune system is the synthesis of immunoglobulins (also
referred to as antibodies), which are globular proteins that
bind foreign substances known as antigens. Immunoglobu-
lins exist as secreted proteins that circulate throughout the
body or as cell membrane–associated receptors that help
mediate B cell survival, activation, and maturation. The
tertiary structure of an antibody molecule consists of a
Y-shaped conformation that contains two functional moi-
eties: two identical variable regions and the constant region
  Supplemental images available on the Expert Consult Premium Edition website.
B Cells
Betty Diamond  • 
Christine Grimaldi
(Fig. 10-2). Each variable region of an antibody molecule
contains an antigen-binding pocket; the constant region
directs immunoglobulin effector functions that mediate the
killing and removal of invading organisms and both the acti-
vation and homeostasis of the immune system. The amino
acid composition of the immunoglobulin variable region
exhibits great diversity, which facilitates the recognition of
a wide array of antigens. As the name implies, the constant
region exhibits far less diversity and can be subdivided into
a few distinct classes known as isotypes.
The fundamental units of an immunoglobulin are the
light (L)-chain polypeptide, which has an apparent molec-
ular mass of 25 kD, and the heavy (H)-chain polypeptide,
which has an apparent molecular mass of 50 to 65 kD.
An immunoglobulin monomer consists of two identical
L-chain molecules covalently linked by disulfide bonds
to two identical H-chain molecules that are also linked
by disulfide bonds (see Fig. 10-2). Early studies analyzing
proteolytic fragments of molecules demonstrated distinct
functional components.1 Cleavage with papain generates
the antigen-binding fragment (Fab); either of two identi-
cal fragments retains the ability to recognize antigen. The
Fab portion is composed of the variable region domains
of the H and L chains, the first H-chain constant region
domain (CH1), and the L-chain constant region domain
(CL). Cleavage with pepsin occurs below the disulfide
linkage of the two H chains and generates a molecule
that contains two disulfide-bonded Fab fragments called
F(ab′)2. The presence of two Fab regions in a single immu-
noglobulin monomer creates a bivalent binding capacity
to interact with repetitive determinants present in mul-
tivalent antigens (i.e., polysaccharide) or two separate
antigen molecules containing the same antigenic determi-
nant. The remaining component, termed fragment crystal-
lizable (Fc), based on its ability to readily undergo crystal
formation, is unable to interact with antigen but contains
the CH2 and CH3 domains of the H chain that mediate
immune effector functions.
Within the variable region of the immunoglobulin
molecule are discrete regions, known as complementarity-
determining regions, that make direct contact with antigen.
The amino acid sequences of the complementarity-deter-
mining regions are highly variable and are flanked by more
conserved amino acid sequences called framework regions.
The H- and L-chain molecules each contain three comple-
mentarity-determining regions and four framework regions
(see Fig. 10-2). The minimal antigenic determinant recog-
nized by the H and L complementarity-determining regions
is known as an epitope, which may be a continuous or
discontinuous region on a protein, carbohydrate, lipid, or
nucleic acid.
177
 178 DIAMOND  |  B Cells
Figure 10-1  Micrograph of a B cell. (Courtesy of Professor Peter
Groscurth, University of Zurich.)
F(ab´)2
FWR
VH
VL
CH1 CDR
CL
Fab
Hinge
CH2
Fc
CH3
Figure 10-2  Schematic of the antibody molecule. An antibody mono-
mer consists of two heavy (H)-chain molecules covalently linked to two
light (L)-chain molecules. The variable region is composed of the VH and
VL domains of the H and L chains, respectively. Within the VH and VL do-
mains are four framework regions (FWRs) and three complementarity-  tosis are called opsonins. Once the complement cascade has
determining regions (CDRs), which together make up the antigen-binding
pocket. Papain digestion generates the Fab portion, which consists of VH,
CH1, VL, and CL domains, and pepsin digestion generates two covalently
linked Fabs, known as the F(ab’)2. The Fc region of the H-chain constant
region, which mediates immune effector functions, consists of the hinge
domain (only in IgG, IgA, and IgD), which increases flexibility, and CH2
and CH3 domains.
IMMUNOGLOBULIN CONSTANT REGION
The specific binding interactions that occur between the
immunoglobulin variable region and the antigen may be
sufficient to block microbial infectivity or neutralize toxins.
However, the ability to eliminate pathogens is mediated by
the Fc portion of the molecule. The Fc regions of antigen-
antibody complexes are made accessible to serum factors or
to cytotoxic and phagocytic cells that mediate the destruction
and removal of pathogens. There are two ways in which the Fc
region directs the destruction of pathogens: activation of the
complement cascade, and engagement of Fc-specific recep-
tors on effector cells such as monocytes, macrophages, neu-
trophils, and natural killer (NK) cells. In mice and humans,
there are five different types of H-chain constant regions, or
isotypes, designated IgM (μ), IgD (δ), IgG (γ), IgA (α), and
IgE (ε); each is encoded by a distinct constant region gene
segment present in the H-chain locus. Each immunoglobulin
molecule is capable of specific effector functions, depending
on its H-chain constant region. The number of CH domains,
presence of a hinge region to increase flexibility between Fab
regions, serum half-life, ability to form polymers, complement
activation, and Fc receptor binding vary among isotypes.
Characteristics of the different immunoglobulin H-chain iso-
types are presented in Table 10-1.2-4 These isotypes may also
differ in the intracellular signaling they initiate when bound
by antigen in their membrane-associated form.
Immunoglobulin M
IgM is the first isotype generated in developing B cells and
the first antibody secreted during a primary immune response.
The secreted form of IgM exists mainly as a pentamer. IgM
also exists as a hexamer, which represents approximately 5%
of total serum IgM.5 Pentameric IgM is linked by a molecule
called the J chain, whereas the hexameric form is not. IgM
antibodies usually exhibit low affinity for antigen because
the process that increases antibody affinity for a particular
antigen (affinity maturation) has not yet been initiated dur-
ing the early stages of a primary immune response. How-
ever, polymeric IgM displays high avidity for antigen. The
presence of multiple Fab regions makes the IgM molecule
suitable to bind large, multimeric antigens. IgM is found
predominantly in serum but is also present in mucosal secre-
tions and breast milk.
The Fc region of IgM (as well as some of the IgG isotypes)
is a potent activator of the classic complement pathway.3
The complement cascade is composed of a series of enzymes
that, on activation, mediate the removal and lysis of invad-
ing organisms. In general, antibody molecules bound to
antigen can activate the classic complement pathway. Depo-
sition of antibody molecules or complement components on
the surface of the antigen facilitates phagocytosis. Proteins,
such as antibody and complement, that enhance phagocy-
been activated, monocytes, macrophages, or neutrophils
engulf opsonized particles through specific receptors present
on phagocytic cells (CD21) that recognize fragments of the
C3 complement component. Activation of the complement
pathway also results in the generation of the membrane
attack complex, which is composed of late complement
components and directly lyses C3-opsonized pathogens.
Immunoglobulin G
IgG is the most common isotype found in serum. IgG anti-
bodies are usually of higher affinity than IgM antibodies and
predominate in a secondary or memory immune response.
There are four subclasses of IgG in humans: IgG1, IgG2,
IgG3, and IgG4. All IgG subclasses exist as monomers. Each
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION 179

Table 10-1  Properties of Human Immunoglobulin (Ig) Isotypes

Characteristic IgM IgG IgA IgE IgD
Valency Pentamer,   Monomer Dimer (IgA2),   Monomer Monomer
hexamer monomer (IgA1)
CH domains 4 3 3 4 3
Serum values (mg/mL) 0.7-1.7 9.5-12.5 1.5-2.6 .0003 .04
Serum half-life (days) 5 23 6 2.5 3
Complement activation   Yes Yes No No No
(classic)
Complement activation   No Yes Yes No Yes
(alternative)
FcR binding No Yes Yes No Yes
FcR-mediated phagocytosis No Yes Yes No No
Antigen-dependent cell-  No Yes No No No
mediated cytotoxicity
Placental transfer No Yes No No No
Presence in mucosal   Yes No Yes No No
secretions

of the four subclasses appears to predominate in different
immune responses. IgG1 and IgG2 antibodies are observed in
responses to polysaccharide antigens; IgG1, IgG3, and IgG4
participate in immune responses to viral and protein anti-
gens; and IgG4 also participates in responses to nematodes.6
IgG1 and IgG3 are more potent activators of the clas-
sic complement pathway than are IgG2 and IgG4. Several
immunoglobulin isotypes, including IgG1, are capable of
initiating the alternative complement pathway (see also
Chapter 19). The mechanisms by which antibody activates
the alternative pathway are poorly understood, but specific
binding sites for the C3 complement component have been
identified on some immunoglobulin isotypes.7,8
All IgG subclasses engage specific Fc gamma receptors
(FcγRs) present on macrophages, neutrophils, and NK cells
to mediate phagocytosis of antibody-coated antigens and
to initiate antibody-dependent cell-mediated cytotoxicity.
The FcγRs on phagocytic cells, such as monocytes, macro-
phages, and neutrophils, mediate the removal of immune
complexes.4 Engagement of FcγRs with immune complexes
results in receptor activation. This, in turn, stimulates
phagocytic cells to ingest opsonized antigens and destroy
them in the phagosome compartment. The FcγRs on NK
cells mediate the killing of antibody-coated cells by the
antibody-dependent cell-mediated cytotoxicity pathway,
resulting in the release of granules that contain perforin, a
pore-forming protein, and enzymes known as granzymes that
induce programmed cell death (apoptosis) of target cells.9
Another important role of IgG is that it is the only
source of maternal antibodies for a developing fetus during
pregnancy. The transcytosis of maternal IgG antibodies into
the fetal blood supply is mediated by two other Fc receptors,
FcRn and FcRγIIB2.10,11 FcRn expressed on endothelial
cells regulates the half-life of serum IgG by blocking IgG
catabolism.10
Immunoglobulin A
There are two subclasses of IgA in humans: IgA1 and IgA2.3
IgA1 exists mainly as a monomer and is present in serum;
IgA2 exists as a dimer linked by the J chain and is the
p­ redominant isotype in saliva, tears, colostrum, breast milk,
and respiratory and vaginal secretions. Secretory IgA (and
IgM) is produced by B cells in the lamina propria, which
is beneath the mucosal lining of polarized epithelial cells.
Specific receptors for secretory immunoglobulin, termed
polymeric immunoglobulin receptors, are expressed on the
basolateral surface of epithelial cells and mediate the trans-
port of immunoglobulin polymers across the epithelium.12
The polymeric immunoglobulin receptor binds dimeric
IgA with higher affinity than IgM. During transport, the
polymeric immunoglobulin receptor undergoes proteo-
lytic cleavage to generate a fragment called the secretory
component. Before transcytosis is complete, the IgA dimer
associates with the secretory component, which renders it
resistant to enzymatic degradation.
It has been suggested that secretory IgA plays an impor-
tant role in protecting against foreign organisms by directly
blocking infection and neutralizing toxins in the mucosa.
IgA is unable to activate complement through the classic
pathway, but IgA1 can activate the alternative complement
pathway.8 Patients with IgA deficiency have reduced lev-
els of both serum and secretory IgA, and at least half these
patients experience increased respiratory and diarrheal
infections.13 An increase in autoimmune disorders has also
been observed in patients with IgA deficiency. An IgA-
specific FcαR has been identified. The physiologic role of
this receptor is not well understood, but there is evidence
that the FcαR expressed on monocytes, macrophages, neu-
trophils, and eosinophils mediates the phagocytosis of IgA-
coated pathogens.14
Immunoglobulin E
IgE exists as a monomer. Only a small amount of IgE is
detectable in serum. IgE triggers immune responses associ-
ated with allergic reactions.6 The Fc region of IgE interacts
with the high-affinity IgE Fc receptor, FcεR, which is found
on the surface of mast cells and basophils.4 When multi-
meric antigen cross-links the variable region of IgE mol-
ecules bound to FcεR molecules, mast cells and basophils
degranulate and release vasoactive molecules ­ associated
 180 DIAMOND  |  B Cells
with anaphylaxis, such as histamine, prostaglandin D2, and
leukotrienes. IgE has been implicated in the protection
against parasitic infections, because cross-linking of FcεR
results in the activation of mast cells, which are involved
in immune responses to parasites and help skew the T cell
response to parasites to a Th2 cytokine profile through the
production of IL-4 (see Chapter 9).
Immunoglobulin D
Little is known about the function of IgD. The membrane
form of IgD is coexpressed with the membrane form of IgM.
Surface expression of IgD occurs during the later stages of B
cell development (see “B Cell Development”). The role of
IgD in a humoral immune response is unclear. Unlike the
other four H-chain isotypes, IgD is not secreted from acti-
vated B cells and is therefore unlikely to play a protective
role against infection. Studies performed with IgD trans-
genic mice suggest that surface IgD protects against toler-
ance induction because autoreactive IgM+, IgD+ B cells, but
not IgM+, IgD– B cells, are resistant to deletion by antigen.15
Targeted disruption of the IgD gene in mice, however, does
not appear to interfere with normal immune functions.
Although the total number of B cells is slightly reduced and
affinity maturation is delayed, there is no evidence of auto-
reactivity.16,17
LIGHT CHAINS
There are two distinct L-chain polypeptides designated
kappa (κ) and lambda (λ). L chains contain a variable
region and a single constant region domain. Amino acid
residues in the L-chain variable region interact with resi-
dues in the H-chain variable region to create the antigen-
binding cleft. Even though there are two L-chain isotypes,
there is no known function associated with the L-chain
constant region. The κ chain is used more often than the λ
chain in human (65%) and mouse (95%) immunoglobulin
molecules.18
IMMUNOGLOBULIN VARIABLE REGION
To allow the recognition of a virtually unlimited number
of antigens, immunoglobulin molecules must be generated
that possess different antigenic specificities. The molecular
basis of immunoglobulin diversity is now well understood.
Immunoglobulin H- and L-chain genes are encoded by dis-
tinct gene segments residing on separate chromosomes; the
H-chain locus is on human chromosome 14, the κ-chain
locus is on chromosome 2, and the λ-chain locus is on
chromosome 22. Individual genes encode the variable and
constant regions of an immunoglobulin molecule. A lim-
ited number of separate H- and L-chain variable region
gene segments undergo somatic rearrangement to generate
a multitude of immunoglobulin molecules bearing differ-
ent antigenic specificities.19 The H-chain variable region is
composed of a variable (VH), a diversity (DH), and a joining
(JH) gene. The L chain is composed of either Vκ and Jκ or Vλ
and Jλ genes; it does not contain D genes. Immunoglobulin
genes can exist as functional gene segments, which can be
expressed as H- or L-chain polypeptides, or as pseudogenes,
which are unable to be expressed. The human H-chain locus
contains approximately 51 VH, 30 DH, and 6 JH functional
genes. The κ-chain locus contains approximately 32 Vκ
genes and 5 Jκ functional genes; the λ-chain locus contains
29 Vλ genes and 4 Jλ functional genes.20
Variable Region Gene Rearrangement
During VDJ rearrangement, different VH, DH, and JH or VL
and JL gene segments are randomly combined to generate a
large number of different immunoglobulin molecules (Fig.
10-3). VDJ recombination occurs in the absence of antigen
stimulation. The molecular mechanism that regulates VDJ
rearrangement is activated during B cell maturation in pri-
mary lymphoid tissue (see “B Cell Development”). Specific
DNA sequences that flank the V, D, and J gene segments are
recognized by components of the recombination machinery.
These highly conserved sequences that compose the recom-
bination signal sequences are either 7 base pairs (heptamer)
or 9 base pairs (nonamer) in length, followed by DNA
spacers that are 12 or 23 base pairs in length.21 Specific
enzymes termed recombination-activating gene 1 (RAG-1)
and recombination-activating gene 2 (RAG-2) initiate
VDJ gene rearrangement at the H-chain or L-chain loci by
generating double-stranded DNA breaks at recombination
signal sequence sites. The cleaved V, D, and J segments are
joined by a complex of several polypeptides that includes
Ku70, Ku80, and DNA-dependent protein kinase.22 The
same recombination machinery also mediates the somatic
rearrangement of gene segments for the T cell receptor (see
Chapter 9).
Variable Region Diversity
The random recombination that occurs among V, D, and
J gene segments can generate a diverse immunoglobulin
repertoire without the need for a large number of germline
H- and L-chain genes. During H-chain recombination,
nucleotides may be added at VHDH and DHJH junctions by
the enzyme terminal deoxynucleotidyl transferase. These
non-germline-encoded sequences are known as N addi-
tions. As long as these nucleotide changes do not disrupt
the reading frame or lead to the incorporation of premature
stop codons, the random addition of N sequences increases
the diversity of the amino acid sequence. Further, imprecise
ligation at the coding junctions may result in the loss of
nucleotides, thereby altering the amino acid sequence. The
expression of different H- and L-chain combinations also
contributes to the creation of diverse binding specificities.3
A process known as somatic mutation can further
increase variable region diversity.23 Somatic mutations are
point mutations that occur at a high frequency (approxi-
mately 10−3 per base pair per cell division) in the variable
region of H- and L-chain genes. Unlike the other genetic
events that increase diversity in developing B cells, somatic
mutations occur in mature, antigen-stimulated B cells pres-
ent in secondary lymphoid tissue in discrete regions called
germinal centers (see “B Cell Activation”). Nucleotide
changes that occur in the complementarity-determining
region can increase the affinity of the immunoglobulin
variable region for a particular antigen or, in some cases,
change the specificity from one antigen to another.24 Anti-
gen selection results in more replacement mutations in the
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION 181

VH1 VH2 VH3 VHn DH1 DH2 DH3 D Hn JH1 JH2 JH3 JHn µ δ γ3 γ1 α1 γ2 γ4 ε α2

VH3 D H2 JH3 µ δ γ3 γ1 α1 γ2 γ4 ε α2

D H2 D H3 JH2 J H3
VH3 D H2 JH3 µ VDJ-Cµ transcript

D H3 JH2

D H2 JH3

DH2 JH3

Figure 10-3  VDJ recombination at the immunoglobulin gene locus. VDJ recombination at the heavy (H)-chain locus is depicted at the top. A single
VH gene segment randomly recombines with a DH and JH gene segment residing on the same chromosome. Following VHDHJH recombination, a tran-
script containing the IgM H-chain constant region gene (Cμ) is generated. The inset represents an example of VDJ recombination occurring between
a single DH and JH gene segment. The white squares represent the heptamer, and the black squares represent the nonamer recombination recognition
sequences. Following recognition and cleavage of these sequences, the coding junctions of the rearranged DH and JH gene segments are ligated. A VH
gene segment then recombines with the rearranged DHJH segment. Light (L)-chain rearrangement is mediated by the same mechanism.

complementarity-determining regions and fewer in the
framework regions.
B CELL DEVELOPMENT
The development of undifferentiated hematopoietic stem
cells into mature B lymphocytes is divided into two phases:
the early stages of B cell lymphopoiesis take place in primary
lymphoid tissue, whereas differentiation into B cells that
either secrete large quantities of immunoglobulin or persist
as long-lived memory cells occurs in secondary or peripheral
lymphoid tissue. Primary lymphoid tissue includes the fetal
liver and the bone marrow. One of the major events associ-
ated with B cell lymphopoiesis in these tissues is immuno-
globulin gene rearrangement. Mature, naive B cells exit the
primary lymphoid tissue and home to secondary lymphoid
tissue, such as the spleen, lymph nodes, tonsils, and Peyer’s
patches of the intestine. It is at these sites where B cells
interact with foreign antigens and specific humoral immune
responses are activated.
SITES OF B CELL LYMPHOPOIESIS
Hematopoiesis begins during fetal development and persists
throughout adulthood.25 Studies of mouse embryonic devel-
opment show that the first anatomic sites of hematopoiesis
are the yolk sac and the aorta-gonad-mesonephros begin-
ning at day 7 post conception. At day 12, hematopoiesis
begins in the fetal liver, and before birth, hematopoesis
takes place in the bone marrow, which becomes the exclu-
sive site of hematopoiesis after birth and throughout adult-
hood. Chemokines and adhesion molecules are believed to
direct the temporal and spatial changes in hematopoiesis
that occur during embryogenesis. Data suggest that the fetal
liver and bone marrow are able to give rise to all B cell sub-
sets, although this is still an area of controversy (see “B Cell
Subsets”).
B CELL SUBSETS
Two subsets of B lymphocytes exist: B1 and B2. The B1 sub-
set is produced earlier in ontogeny than are the B2 cells; the
B2 subset includes “conventional” B cells that are replen-
ished from precursor cells in the bone marrow.
B1 Cells
B1 cells represent a minor population of B cells that reside
predominantly in the pleural and peritoneal cavities. B1
cells can be distinguished from B2 cells on the basis of their
expression of specific cell surface molecules and the types
of humoral immune responses in which they participate
(Table 10-2). B1 cells are subdivided into B1a and B1b
cells. One of the characteristic markers expressed on B1a
cells is constitutive expression of the CD5 surface molecule,
which is absent on B1b cells and can be induced on B2 cells.
The CD5 molecule is also a common marker for chronic
lymphocytic leukemia, which is a B cell-derived tumor.26
Data from mouse studies suggest that B1a cells provide a
preexisting source of natural antibody against pathogens,
whereas B1b cells produce protective antibodies in response
to foreign antigens. There is strong evidence that CD19
and CD21, which form the B cell coreceptor complex, are
important for B1 maintenance, because mice deficient in
the genes that encode this complex have a reduced number
of B1 cells.27
 182 DIAMOND  |  B Cells
Table 10-2  Characteristics of B Cell Subsets
Characteristic B1a B1b
Surface IgM High High
Surface IgD Low Low
CD5 + –
CD21 – –
CD23 – –
CD11b/CD18* + +
Bone marrow progenitors – +
Self-renewal capacity + +
Response to T cell–  + + +/–
independent antigens
Response to T cell–  +/– +/–
dependent antigens
Predominant isotype IgM IgM
Anatomic locations Peritoneum,   Peritoneum,   Spleen, lymph nodes,   Spleen, tonsils
pleura, spleen pleura, spleen
Ig, immunoglobulin.
*B1 cells in the spleen do not express CD11b/CD18.
The origin of B1 cells has been debated, but data sug-
gest that most B1 cells arise predominantly from a distinct
developmental lineage present in the fetal liver and, to a
lesser extent, from precursor cells present in the postnatal
bone marrow. Some may arise in the adult bone marrow as
well.28 The immunoglobulin repertoire of B1 cells appears
to be more restricted than that of B2 cells. Another feature
of B1 cells is that terminal deoxynucleotidyl transferase is
not expressed in this subset, which is consistent with the
absence of N nucleotide additions at the VHDH and JHDH
junctions observed in H-chain genes of B1 cells.
B2 Cells and the Bone Marrow Microenvironment
In humans and mice, B2 cells first arise in the fetal liver, and
after birth, they are continually renewed throughout adult-
hood. Self-renewing pluripotent progenitor cells present in
the bone marrow, which give rise to all blood cell types,
generate B2 cells. The microenvironment of the bone mar-
row stroma—which consists of several different cell types,
including reticular cells, endothelial cells, macrophages,
osteoblasts, and adipose tissue surrounded by extracellu-
lar matrix components—provides the environmental cues
required for B2 cell development.
Hematopoietic stem cells fail to differentiate in vitro in
the absence of cultured stromal cells. Thus, the reconstitu-
tion of B cell differentiation in vitro using stromal cell lines
has led to the discovery of a number of cell surface molecules
and soluble factors that are required for B cell lymphopoiesis.
Some of these cell surface interactions include the follow-
ing: vascular adhesion molecule 1 (VCAM-1), expressed on
endothelial cells and macrophages, with very late antigen 4
(VLA-4), expressed on B cell progenitors; intercellular
adhesion molecule, expressed on stromal cells, with VLA-5,
expressed on B cell progenitors; hyaluronate, present on
stromal cells, with CD44, expressed on B cell progenitors;
and neural cell adhesion molecule (NCAM), expressed on
stromal cells, with the membrane proteoglycan syndecan,
Follicular Marginal Zone
Low High
High Low
– –
+ ++
+ –
– –
+ +
– –
+
+ +/–
IgG IgM
Peyer’s patches,  
tonsils, peripheral blood
expressed on B cell progenitors.20,29 Although many of the
soluble factors important in B cell lymphopoiesis have yet
to be definitively identified, there is evidence that CXC-
chemokine ligand 12 (CXCL12), fms-related tyrosine kinase 3
ligand (Flt3L), stem cell factor (SCF), receptor activator
of nuclear factor κB ligand (RANKL), interleukin (IL)-7,
and sex hormones play a role in human B cell develop-
ment.30,31 As B cells begin to express surface IgM molecules,
they migrate toward the center of the bone marrow cavity
and become less dependent on direct interaction with the
stroma.
BONE MARROW STAGES
The stages of B cell development are defined, in part, by the
differential expression of surface markers and genes associ-
ated with immunoglobulin gene rearrangement. In the labo-
ratory, these markers are identified by flow cytometry with
specific antibodies or by reverse transcription polymerase
chain reaction. Many of the cell surface markers used to
define particular B cell subsets mediate important signal
transduction events associated with maturation and activa-
tion, whereas others have no known function at present.
Because the events associated with B cell development are
not strictly linear, the nomenclature and classification of
particular stages vary slightly among the different laborato-
ries working in this field. For simplicity, we have divided the
stages of B cell lymphopoiesis into pro-B, pre-B, immature,
transitional, and mature cell stages (Table 10-3).
Pro-B Cell Stage
In humans, the earliest hematopoietic stem cells express the
CD34 surface marker. The progression from CD34+ cells to
B cell progenitors is regulated by specific transcription fac-
tors, including PU.1, Ikaros, E2A, EBF, PAX-5, and Lef-1.32
Pro-B cells continue to express CD34 as well as the surface
markers CD19, CD10, CD20, CD21, CD22, CD38, CD40,
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION 183

Table 10-3  Human B Cell Maturation Markers during Early B Cell Development
Transitional Transitional
Marker HSC Pro-B Pre-B Immature Type 1 Type 2
CD34 + + – – – –
CD19 – + + + + +
CD10 – + + + + +
CD20 – + + + + +
CD21 – – – – – +
CD22 – – + + + +
CD23 – – – – – +
CD38 – + + + + +
CD40 – + + + + +
CD45 – + + + + +
RAG-1 – + + +/– +/– +/–
RAG-2 – + + +/– +/– +/–
Tdt – + + – – –
Igα – + + + + +
Igβ – + + + + +
Heavy chain – – (DH-JH) + (VH-DH-JH) + + +
Pre-BCR – – + – – –
Surface IgM – – – + + +
Surface IgD – – – – – +
Light chain – – + (Vк-Jк Vλ-Jλ) + + +
BCR, B cell receptor; HSC, hematopoietic stem cell; Ig, immunoglobulin.

and CD45RB and the major histocompatibility complex
(MHC) class II. The surface immunoglobulin accessory mol-
ecules Igα and Igβ are also expressed during the pro-B stage.
Pro-B cells are dependent on interactions with endothelial
cells present in the stroma. The VLA-4 integrin receptor
and the CD44 molecules, which mediate adhesion to stro-
mal cells, are highly expressed at this stage and are believed
to be important for continued development.20 Pro-B
cells also express high levels of Bcl-2. As will be discussed
later, this molecule plays a pivotal role in protecting against
apoptosis.
At the onset of pro-B cell development, the variable gene
segments on both H- and L-chain loci are in the unrear-
ranged germline configuration. Before VDJ rearrangement,
the genes required for recombination (e.g., those encoding
RAG-1, RAG-2, terminal deoxynucleotidyl transferase,
and the Ku complex) are expressed. After expression of
the recombination machinery, a DH gene segment on one
H-chain chromosome rearranges with a JH gene segment
residing on the same chromosome (see Fig. 10-3), often with
the inclusion of nontemplate nucleotides at the junction.
Pre-B Cell Stage
During the pre-B cell stage of development, a VH gene rear-
ranges to the DHJH gene fragment. Completion of VHDHJH
gene rearrangement leads to the generation of an H-chain
transcript that contains the IgM constant region (Cμ), the con-
stant region gene most proximal to the variable region genes
on the chromosome (see Fig. 10-3). The different H-chain
transcripts encode either a transmembrane molecule or a
secretory molecule, determined by alternative splicing. In
pre-B cells, however, only transmembrane Cμ is produced.
A critical developmental checkpoint in B cell lym-
phopoiesis is the cell surface expression of the pre-B cell
receptor (pre-BCR) complex at the pre-B cell stage. Before
L-chain gene rearrangement, the transmembrane form of
the Cμ polypeptide expressed in pre-B cells associates with
two other polypeptides, Vpre-B and lambda 5 (λ5), which
form the surrogate L chain.33,34 The genes for these mole-
cules are located on human chromosome 22, which also car-
ries the λ-chain locus. There are three separate genes for λ5,
known as 14.1, 16.1, and Fλ1; however, only 14.1 encodes
a protein. A disulfide-linked complex is formed between
the Cμ and the surrogate L chain, which is expressed on
the cell surface in association with the accessory molecules
Igα and Igβ. It is believed that signals transmitted by the
surface pre-BCR complex play an important role in B cell
maturation. Surface expression of the pre-BCR transduces
a signal to the developing pre-B cell that VDJ rearrange-
ment was successful and halts recombination of the second
H-chain allele. This process of allelic exclusion ensures that
all immunoglobulin molecules generated within each B cell
are identical and have the same antigenic specificity. If rear-
rangement of the first allele is nonproductive (e.g., genes
contain a premature stop codon and cannot be translated
into a full-length polypeptide), the absence of pre-BCR sig-
naling permits rearrangement of the second H-chain allele.
If the second rearrangement also results in a nonproductive
H-chain molecule, the absence of pre-BCR-mediated sig-
nal induces apoptosis. Because it has been estimated that
about two thirds of VDJ rearrangements are nonproduc-
tive, the inability to express the pre-BCR complex ensures
that B cells without a productive H chain will not undergo
further differentiation. Targeted disruption of genes encod-
ing the pre-BCR complex, such as the IgM transmembrane
 184 DIAMOND  |  B Cells
constant region domain, λ5, or the Igα and Igβ accessory
molecules, results in a profound decrease in developing B
cells. Also, defects in the adapter molecule BLNK or the
tyrosine kinase Btk block pre-B cell maturation.
After expression of the H-chain polypeptide, L-chain
rearrangement occurs. Because terminal deoxynucleotidyl
transferase expression is reduced at this stage, L chains do
not usually contain N sequences at the VLJL junction. Igα,
Igβ, CD10, CD19, CD20, CD21, CD22, CD38, CD40,
CD45RB, and MHC class II expression continues through-
out the pre-B cell stage; the antiapoptotic molecule Bcl-XL
is upregulated in pre-B cells, whereas expression of Bcl-2 is
downregulated.
Immature B Cell Stage
L-chain gene rearrangement marks the transition from the
pre-B cell stage to the immature B cell stage. The rearranged
transmembrane Cμ and L-chain polypeptides assemble into
functional immunoglobulin molecules. At this stage, surface
immunoglobulin, which is also known as the B cell recep-
tor (BCR), is expressed. It is believed that surface expres-
sion of the BCR on immature B cells transduces signals that
enforce allelic exclusion at the L-chain locus and downregu-
late expression of the RAG genes. As discussed later in this
chapter, one of the functions of surface immunoglobulin is
to mediate negative selection of autoreactive B cells arising
in the bone marrow.
Despite the necessity to express a single H- and L-chain
molecule in each B cell, circumstances exist that permit the
rearrangement of a second H- or L-chain allele. Autoreac-
tivity generated during B cell development in the bone mar-
row can arise by the random expression of different H and
L chains. One of the mechanisms that prevents the survival
of potentially autoreactive B cells is a process called recep-
tor editing. Re-expression of the RAG genes in immature
B cells permits rearrangement and expression of the second
H- or L-chain allele in autoreactive B cells, thus altering
antibody specificity. Expression of the antiapoptotic mol-
ecule Bcl-XL has been implicated in receptor editing.35
This molecule may prevent apoptosis of autoreactive B cells
and extends the window for receptor editing to occur. The
mechanism of receptor editing and its role in the regula-
tion of autoreactivity are discussed later in more detail (see
“Negative Selection”).
Transitional B Cell Stage
Murine studies have demonstrated that the later stages of B
cell maturation occur in the spleen. Immature B cells dif-
ferentiate into a phenotypically distinct population known
as transitional type 1 (T1) B cells and migrate to the spleen
to undergo further maturation to transitional type 2 (T2)
B cells. It has not been clearly established whether T2 B
cells are direct precursors of mature B cell subsets or whether
they undergo additional maturation steps to generate mature
B cell precursor cells. The B cell survival factor known as
B cell–activating factor of the tumor necrosis factor family
(BAFF; also known as Blys) appears to play an important
role in T2 B cell development, because blockade of BAFF
leads to arrested development at the T2 stage.36 During the
transitional B cell stage, an IgD (Cδ) H chain is coexpressed
that bears the same variable region as the previously rear-
ranged Cμ H chain and associates with the same L chain.
The switch from IgM+, IgD– to IgM+, IgD+ occurs at the
level of transcription. A long transcript containing both
Cμ and Cδ genes undergoes alternative splicing to generate
both IgM and IgD H chains. The existence of T1 and T2 B
cell subsets has also been identified in humans, and these
display similar properties as the murine subsets.37
Mature B Cell Stage
The final stages of maturation that occur in the spleen and
give rise to naive B cell subsets have not been fully eluci-
dated, but the prevailing theory is that T2 B cells give rise
to the mature B2 cell populations—follicular and marginal
zone B cells. These mature subsets are both phenotypically
and functionally distinct and thus respond to different types
of antigens (see “B Cell Activation”).
Follicular B cells are IgM+, IgD+, CD23+ and display inter-
mediate expression of CD21. The follicular B cell is the most
predominant B cell subset in the spleen, and it recirculates
throughout the body. As discussed later, follicular B cells
require the help of antigen-specific T cells to undergo activa-
tion and further maturation into antigen-responsive B cells.
Marginal zone B cells are IgM+, IgD–, CD23– and dis-
play high expression of CD21. The population is restricted
mainly to the marginal sinuses of the spleen. This B cell
subset responds to a distinct set of antigens and does not
require antigen-specific T cell help. Unlike follicular B cells,
marginal zone B cells are absent in humans younger than
2 years; therefore, these young children are unable to gener-
ate immune responses to certain antigens.
GERMINAL CENTER STAGE
Following T cell–dependent B cell activation, B cells
undergo further maturation into germinal center cells. Ger-
minal center B cells form the germinal centers present in
secondary lymphoid tissue, which are the sites where the
final stages of T cell–dependent B cell maturation occur.
Following activation, B cells mature into centroblasts. Cen-
troblasts undergo rapid proliferation, and it is at this devel-
opmental stage that the process of somatic hypermutation
and isotype class switching occurs. Centroblasts develop into
centrocytes, which are quite susceptible to programmed cell
death unless they receive survival signals from specialized
helper T cells and follicular dendritic cells (FDCs) present
in the germinal center. Cells that emerge from the centro-
cyte stage develop into effector memory B cells or plasma
cells. Development into memory or plasma cells is regulated
by different sets of transcription factors. Memory B cells
leave the germinal center and recirculate throughout the
body, where they can undergo antigen-dependent activa-
tion. Plasma cells home to the bone marrow and secondary
lymphoid tissue such as lymph nodes and tonsils, where they
secrete copious amounts of antigen-specific antibody.
Memory B Cells
Memory B cells typically express immunoglobulin genes that
have undergone isotype class switching and possess somatic
mutations. It is believed that the CD40-CD40L interaction
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
directs centrocytes to undergo maturation into long-lived
memory B cells. The exact life span of memory B cells is
unknown, but it has been postulated that these B cells may
persist throughout the lifetime of the host.38
Memory B cells circulate throughout the body in a qui-
escent state until specific antigen is re-encountered and
triggers a potent secondary immune response. Much less
antigen is required to activate a secondary response than
a primary response. Further, a secondary immune response
is generated more quickly than a primary one. Memory B
cells ingest antigen and express peptide–MHC class II frag-
ments. After antigen presentation of peptide to helper T
cells, memory B cells are activated to undergo expansion
and maturation, giving rise to a second wave of plasma cells
or memory B cells.
Follicular, marginal zone, and B1 cells can generate
memory responses. Follicular B cells follow the pathway
outlined earlier in which T cells drive their expansion and
differentiation into centroblasts, followed by competition
for antigen as centrocytes. Evidence for T cell–independent
memory responses also exists.39 Marginal zone B cells play
an important role in immunity against blood-borne patho-
gens to generate memory in response to bacterial compo-
nents.40 B1b cells induce memory responses to carbohydrate
antigens.41 Despite the ability of marginal zone and B1b
subsets to mediate a memory response, unlike follicular
B cells, these T cell–independent subsets do not undergo
somatic mutation or affinity maturation. Thus, the preim-
mune repertoire of marginal zone and B1 cells contributes
to the “natural” or “innate” memory response.
Plasma Cells
The B cell maturation cascade ends with the generation of
plasma cells, which are factories for the secretion of solu-
ble antibody molecules. B cells undergoing differentiation
into plasma cells exit the lymphoid follicles and migrate to
extrafollicular regions of secondary lymphoid tissue or to the
bone marrow, where the final stage of plasma cell maturation
occurs. There is evidence that IL-5 and IL-6 help induce
plasma cell differentiation, whereas engagement of CD40-
CD40L molecules blocks this differentiation pathway. The
transcriptional repressor known as B lymphocyte–induced
maturation transcription factor (Blimp-1) plays a critical
role in the differentiation into plasma cells.42 Plasma cells
are terminally differentiated and have a finite life span,
which can be quite long.43 Studies performed with immu-
nized mice demonstrate that long-lived plasma cells gener-
ated within germinal centers have a half-life of more than
100 days; short-lived plasma cells that arise from extrafol-
licular B cells have a half-life of less than 10 days.44
B CELL HOMING
Lymphoid tissues contain the microenvironment necessary
for the homing, retention, and activation of B cells. As men-
tioned earlier, several different types of secondary lymphoid
tissue exist, including the spleen, lymph nodes, and mucosa-
associated lymphoid tissue (e.g., Peyer’s patches, appendix,
tonsils). Secondary lymphoid tissues are well adapted to trap
circulating antigen. Peripheral lymphoid tissue contains
specialized antigen presenting cells known as dendritic cells
185
(see also Chapter 8). The Peyer’s patches of the intestines
collect foreign antigen in specialized epithelial cells known
as M cells. Even though peripheral lymphoid tissues vary in
structure and cellular organization, they all possess antigen
presenting cells and B cell–containing follicles surrounded
by T cell–rich zones. As explained in the following sec-
tion, antigen, T cells, and dendritic cells are required for
B cell activation and differentiation into immunoglobulin-
secreting plasma cells or memory B cells.
B1 cells typically home to the peritoneal and pleural
cavities and, to a lesser extent, the spleen. Follicular B cells
enter the peripheral circulation by passing through the
endothelial lining of the sinusoids of secondary lymphoid
tissue and recirculate throughout the follicles of secondary
lymphoid tissues. Because follicular B cells require anti-
gen-specific T cell help for activation, the localization of
this subset near a T cell zone of B cell follicles facilitates
the chance encounter between antigen-specific B cells and
cognate T cells. Conversely, marginal zone B cells respond
to antigen without the help of cognate T cells. They local-
ize exclusively in the spleen of mice and in the spleen and
tonsils of humans owing to interactions between adhesion
molecules and chemokine receptors that sequester marginal
zone B cells within the marginal sinuses. The location of
marginal zone B cells makes them well suited to capture
blood-borne antigens.
As mentioned earlier, chemokines play a major role in
the formation of secondary lymphoid tissue and the localiza-
tion and retention of B cells in environmental niches.45,46
The chemokines CCL19 and CCL21, which bind to the
CCR7 receptor on T cells and dendritic cells, mediate the
formation of the T cell zones where cognate T cell–B cell
interactions occur. The CXCR5 molecule expressed on B
cells mediates the migration to follicles in response to the
chemokine CXCL13, which is regulated in turn by the
cytokine lymphotoxin a, made by B cells. In mucosal tis-
sue, CCL20 is responsible for the recruitment and retention
of CCR6-positive B cells, and CCL25-CCR9 interactions
play a role in localizing plasma cells to the small intestine.
Dysregulation of the chemokine response is thought to play
a role in the formation of ectopic lymphoid aggregates that
resemble secondary lymphoid tissue and occur in several
inflammatory diseases, including rheumatoid arthritis and
Sjögren’s syndrome.
Chemokines also play an important role in germinal cen-
ter responses. The chemokine CXCL12 retains centroblasts
in the dark zone during the process of somatic hypermuta-
tion and isotype class switching. CXCL13 regulates migra-
tion to the light zone, where survival and selection events
are mediated by interactions with CXCR5-expressing fol-
licular helper T cells and FDCs. In addition, CXCL12 pro-
motes the migration of plasmablasts to the bone marrow,
where they undergo further development into long-lived
plasma cells.
B CELL ACTIVATION
REGULATION OF B CELL ACTIVATION
Engagement of surface immunoglobulin by antigen triggers a
series of cellular events that regulate B cell proliferation and
differentiation. Receptor cross-linking leads to relocation of
 186 DIAMOND  |  B Cells

the BCR to microdomains known as lipid rafts, which leads
to the rapid activation of proximal mediators of the BCR sig-
nal transduction pathway.47 This results in the activation of
second messengers such as phospholipase C, phosphatidylino-
sitol 3-kinase, and Ras pathways. Induction of these pathways
ultimately transmits signals to the nucleus, initiating new
gene expression. Depending on the type of signal delivered
and the stage of maturation, B cells can undergo either differ-
entiation into memory B cells and plasma cells or apoptosis.
Along with surface immunoglobulin, several other membrane
receptors modulate antigen-induced signal transduction.
POSITIVE REGULATORS
The BCR complex is composed of surface immunoglobulin,
along with the accessory molecules Igα and Igβ. The role
of surface immunoglobulin is to recognize foreign antigen;
the Igα and Igβ molecules are responsible for the induction
of signal transduction pathways required for B cell activa-
tion. The cytoplasmic domains of Igα and Igβ contain a
specific signaling motif known as the immunoreceptor tyro-
sine-based activation motif (ITAM). The ITAM amino acid
sequence contains two tyrosine residues that are critical for
signaling. After phosphorylation of these tyrosine residues,
the ITAM acts as a docking site to recruit other signaling
molecules.
The binding of antigen to surface immunoglobulin results
in BCR clustering on the cell surface and lipid raft formation.
This triggers a cascade that first leads to kinase activation,
followed by the activation of second messenger pathways.
Once the receptors are cross-linked, the BCR-associated
tyrosine kinases mediate phosphorylation of the Igα and Igβ
ITAM tyrosine residues (Fig. 10-4). Signaling through the
BCR leads to numerous events, such as B cell activation and
endocytosis of antigen-antibody complexes, B cell prolifera-
tion, B cell differentiation, and apoptosis. Many molecules
can modulate BCR signal transduction, either enhancing or
diminishing the signal transduced by antigen; B cell core-
ceptor complex (CD19/CD21/CD81/Leu-13), CD45, SHP-
1, SHP-2, SHIP, CD22, FcγRIIB1, CD5, CD72, PIR-B, and
PD-1 (see Fig. 10-4) determine the threshold for activation
as well as the strength of the BCR signal.
CD45
Receptor tyrosine kinases require activation by phosphory-
lation of specific tyrosine residues; they also require inacti-
vation. Their inactivation in resting B cells is maintained

Activation Inhibition

BCR
CD21 CD45 CD22 PIR-B CD72 PD-1 FcγR CD5
CD19 CD81 Leu-13 IIB-1
Igα Igβ

Blk I
I I Fyn T
T T Lyn A
A A M
M M
Lyn I I I I I
Vav Syk T T T T T SHP-1
Fyn and I I I I I
Lyn Btk M M M M M
SHP-1 SHP-1 SHP-1
SHP-1 SHP-2 SHIP
SHP-2
BLNK

PLC/PKC/Ras

P-Tyr
MAPK Tyr

Nucleus Gene expression

Figure 10-4  Molecules that regulate the activation state of B cells. Coligation of surface immunoglobulin results in tyrosine phosphorylation at spe-
cific tyrosine residues present in the immunoreceptor tyrosine activation motif (ITAM) of Igα and Igβ cytoplasmic domains. This occurs after the removal
of the inhibitory tyrosine residues of the B cell receptor (BCR)–associated cytoplasmic kinases such as Blk, Fyn, and Lyn, which is mediated by CD45. The
phosphorylated ITAMs recruit and activate the Syk and Btk kinases, which in turn activate a series of second messenger pathways (PLC, PKC, and Ras)
that result in the upregulation of genes required for B cell activation and survival. Coligation of the pre-BCR complex (CD19, CD21, CD81, and Leu-13)
results in phosphorylation of tyrosine residues residing in the cytoplasmic domain of CD19. Cytoplasmic kinases, including Vav, Fyn, and Lyn, become
activated and enhance the signaling mediated by the BCR. Following the activation of distal mediators of BCR signaling such as PLC, PKC, and Ras,
molecules of the MAPK pathway become activated and translocate to the nucleus to regulate gene expression. Signals mediated by CD22, PIR-B, CD72, 
PD-1, FcγRIIB1, and CD5 deliver negative signals that block the activation of distal molecules. Following phosphorylation of the immunoreceptor tyro-
sine inhibition motif (ITIM), present in the cytoplasmic tail of these molecules, the phosphatases SHP-1, SHP-2, and SHIP are recruited and activated.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
by the phosphorylation of specific inhibitory tyrosine resi-
dues. For B cell activation to occur, phosphate groups must
first be removed from the inhibitory tyrosine residues of the
BCR-associated Src tyrosine kinases by CD45. CD45 has
an extensive extracellular domain, with no clearly defined
ligand specificity, and a cytoplasmic domain that possesses
tyrosine phosphatase activity. Mice deficient for CD45 dis-
play a diminished response to antigen and have a decreased
number of mature B cells, indicating that some level of BCR
activation is required for the transition from immature to
mature B cells during lymphopoiesis.48 CD45 is generally
considered to be a positive regulator of BCR signaling, but it
can also act as a negative regulator by initiating a feedback
loop to limit the extent of BCR activation (see “CD22”).
CD19 and CD21
The B cell coreceptor complex is composed of CD19,
CD21, CD81, and an interferon-inducible molecule called
Leu-13.5 CD19 has also been shown to associate with surface
immunoglobulin.49 After antigen cross-linking of surface
immunoglobulin, specific tyrosine residues contained within
the CD19 cytoplasmic domain rapidly become phosphory-
lated. To date, the natural ligand for CD19 is not known. In
vitro studies have demonstrated that ligation of CD19 with
anti-CD19 antibody lowers the threshold required for BCR-
mediated B cell activation and enhances the proliferative
effect of anti-IgM treatment on B cells.50 A role for CD19
in B cell activation has been clearly defined in mice that
either are deficient in or overexpress CD19.27 The CD19
molecule is required for germinal center formation and
humoral responses to T cell–dependent antigens and pos-
sibly T cell–independent antigens.
The CD21 molecule serves as a receptor for cleavage
fragments of the C3 component of complement—iC3b,
C3dg, and C3d. Mice deficient in CD21 also have impaired
responses to T cell–dependent and T cell–independent anti-
gens and a defect in germinal center formation.27,51 A pro-
posed mechanism for the function is that co-cross-linking
of the BCR and CD21 with complement-coated antigen
triggers the activation of the cytoplasmic tail of CD19.
The functions of the remaining two components of the B
cell coreceptor complex, CD81 and Leu-13, have not been
characterized, although it has been suggested that these
molecules may mediate homotypic cell adhesion.
INTRACELLULAR KINASES AND DOWNSTREAM
PATHWAYS
The signal transduction events that occur after BCR cross-
linking are mediated by the subsequent recruitment and acti-
vation of intracellular kinases, including Lyn, Fyn, Btk, and
Syk. The most proximal event following BCR cross-linking
is the activation of Lyn, which results in the activation of
CD45 to remove the inhibitory phosphates on the ITAMs
of Igα and Igß; the activation of Lyn leads to the activa-
tion of Syk and Btk.52 There is evidence that ligation of
CD19 leads to recruitment and activation of Vav, phospha-
tidylinositol 3-kinase, Fyn, Lyn, and Lck.53 Subsequently,
the tyrosine kinases Syk and Btk interact with the phos-
phorylated ITAM and are activated by tyrosine phosphory-
lation. The phosphorylation of Syk triggers the activation of
187
phospholipase C, phosphatidylinositol 3-kinase, and Ras
pathways. The activation of Syk appears to be absolutely
critical for BCR-mediated signal transduction, because Syk-
deficient cell lines exhibit a loss of BCR-induced signaling.
Btk also appears to be required for the activation of second
messenger pathways. In patients with X-linked agamma-
globulinemia, a mutation in the Btk gene results in impaired
BCR signaling at the pre-B cell stage.52 As a consequence,
these patients have a greatly reduced number of mature B
cells and generate poor antibody responses. In mice, how-
ever, a mutation in Btk leads to a disease known as X-linked
immunodeficiency. B cell development is impaired at the
transitional T2 stage, and B cells that do go on to matu-
rity are unable to respond to certain T cell–independent
antigens.
Following recruitment and activation of the intracellu-
lar kinases, downstream pathways are initiated. Btk, Syk,
and the adapter molecule BLNK are required for the phos-
pholipase C gamma. This leads to breakdown of phosphati-
dylinositol 4-phosphate to DAG and IP3 to trigger calcium
release from intracellular stores and the subsequent trans-
location of nuclear factor of activated T cells (NFAT) to
the nucleus. In addition, Btk activates Ras, which leads to
nuclear translocation of the transcription factor activator
protein-1 (AP-1). BCR cross-linking also activates nuclear
factor κB (NFκB) via the degradation of IKKα and ERK
MAP kinases; ultimately, NFκB regulates cellular processes
such as activation apoptosis.
NEGATIVE REGULATORS
CD22
The CD22 receptor is a surface molecule that can also asso-
ciate with the BCR, presumably through interaction with
α2,6-sialylate sugars present on IgM.54 Although CD22 con-
tains an ITAM and is able to recruit Src tyrosine kinase to
its cytoplasmic domain,55 CD22 is primarily a negative reg-
ulator of BCR activation. Within the cytoplasmic domain
of CD22 is a specific motif known as the immunoreceptor
tyrosine-based inhibition motif (ITIM). As with the ITAM,
a critical tyrosine residue resides in the ITIM that mediates
signal transduction events. After activation of Lyn by CD45,
it is believed that the ITIM of CD22 is phosphorylated by
Lyn, leading to the recruitment of intracellular phospha-
tases such as SHP-1.54 B cells of mice deficient for CD2255
and Lyn56 have a phenotype similar to the SHP-1–deficient
viable moth-eaten mice (see later).
FCγRIIB1
FcγRIIB1 appears to be expressed exclusively on B cells,
and unlike other types of FcγR, it is unable to mediate
phagocytosis. The simultaneous ligation of both the BCR
and FcγRIIB1 sends an inhibitory signal to prevent antigen
activation of naive B cells. In the presence of high levels of
circulating immune complexes, this inhibitory signal pro-
vides a negative feedback mechanism to attenuate an anti-
gen-induced antibody response. Recruitment and activation
of SHIP by FcγRIIB1 is required for downregulation of BCR
signaling.57 After coligation of FcγRIIB1 and the BCR, it
is believed that Lyn phosphorylates FcγRIIB1.56 SHIP then
 188 DIAMOND  |  B Cells
associates with the FcγRIIB1 and mediates the dephos-
phorylation of CD19, thereby terminating BCR signaling.58
Depending on the genetic background, mice deficient in
FcγRIIB1 display a lupus-like phenotype.59
CD5
The role of CD5 in B1a cell function is not well understood.
After BCR cross-linking, CD5 is thought to mediate signals
that induce apoptosis and block proliferation.60 Cross-linking
of CD5 with an anti-CD5 monoclonal antibody results in
apoptosis. There is some evidence that CD5 recruits the
inhibitory phosphatase SHP-1 to its cytoplasmic domain.
However, unlike CD22 and FcγRIIB1, CD5 does not con-
tain a strong ITIM consensus sequence and may recruit
SHP-1 indirectly.61 The ligand-binding region of CD5
remains to be elucidated, but recent evidence demonstrates
that CD5 is a ligand for another negative regulator of BCR
signaling, CD72.
CD72
CD72 is a transmembrane receptor that is expressed as a
homodimer. The cytoplasmic tail of CD72 contains ITIMs,
and experiments have shown that CD72 recruits SHP-1.
Mice with a targeted disruption of the CD72 gene reveal
that CD72 plays a negative role in B cell activation. The
B cells of CD72-deficient mice are similar to those of viable
moth-eaten mice. They have an expansion of B1 cells and
B cells that are hyperresponsive to BCR cross-linking and
are more resistant to BCR-mediated apoptosis.62 There
are several putative ligands for CD72, including CD5 and
CD100.
PIR
Paired immunoglobulin-like receptor (PIR)-A and PIR-B
are expressed in a pair-wise fashion, as the name implies.
These receptors are believed to have opposing functions,
with PIR-A inducing an activation signal and PIR-B induc-
ing an inhibition signal. The ligands for PIR-A and PIR-B
remain to be elucidated. Although little is known about the
role of PIR-A in B cell activation, recent data demonstrate
that PIR-B plays a role in downregulating B cell responses.
The cytoplasmic tail of PIR-B possesses several ITIMs that
recruit inhibitory phosphatases. Mice that harbor a targeted
disruption of the PIR-B gene exhibit a phenotype similar to
mice that are deficient in the other ITIM-bearing inhibitory
receptors, such as an expansion of B1 cells and B cell hyper-
responsiveness.63
PD-1
PD-1 is an inhibitory molecule expressed predominantly on
activated B and T cells. The ligand-binding domain binds
PD-1L, and the cytoplasmic tail of PD-1 contains ITIMs
that recruit SHP-2 to attenuate BCR signals. The B cells
of PD-1–deficient mice are hyperresponsive to BCR sig-
naling, and these mice display an augmented response to
T cell–independent type II antigens. On certain genetic
backgrounds, PD-1 deficiency leads to an autoimmune
phenotype.64
B cells develop without bias for a particular antigenic
specificity, ensuring that a diverse repertoire of different
immunoglobulin molecules is produced. Despite the expres-
sion of antiapoptotic molecules such as Bcl-2, naive B cells
are short-lived unless they are activated in the presence
of antigen and accessory cells, such as dendritic cells and
T cells. Antigen-activated B cells undergo clonal expan-
sion; B cells that do not interact with antigen are destined
to undergo programmed cell death in a matter of days or
weeks. The B1 and B2 cell subsets are regulated by differ-
ent activation mechanisms and are involved in different
immune responses (see Table 10-2).
INHIBITORY PHOSPHATASES
SHP-1
SHP-1, another tyrosine phosphatase, is a potent nega-
tive regulator of BCR signaling. SHP-1 is a cytosolic pro-
tein found in association with transmembrane proteins
such as CD22, FcγRIIB1, CD5, CD72, and PIR-B. SHP-1
antagonizes BCR signaling by inactivating tyrosine kinases
associated with signaling. Potential candidates for SHP-1
dephosphorylation include Igα and Igβ, CD19, and Syk.61
The function of SHP-1 has been extensively studied in mice
that bear a naturally occurring mutation in the SHP-1 gene.
Mice with this genetic defect are known as moth-eaten
mice because of the appearance of their fur. These mice
have a decreased number of conventional B cells and an
expansion of B1 cells, accompanied by high titers of auto-
reactive IgM antibodies.65 This defect in B cell regulation
underscores the importance of SHP-1 in limiting the extent
of BCR signaling.
SHP-2
The intracellular tyrosine phosphatase SHP-2 is structurally
similar to SHP-1. As with SHP-1, SHP-2 is recruited to the
ITIM of inhibitory receptors. Mice deficient in SHP-2 are
not viable, suggesting that SHP-2 plays a role in embryonic
development.66 Studies of chimeric mice suggest that SHP-2
plays a role in hematopoiesis and that SHP-1 and SHP-2 act
in an antagonistic fashion.67
SHIP
SHIP is an inositol phosphatase that inhibits B cell activa-
tion by hydrolyzing the 5' phosphate from phosphatidylino-
sitol 3,4,5-triphosphate, a critical component of numerous
signaling pathways. As with SHP-1 and SHP-2, SHIP is
recruited to ITIMs following BCR cross-linking. The role
of SHIP in downregulating BCR-induced signals is dem-
onstrated by its association with FcγRIIB1. Mice deficient
in SHIP display splenomegaly and elevated levels of serum
antibody.68
SIGNAL TRANSDUCTION IN IMMATURE VERSUS
MATURE B CELLS
An important event in BCR signaling is the recruitment
of signaling components to lipid rafts, which are lipid-rich
microdomains of the membrane. In the resting state, the
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
BCR is excluded from lipid rafts, but following antigen
engagement, the BCR translocates into rafts, and the BCR
signaling cascade ensues due to the clustering of signaling
components. In addition to activating signals, however,
inhibitory signals are mediated by recruitment of FcγRIIb
into lipid rafts. As discussed later, BCR ligation mediates
negative selection during the immature and transitional B
cell stages and B cell activation during the mature B cell
stage. The reason for these two distinct outcomes is not
clear, because the same signal components are present; how-
ever, differences in membrane cholesterol limit recruitment
of the BCR to lipid rafts in immature B cells.69 There is also
evidence that the signal strength and duration and stage-
specific expression patterns of signaling elements may differ
between immature and mature B cells.70
B1 CELL ACTIVATION
B1 cells present in the pleural and peritoneal cavities respond
to T cell–independent antigens. There are two classes of
T cell–independent antigens: type I, which includes lipo-
polysaccharide, and type II, which includes large multivalent
antigens with repetitive epitopes, often found on the surface
of bacteria. T cell–independent antigens can directly acti-
vate B cells, resulting in the secretion of antibody. Soluble
factors, such as IL-5 and IL-10, also appear to be involved in
the maintenance and activation of B1 cells.
B1 cells do not require activation by antigen-specific
T cells. However, activated T cells and macrophages may
augment B1 cell activation, enhance immunoglobulin pro-
duction, and influence isotype class switching. The main
H-chain isotypes produced by B1 cells in response to T cell–
independent antigens are IgM, IgA, and IgG.
MARGINAL ZONE B CELL ACTIVATION
Marginal zone B cells are similar to B1 cells, in that they
do not require cognate T cell help for activation. Marginal
zone B cells respond to T cell–independent type II antigens
and play an important role in early protection against par-
ticulate blood-borne pathogens. They are situated in the
marginal sinuses, where specialized macrophages trap and
remove antigen from the circulation. Soluble factors, such
as BAFF, and T cell–derived cytokines appear to be impor-
tant for marginal zone B cell activation. Following activa-
tion by antigen, marginal zone B cells differentiate rapidly
into antibody-secreting plasma cells. Marginal zone B cells
secrete predominantly IgM antibodies and, to a lesser extent,
IgG antibodies.
FOLLICULAR B CELL ACTIVATION
As naive follicular B cells leave the circulation and con-
tact antigen and antigen-specific Th cells, those cells with
a BCR specific for antigen are sequestered and activated.
Engagement of the BCR by antigen signals the B cells to
engulf the antigen, process it intracellularly, and express
peptide fragments bound to MHC class II molecules on the
B cell surface.
After the expression of peptide fragments bound to MHC
class II molecules, antigen-activated B cells present peptide
to primed helper T cells. B cells and T cells interact through
189
Antigen
BCR
CD40 CD40L
CD4
Naive
MHC II Peptide Activated TH
B cell
TCR
B7 CD28
Cytokines
IL-2
Activated IL-3
B cell IL-4
IL-5
IL-10
IFN-γ
Figure 10-5  B cells as antigen presenting cells. Antigen bound to
surface immunoglobulin on naive B cells triggers endocytosis and intra-
cellular processing of the antigen. B cells engage antigen-specific helper 
T (TH) cells through the recognition of foreign peptide by the T cell recep-
tor (TCR) and through the binding of a conserved region of the MHC II
molecule by CD4. Along with antigen binding, coligation of CD40 and
B7 (expressed on B cells) with CD40L and CD28 (expressed on T cells)
provides critical costimulatory signals and the secretion of cytokines re-
quired for B cell activation.
T cell receptor recognition of a peptide-MHC complex on
the B cell and through engagement of costimulatory mol-
ecules B7 and CD40 (Fig. 10-5). The B7 molecule, which
interacts with CD28 on helper T cells, is not constitutively
expressed on B cells but is induced after antigen uptake by
B cells. Helper T cells secrete cytokines, such as IL-3, IL-4,
IL-5, and IL-10, and provide costimulatory signals that are
important for B cell maturation and differentiation.71 B cell
activation during a T cell–dependent immune response also
depends on engagement of the CD40 receptor expressed
on B cells with the CD40 ligand (CD40L) expressed on
T cells.72 The importance of signal transduction events medi-
ated by CD40-CD40L engagement is evident from studies
of patients with a type of X-linked hyper-IgM syndrome, an
immunodeficiency disease resulting from a defect in CD40L.
These individuals do not mount strong immune responses to
T cell–dependent antigens; they have high concentrations
of circulating IgM but only trace amounts of IgG isotypes
and no affinity maturation of the antibody response.
Activated follicular B cells can undergo two different
fates. Some of the activated B cells begin to proliferate to
form primary foci.73 These B cells can differentiate into
short-lived plasma cells that secrete an immediate supply of
antigen-specific antibody during a primary immune response
(i.e., the first time a particular antigen is encountered by
the immune system). The antibodies produced by these
short-lived plasma cells are usually of the IgM isotype and
are of low affinity because of the absence of somatic muta-
tions. Alternatively, the proliferating B cells, along with
the activating antigen-specific helper T cells, can migrate
to the primary follicles of secondary lymphoid tissue. Dur-
ing a primary immune response, antigen-activated follicu-
lar B cells form discrete structures in the primary follicles,
 190 DIAMOND  |  B Cells
known as germinal centers. Isotype class switching, affinity
­maturation, and differentiation into memory B cells or long-
lived plasma cells are part of the germinal center reaction.
MATURATION IN GERMINAL CENTERS
Germinal centers can be subdivided into separate regions
where the different stages of B cell maturation take place
(Fig. 10-6). The dark zone is composed of rapidly dividing
cells called centroblasts, which develop into cells known as
centrocytes. Centrocytes migrate to the light zone, where
they encounter a dense network of FDCs and follicular
helper T cells. B cells that do not express moderate- to
high-affinity BCRs are excluded from the light zone; B cells
that receive vital survival signals differentiate into plasma
or memory cells. Each stage of maturation is character-
ized by the differential expression of B cell surface markers
(Table 10-4).
Centroblasts in the dark zone are rapidly proliferating
cells derived from a relatively small number of antigen-acti-
vated B cells. Expression of the antiapoptotic Bcl-2 protein
is low in these cells, whereas expression of the proapoptotic
Fas protein is upregulated.74 Low levels of Bcl-2 expression
render developing B cells sensitive to apoptosis, but these
cells can be rescued by antigen and CD40-CD40L interac-
tions provided by antigen-specific helper T cells.
The process of somatic mutation is activated during the
centroblast stage. As discussed earlier, somatic mutation is
a process that introduces nucleotide base-pair changes in
the DNA sequence of immunoglobulin genes, giving rise
to antibodies with a potentially higher affinity for anti-
gen. The mechanisms that regulate the somatic mutation
process are poorly understood. Current data show that
(1) somatic mutations are introduced only into rearranged
immunoglobulin genes, (2) cis-acting DNA elements that
flank immunoglobulin genes may help target the variable
region genes for mutation, and (3) immunoglobulin gene
transcription is required.23 Activation-induced cytidine
deaminase (AID) plays a critical role in somatic muta-
tion and also isotype class switching (see later), because
the immunoglobulin variable regions of humans and mice
with genetic lesions in the AID gene are largely unmutated
and display no H-chain class switching.75,76 The insertion
of somatic mutations occurs throughout the coding region
of the variable gene segment and within 1 kilobase in each
direction of the coding region between the promoter region
and constant region of the rearranged immunoglobulin
gene. DNA sequences known as “hot spot” motifs have
been identified that appear to be targeted by the somatic
mutation machinery with a higher frequency than are
non–hot spot sequences.77 B cell clones that express surface
immunoglobulin with an increased affinity for antigen are
selectively expanded during the affinity maturation process,
whereas B cells that express somatically mutated immuno-
globulins with low affinity for antigen or novel binding
to self-antigens are targeted for apoptosis or inactivation.
The importance of somatic mutation and affinity matura-
tion during an immune response is underscored by the fact
that patients with mutations in the AID gene are severely
immunocompromised.
Centroblasts give rise to centrocytes located in the
basal region of the germinal center light zone.74 Because
Resting B cell FDC
Centrocyte Centroblast
Follicular
Memory mantle zone
cell
TH
? Apical light
or zone
Plasma TH Basal light
cell
zone
Dark
zone
Figure 10-6  B cell maturation in germinal centers. Following expo-
sure to antigen, B cells in the primary follicles form germinal centers or
migrate to previously formed germinal centers. Centroblasts located in
the dark zone undergo proliferation and acquire somatic mutations. A
small number of proliferating centroblasts can give rise to a larger num-
ber of centrocytes present in the basal light zone. As these cells pass
through a dense network of follicular dendritic cells (FDCs) and helper
T cells (TH), centrocytes bearing surface immunoglobulin receptors with
high affinity for antigen undergo positive selection. Centrocytes in the
apical light zone are nondividing cells that undergo differentiation into
memory B cells or plasma cells. It has been suggested that centrocytes
may return to the dark zone, where additional somatic mutations may
be acquired. Resting B cells that are not activated by antigen are pushed
aside to form the follicular mantle zone.
centrocytes are short-lived cells and express high levels of
Fas and low levels of Bcl-2, they require survival signals
from FDCs. FDCs differ from the other types of antigen pre­
senting cells in that they do not process antigen and pre­
sent peptide–MHC class II complexes on the cell surface.
Instead, they trap antigen by collecting antigen-antibody
complexes known as iccosomes (immune complex-coated
bodies) on the cell surface. Iccosomes bind to FcγR present
on FDCs and deliver an antigen-specific signal to B cells
through the BCR (Fig. 10-7). Centrocytes with specificity
for antigen on FDCs are saved from apoptosis by upregula-
tion of the Bcl-2 molecule. Engagement of the complement
receptors CR1 and CR2 (CD21 and CD35, respectively)
on B cells by components of the C3 complement protein
(iC3b, C3dg, and C3d) bound to FDCs may mediate a sec-
ondary costimulatory signal.78 If centrocytes do not receive
these positive selection signals, they rapidly die. As antigen-
selected centrocytes move into the apical light zone, they no
longer divide but continue maturing into memory B cells or
plasma cells.
Concurrent with the positive selection of antigen-spe-
cific centrocytes in germinal centers, signaling events medi-
ated by CD40L-CD40 and helper T cell–derived cytokines
promote isotype class switching in B cells. Helper T cells
secrete IL-4, which mediates class switching to IgG4 and
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION 191

Table 10-4  Markers of Antigen-Activated B Cells in Secondary Lymphoid Tissue

Marker Naive Centroblast Centrocyte Memory Plasma
Surface IgD + – – – –
Surface IgM, IgG, IgA,   + – + + –
or IgE
CD10 – + + – –
CD20 + + + + –
CD38 – + + – +
CD77 – + – – –
Presence of somatic   – + + + +
mutation
Isotype class switch – – + + +
Bcl-2 + – +/–* + +
Fas + + + + –
AID – + – - –
Blimp-1 – – – ? +
AID, activation-induced cytidine deaminase; Ig, immunoglobulin.
*Bcl-2 is expressed in centrocytes only after interaction with follicular dendritic cells.

GC
B cell
α β
CD21
BCR
CD21L
Antigen-antibody (C3 fragments)
complex
FcR CD21
FDC
Figure 10-7  Engagement of B cells with follicular dendritic cells. In­
teraction between follicular dendritic cells (FDCs) and B cells results in
signals that mediate the positive selection of B cells in germinal centers
(GCs). Antigen-antibody complexes trapped on the FDC surface deliver
a signal to the B cell receptor (BCR). A second signal is delivered by the
binding of CD21 on B cells to C3 complement components on the surface
of FDCs.
IgE and IL-10, which mediates class switching to IgG1 and
IgG3. Switching occurs by a deletional mechanism that
brings a downstream constant region gene in juxtaposition
with the VDJ gene segment.6 Upstream of each constant
region gene segment are switch recombination sequences
that contain binding sites for proteins known as switch
factors. A putative enzyme known as switch recombinase
catalyzes the removal of the upstream constant region gene
sequences. The AID molecule, which plays a critical role in
somatic mutation, is also required for isotype class switch-
ing, and patients and mice with mutations in the AID
gene exhibit a hyper-IgM syndrome due to a defect in class
switching.76
Although it is clear that follicular B cells participate in
a germinal center reaction, some data suggest that marginal
zone B cells can form germinal centers in response to a
T cell–dependent antigen.79
REPERTOIRE SELECTION
NEGATIVE SELECTION
An important feature of the immune system is the discrimi-
nation between foreign and self-antigens. Self-antigens are
molecules derived from intracellular or extracellular com-
ponents of the host. B cells with surface immunoglobulin
receptors that recognize self-components are subjected to a
process known as negative selection, or tolerance induction,
to avoid autoreactivity. The immune system has evolved
several mechanisms to selectively inactivate B cells that
recognize self-antigen while still permitting the activation
and expansion of B cells that recognize foreign antigen. In
an intact immune system, autoreactive B cells that are gen-
erated in either the bone marrow or the periphery can be
regulated by various mechanisms; the tolerance mechanism
activated depends on the strength of the BCR signal deliv-
ered by the self-antigen and the developmental stage of the
B cell. The concentration of self-antigen and the affinity of
the antibody for self-antigen determine the degree of recep-
tor occupancy of surface immunoglobulin and the strength
of the BCR signal. If there is little receptor cross-linking,
either because antigen concentration is low or because the
affinity of the antibody is weak, there is no BCR signaling,
and the autoreactive B cells are not tolerized. If receptor
cross-linking reaches the threshold necessary to trigger a
signaling cascade, an autoreactive B cell (in the absence of
T cell help) undergoes tolerance induction. Three mecha-
nisms are currently believed to mediate tolerance induction:
receptor editing, anergy, and deletion. When mechanisms
that regulate autoreactive B cells fail, the breakdown of
self-tolerance can lead to the development of autoimmune
disease.
CENTRAL AND PERIPHERAL TOLERANCE
There are two stages in B cell development when autoreac-
tive B cells can be generated. The first wave of autoreactiv-
ity arises in the bone marrow after VDJ rearrangement and
 192 DIAMOND  |  B Cells
expression of surface immunoglobulin on immature B cells.
Because of the vast number of different antibody molecules
that can be formed through the random recombination of
H- and L-chain variable region genes, all individuals gener-
ate autoreactive B cells. The process that prevents the exit
of autoreactive B cells from the bone marrow is known as
central tolerance. Immature B cells are particularly sensi-
tive to tolerance induction, probably owing to the absence
of Bcl-2 expression and the lack of costimulation by helper
T cells. Peripheral tolerance refers to the downregulation
of autoreactive B cells in peripheral lymphoid tissue. Tran-
sitional B cells may undergo peripheral tolerance if they
encounter autoantigen for the first time in the periphery.
Antigen-activated B cells that have acquired autoreactivity
by somatic mutation in germinal centers may also undergo
peripheral tolerance. At this developmental stage, it is cru-
cial that the maturation of autoreactive B cells into memory
B cells or plasma cells be blocked.
RECEPTOR EDITING
Cross-linking of the BCR on autoreactive B cells with a
high affinity for self-antigen results in tolerance induction.
However, these B cells can be salvaged if the original auto-
specificity is modified by a process known as receptor edit-
ing. In this process, the immunoglobulin surface receptor
undergoes revision to acquire a new, nonautoreactive speci-
ficity. This occurs by secondary gene rearrangement events,
such as intrachromosomal deletion accompanied by VH or
VL replacement, novel intrachromosomal rearrangement of
upstream VL genes and downstream JL genes, or rearrange-
ment of H- and L-chain genes on an unrearranged locus.80
Studies from transgenic mice suggest that receptor edit-
ing occurs in both immature and transitional B cells and
possibly in germinal center B cells as well. Autoreactive B
cells in transgenic mice expressing self-reactive specificities,
such as anti–double-stranded DNA81 or anti–MHC class I
haplotype,82 encounter self-antigen in the bone marrow.
They continue to express RAG-1 and RAG-2 and gener-
ate DNA excision products resulting from secondary immu-
noglobulin gene rearrangement events. In these models,
survival of edited B cells depends on the expression of a
second L-chain molecule that is able to associate with the
transgene-encoded H chain and displace the initial L chain.
There is evidence that H-chain receptor editing can also
occur in the bone marrow.83,84 It remains controversial
whether mature B cells in the periphery can also undergo
receptor editing during the germinal center stage of B cell
development.
ANERGY
Some autoreactive B cells enter a hyporesponsive state
known as anergy. Anergy is thought to be induced in imma-
ture B cells when they undergo a modest degree of BCR
cross-linking. Anergic B cells downregulate surface immu-
noglobulin receptors and display a desensitization of the
BCR, blocking activation of downstream mediators of sig-
naling. Further, anergic B cells are short-lived. Goodnow
and colleagues85 performed classic studies on B cell toler-
ance induction in mice engineered to express an anti–hen
egg lysozyme (HEL) antibody, along with soluble HEL,
which acts as a self-antigen. In the anti-HEL transgenic
mouse model, B cells that encounter soluble, monovalent­
HEL are anergized. These B cells populate secondary lym-
phoid tissue but do not secrete anti-HEL antibody and
cannot be recruited into a germinal center response. This
phenomenon is known as follicular exclusion.86
It appears that anergy can be reversed under certain
conditions. Experimentally, anergy is broken in vitro by
treatment with lipopolysaccharide or anti-CD40 antibody
and IL-4. Exposure of anergic B cells in vivo to multivalent
antigen in the presence of activated helper T cells may also
lead to their activation.87 It has been suggested that anergic
B cells serve as a potential source of autoantibody and may
be activated in inflammatory conditions.
DELETION
Extensive BCR cross-linking in the absence of helper T cell
costimulation leads to a type of tolerance induction called
deletion. In the transgenic model described earlier, when
anti-HEL B cells encounter multivalent membrane-bound
HEL autoantigen in the bone marrow, they are deleted.88
These results suggest that when antigen binding exceeds the
threshold for anergy induction, cell death is triggered. Dele-
tion of autoreactive B cells also occurs in the periphery and
may be a means to control B cells expressing somatically
mutated, high-affinity autoantibodies.
B cells are deleted from the immune system by a process
known as apoptosis, or programmed cell death. Apoptosis is
a highly regulated event that is distinct from necrotic cell
death, which results from destruction of the plasma membrane
(see Chapter 24). Apoptotic cell death is mediated primar-
ily through the activation of a series of endogenous prote-
ases. Once apoptosis is triggered, characteristic morphologic
and cellular changes take place, including a decrease in cell
volume, membrane blebbing, movement of phosphatidylser-
ine to the outer leaflet of the plasma membrane, chromatin
condensation, and DNA fragmentation. The pathways that
regulate apoptosis at the different stages of B cell develop-
ment are not entirely understood, but at least two pathways
have been shown to play an active role in regulating B cell
death: the Fas pathway and the Bcl-2 pathway.
Fas (also known as CD95 or Apo-1), a member of the
tumor necrosis factor receptor gene family, and Fas ligand
are transmembrane proteins expressed on a variety of cell
types. Because Fas ligand is a homotrimeric molecule, it can
bind three Fas molecules. Clustering of Fas on the cell sur-
face, which occurs when Fas molecules bind Fas ligand, acti-
vates a cascade of intracellular enzymes, known as caspases,
that mediate apoptosis.89
It appears that when B cells engage CD40L expressed on
helper T cells in the absence of BCR ligation, Fas signal-
ing induces apoptosis.90,91 Other triggers for Fas-mediated
death have not been clearly identified. Mutations in Fas
(lpr) or Fas ligand (gld) in mice result in a systemic lupus
erythematosus–like syndrome characterized by the produc-
tion of pathogenic autoantibodies and lymphadenopathy.
In humans, similar mutations lead to lymphadenopathy and
antierythrocyte antibodies, but anti-DNA antibodies and
glomerulonephritis are not present in these individuals.92
The Bcl-2 gene family is composed of molecules that
either protect against or induce apoptosis in many cell
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
types. Relative levels of these molecules dictate cell fate.
For example, excess Bcl-2 promotes cell survival, whereas
excess Bax induces cell death.93
The expression pattern of the antiapoptotic molecules
Bcl-2 and Bcl-XL during B cell development suggests that
they may play an important role in B cell survival. Levels
are low at times of repertoire selection in the bone marrow
or germinal centers and increase concomitantly with posi-
tive selection. B cells from mice deficient in Bcl-2 undergo
spontaneous apoptosis,94 whereas certain mouse strains that
overexpress Bcl-2 in B cells produce autoantibodies.95
ACTIVE TOLERANCE
Recently it has been shown that B cells can be blocked from
activation if they are chronically exposed to antigen and
IL-6. If IL-6 is removed from the microenvironment, those
chronically activated B cells will secrete antibody.96
B CELL AUTOIMMUNITY
B cell activation must be tightly regulated at multiple levels
to prevent the development of autoantibody-mediated auto-
immune diseases. Many autoimmune disorders, including
several rheumatologic diseases, involve the production of
autoantibodies. The specific autoantigens and the affected
organ systems vary. It has been speculated that B cell–
associated autoimmune diseases may be linked by a common
defect in the machinery that regulates B cell tolerance, but
the cause of most autoimmune diseases remains unknown.
ORIGIN OF AUTOREACTIVE B CELLS
One of the fundamental questions surrounding B cell
autoimmunity is the origin of autoreactive B cells. There
is evidence that both B1 and B2 cell subsets contribute to
autoimmunity.97 B1 cells have been suggested as a source
of autoantibodies because this subset is known to secrete
high levels of polyreactive antibody and can be directly
activated by BCR cross-linking with multivalent antigens
in the absence of antigen-specific T cell help.98 Despite the
production of autoreactive antibodies by this population, it
is not clear whether these antibodies contribute to disease.
B1 cells in nonautoimmune individuals produce low-affinity
autoantibodies that are nonpathogenic. It has been proposed
that these autoantibodies may actually play a protective
role against the tissue damage from more pathogenic auto-
antibodies. There is also some evidence that marginal zone
B cells can secrete autoantibodies; however, there is no
direct evidence that these antibodies are pathogenic. Fur-
ther, it is not clear whether B1 and marginal zone B cells
can also secrete high-affinity autoantibodies in autoimmune
individuals. However, a number of recently described genet-
ically engineered mice have increased numbers of marginal
zone B cells, produce autoantibodies, and exhibit autoanti-
body-mediated tissue damage.
Antibody production by the follicular subset requires the
involvement of T cells. These B cells undergo H-chain class
switching and somatic mutation in germinal centers. Analy-
sis of autoantibodies produced in both NZB/NZW F1 mice
and MRL/lpr mice that spontaneously develop systemic lupus
erythematosus demonstrates extensive somatic ­mutation and
193
class switching to IgG isotypes, suggesting that they arise
from follicular B cells.99,100 The relative importance of each
B cell subset in the generation of autoreactive B cells is still
being debated.
INITIATION AND PROPAGATION
OF AUTOIMMUNITY
There are several prevailing theories that attempt to explain
the activation and expansion of B cells that should normally
be silenced. Autoimmunity is thought to arise by a combi-
nation of environmental factors, such as infectious agents
that initiate an autoimmune response, and genetic defects
that alter B cell regulation. Proposed models for autoimmu-
nity include (1) cross-reactivity of foreign antigen with self-
antigen, (2) inappropriate costimulation, and (3) altered
thresholds for BCR signaling.
Much of our understanding of the breakdown of self-
tolerance and the progression of autoimmunity comes from
the examination of mouse models. Autoimmune mouse
models can be divided into three broad categories: induced
autoimmunity, spontaneously occurring autoimmunity, and
genetically manipulated mice. Even though the progression of
autoimmunity in humans is thought to be a highly complex
process that involves multiple genetic and environmental
factors, these animal models have provided much information
about the molecular events that maintain self-tolerance.
MOLECULAR MIMICRY
One proposed model for the initiation of autoreactivity
is that cross-reactive anti–self-, antiforeign B cells escape
central tolerance because self-antigen is present at too low
a concentration to trigger tolerance induction or because
the affinity of the antibody for autoantigen is below the
signaling threshold. These B cells become activated in the
periphery by foreign pathogens resembling self-antigen and
produce antibodies that bind both foreign and self-antigen.
This cross-reactivity is known as molecular mimicry. Molec-
ular mimicry is a popular model to explain the induction
of many autoimmune disorders.101 Once the pathogen is
cleared, the autoantibody response is terminated because
antigen-specific T cell help is no longer present. In the case
of autoimmune-prone individuals, it is possible that intrinsic
B cell defects prevent the downregulation of autoantibody
production, even after foreign antigen disappears.
The data that support molecular mimicry as a trigger for
B cell–mediated autoimmunity are circumstantial; antigens
from infectious agents have been identified that cross-react
with self-molecules associated with specific autoimmune
diseases101-105 (Table 10-5). The strongest evidence for
molecular mimicry as a trigger for autoimmune disease is the
cross-reactivity observed between the M protein of group A
streptococcus and cardiac myosin in rheumatic heart disease.
There is also experimental evidence that phosphorylcholine,
a component of the cell wall found in many bacterial strains,
mimics the structure of double-stranded DNA. Immuni-
zation of a nonautoimmune mouse strain with phosphor-
ylcholine coupled to a protein carrier routinely generates
anti–double-stranded DNA antibodies in germinal center
B cells.106 However, these B cells are normally downregu-
lated before contributing to a serum response.
 194 DIAMOND  |  B Cells
Table 10-5  Evidence for Antibody Cross-reactivity   is that foreign antigen acts as a molecular trigger to initiate
between Foreign and Self-Antigens
Foreign Antigen Self-Antigen
Yersinia, Klebsiella, Streptococcusa DNA
Epstein-Barr virus nuclear antigen 1a Ribonucleoprotein SmD
Streptococcus M proteinb Cardiac myosin
Coxsackie B3 capsid proteinc Cardiac myosin
Klebsiella nitrogenased HLA B27
Yersinia lipoproteine Thyrotropin receptor
Mycobacteria heat shock proteinf Mitochondrial components
Escherichia, Klebsiella, Proteusg Acetylchloline receptor
gpD derived from herpes   Acetylcholine receptor
simplex virusg
Autoimmune disorders exhibiting cross-reactive antibodies: a, systemic
lupus erythematosus; b, rheumatic fever; c, myocarditis; d, ankylosing spondyli-
tis; e, Graves’ disease; f, primary biliary cirrhosis; g, myasthenia gravis.
In general, an initial immune response is generated
against a dominant set of epitopes, followed by a later
response to secondary or “cryptic” epitopes, a process known
as epitope spreading.107 Epitope spreading is an important
aspect of a protective immune response, because the ability
to recognize multiple antigenic determinants increases the
efficiency of the neutralization and removal of pathogens.
When an autoimmune response has been triggered, epitope
spreading can lead to the production of additional autoanti-
bodies with specificity for multiple self-antigens. There are
several proposed mechanisms by which epitope spreading
triggers a cascade of T and B cell activation. For instance,
antigen presenting cells present a foreign peptide that mim-
ics a self-peptide to T cells (Fig. 10-8A). These cross-reactive
T cells become activated and provide costimulation to
autoreactive B cells that recognize self-antigen. This results
in the production of autoantibodies specific for the anti-
gen recognized by the T cell. After internalization of the
self-antigen by the autoreactive B cells, the autoantigen is
processed, and new cryptic epitopes of the self-antigen are
presented to T cells. A B cell binding to the self-antigen
internalizes not only that self-antigen but also any complex
of molecules that includes the self-antigen. The B cell may,
therefore, present cryptic epitopes of many self-antigens and
activate autoreactive T cells with multiple autospecificities.
In the periphery, T cells are present that have not been toler-
ized to these epitopes and thus are activated by self-peptide.
These activated T cells in turn help provide costimulation
and activate other autoreactive B cells.
Alternatively, cross-reactive B cells may be activated
first after exposure to foreign antigen and T cell help (Fig.
10-8B). These B cells internalize self-antigen and present
cryptic peptides to T cells that have not been tolerized,
leading to activation of autoreactive T cells and initiation
of the cascade. Thus, molecular mimicry and epitope spread-
ing can lead to the activation of T cells and B cells specific
for multiple autoantigens so long as the autoantigens form a
complex in vivo.
Both nonautoimmune and autoimmune-prone individu-
als have the capacity to generate autoantibodies. Therefore,
it is not likely that cross-reactivity between foreign and
self-antigens is solely responsible for the breakdown of tol-
erance that leads to autoimmunity. A plausible explanation
an autoimmune response to self-molecules, and a defect in
the mechanism that regulates B cell activation leads to the
propagation of an autoimmune response.
COSTIMULATION
It is evident that costimulatory signals provided by T cells
play a critical role in B cell activation. Therefore, inap-
propriate costimulation could lead to the propagation of
an immune response directed against a self-antigen. The
interaction between B7 on B cells and CD28 on T cells
is crucial for the activation of antigen-specific T cells and
B cells. When a genetically engineered protein that inhib-
its B7-CD28 interactions is administered to NZB/NZW
F1 lupus-prone mice, progression of disease is blocked.108
Reciprocally, autoreactive B cells present in mice that con-
stitutively overexpress B7 are not sensitive to Fas killing and
display high serum autoantibody titers.109 Overexpression of
CD40 or CD40L may also activate autoreactivity. In vitro
studies have demonstrated that CD40-CD40L ligation in
the presence of IL-4 activates anergic cells. It has been sug-
gested that CD40L may be overexpressed in lymphoid cells
of patients with systemic lupus erythematosus.110,111
Roquin, a recently identified member of the ubiquitin
ligase family, regulates the function of a distinct subset of
helper T cells known as follicular helper T cells.112 The role
of these cells is to select for somatically mutated B cells with
high affinity for antigen during an immune response. Recent
studies suggest that roquin is essential for the negative selec-
tion of autoreactive B cells in the germinal center. Roquin
belongs to a family of RING-type ubiquitin ligases involved
in the post-translational regulation of gene expression and
appears to repress the expression of ICOS, which plays an
important role in follicular helper T cell function. Mice har-
boring a mutation in the roquin gene display high-affinity
dsDNA antibodies owing to increased numbers of germinal
centers and follicular helper T cells.
Interferon regulatory factor-4 binding protein (IBP) has
also been shown to regulate T cell costimulatory signals.113
IBP is a novel regulator of Rho GTPases and is recruited to
the immunologic synapse following T cell receptor cross-
linking to mediate the reorganization of the cytoskeleton.
Mice deficient in IBP exhibit an autoimmune phenotype
characterized by the production of dsDNA antibodies
and glomerulonephritis. IBP plays an important role in
the survival and effector function of memory T cells and
underscores a novel role for Rho GTPases in regulating
interactions between T cells and autoreactive B cells.
Toll-like receptors (TLRs) belong to a family of pattern
recognition receptors that initiate innate immune responses
to various components of pathogens. TLR7, which recog-
nizes RNA, and TLR9, which recognizes unmethylated
CpG-containing nucleic acid sequences, are expressed on
B cells and have been implicated in autoimmunity. There
are numerous data suggesting that co-cross-linking of the
BCR and TLR with autoimmune complexes containing
nuclear antigens triggers the activation of antinuclear B
cells, implying that TLR 7 and TLR9 enhance the activa-
tion of autoreactive B cells.114-116
Cytokines such as IL-4, IL-6, and IL-10 are important sur-
vival factors for B cells and are secreted primarily by T cells,
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION 195

I.

APC Self-antigen
complex
Antibody #1-specific for self-antigen
MHC II II.

Foreign Self
peptide TCR peptide

MHC II
MHC II
T cell TCR B cell
(foreign/self) (self)
Antibody #2-specific
es

II
id for cryptic epitope

HC
pt
pe

M
MHC II
tic
r yp
C
TCR

T cell B cell

MHC II
TCR
III. (cryptic (cryptic
peptide) epitope)

IV.
A
I.
Self-antigen
Foreign complex
antigen Antibody #1-specific for self-antigen

B cell
(foreign/self)
MHC II

Antibody #2-specific
es
II

for cryptic epitope

HC

id
pt
M

MHC II
pe
tic
yp
Cr

TCR
II.
T cell B cell
MHC II
TCR

(cryptic (cryptic
peptide) epitope)

B III.
Figure 10-8  Epitope spreading. A, Epitope spreading by activation of cross-reactive T cells. I. Following antigen presentation of a foreign peptide 
that is recognized by cross-reactive T cells, costimulatory signals are delivered to B cells with surface immunoglobulin receptors that recognize a self-
antigen as part of a complex of self-molecules. II. The complex is engulfed by a self-reactive B cell, and antibodies specific for self-antigen are generated.
III. Self-reactive B cells process the self-molecules and present cryptic peptide–MHC II complexes on the cell surface. IV. If these cryptic peptides are
recognized by nontolerized autoreactive T cells, B cells specific for these cryptic peptides are activated, and the autoantibody response spreads to other
components of the self-antigen complex. B, Epitope spreading by activation of autoreactive B cells. I. A foreign antigen that mimics a self-molecule can
mediate the endocytosis of a self-molecule that is included in a self-antigen complex. The self-molecules of the complex are processed and expressed
on the cell surface of the B cell as cryptic peptide–MHC II complexes. II. If the cryptic peptides are recognized by nontolerized autoreactive T cells, 
III. These T cells provide costimulation to B cells that recognize cryptic peptides, resulting in the production of additional self-reactive antibodies.
 196 DIAMOND  |  B Cells

monocytes, and dendritic cells. BAFF plays an important
role in B cell development, survival, and antibody secretion.
Enhanced survival and activation of autoreactive B cells have
been demonstrated in mice that overexpress BAFF.117 An
increase in serum levels of BAFF has been observed in some
patients with lupus, rheumatoid arthritis, and Sjögren’s syn-
drome,118 which supports the notion that autoreactive B cells
have a survival advantage in the presence of excess BAFF.
The concept of B effector (Be) cells has been established
recently, with Be1 cells secreting IL-2, IL-12, and inter-
feron-γ and Be2 cells secreting IL-4, IL-5, and IL-10.119 This
raises the possibility that B cells can influence the polariza-
tion of T cells and may also establish a cytokine autocrine
loop that enhances the survival and activation of B cells.
In addition, B cells reportedly secrete transforming growth
factor-β (TGF-ß).120 TGF-ß is known to be immunosup-
pressive, and it has been suggested that B cell production of
TGF-ß induces apoptosis to limit a B cell response. In agree-
ment with these observations, mice deficient in TGF-ß or
the TGF-ß receptor exhibit an autoimmune phenotype.
B CELL SIGNALING THRESHOLDS
The effects of altering the threshold for BCR signaling
have been demonstrated in several mouse models. In trans-
genic mice that overexpress the BCR coreceptor complex
c­ omponent CD19, anergic B cells are activated and secrete
autoantibody.121 These results suggest that a decrease in the
minimal requirement for antigen engagement of the BCR
can lead to inappropriate activation of autoreactive B cells.
Viable moth-eaten mice also develop an autoimmune syn-
drome due to a naturally occurring deficiency in the SHP-1
phosphatase, a potent negative regulator of BCR signaling.65
In these mice, B1 cells are responsible for the production
of IgM anti-DNA antibodies. Transgenic mice deficient in
other signaling molecules that alter threshold activation,
such as CD2255 and Lyn,56 also produce autoantibodies.
Thus, changes in thresholds for antigen-induced B cell acti-
vation can lead to the activation of autoreactive B cells.
SUMMARY
The generation of a diverse repertoire of antibody mol-
ecules provides an important line of defense against micro-
bial infections. The immune system is exquisitely controlled
at multiple levels to allow the maturation of B cells that
produce protective antibodies while attempting to avoid
the production of autoantibodies (Fig. 10-9). Only a small
percentage of B cell precursors generated completes the
maturation scheme. During the pro-B and pre-B cell stages
of development, B cells with aberrantly rearranged H- or
L-chain genes are eliminated. As the remaining precursor

Bone marrow
Spleen
Pro-B
MZ
HC
Negative
rearrangement
selection (2)

Transitional FO
2

Pre-B

LC Transitional Negative
GC selection (3)
rearrangement 1

Plasmablast
Negative
selection (1) Immature

Negative
selection (4)? Memory Periphery
Bone marrow
Plasma

Figure 10-9  Selection checkpoints during B cell maturation. Autoreactive B cells can be censored at multiple developmental checkpoints: (1)
Fol­lowing surface expression of surface immunoglobulin, immature B cells that encounter autoantigen in the bone marrow are subject to negative
selection. (2) B cells that are not eliminated in the bone marrow may under go negative selection during the transitional B cell stage. Transitional B cells
that emerge from this development stage give to rise to follicular (FO) or marginal zone (MZ) B cells. Follicular B cells activated by antigen and the help
of cognate T cells progress to the germinal center (GC) B cell stage. (3) Germinal center B cells that acquire high affinity for autoantigen by the process
of somatic hypermutation may be eliminated in the germinal center to block their further maturation into long-lived plasma cells or memory cells. 
(4) There is evidence that autoreactive plasmablasts may also be subject to negative selection. Long-lived plasma cells that emerge from the selection
process home primarily to the bone marrow, and memory B cells circulate throughout the periphery. HC, heavy chain; LC, light chain.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
cells transit into the immature B cell stage, they are sub-
jected to negative selection; that is, immature B cells bear-
ing autospecificity are either deleted or inactivated, whereas
B cells that can potentially bind foreign antigen are released
into the periphery. B cells that are stimulated by foreign
antigen are selectively expanded and undergo further immu-
noglobulin gene diversification in peripheral lymphoid tis-
sue. During this stage of development, B cells that express
high-affinity immunoglobulin receptors undergo positive
selection, whereas B cells with a diminished affinity or those
that have acquired autoreactivity are eliminated. B cells
that pass through these critical developmental checkpoints
differentiate into long-lived memory B cells or plasma cells.
The underlying causes of B cell–associated autoimmunity
are not well understood, but just as there are multiple check-
points for the survival or activation of autoreactive B cells,
it seems likely that multiple defects in the regulatory mech-
anisms that control B cell maturation and differentiation
contribute to autoimmune disease.
REFERENCES
  1. Janeway JA, Travers P, Walport M, et al: The structure of a typical
antibody molecule. In Immunobiology, 5th ed. New York, Garland,
2001, p 96.
  2. Janeway JA, Travers P, Walport M, et al: Structural variation in
immunoglobulin constant regions. In Immunobiology, 5th ed. New
York, Garland, 2001, p 142.
  3. Frazer JK, Capra JD: Immunoglobulins: Structure and function. In
Paul WE (ed): Fundamental Immunology. Philadelphia, Lippincott-
Raven, 1999, p 37.
  4. Raghavan M, Bjorkman PJ: Fc receptors and their interactions with
immunoglobulins. Annu Rev Cell Dev Biol 12:181, 1996.
  5. Bradbury LE, Kansas GS, Levy S, et al: The CD19/CD21 signal trans-
ducing complex of human B lymphocytes includes the target of anti-
proliferative antibody-1 and Leu-13 molecules. J Immunol 149:2841,
1992.
  6. Snapper CM, Finkelman FD: Immunoglobulin class switching. In
Paul WE (ed): Fundamental Immunology. Philadelphia, Lippincott-
Raven, 1999, p 831.
  7. Shohet JM, Pemberton P, Carroll MC: Identification of a major bind-
ing site for complement C3 on the IgG1 heavy chain. J Biol Chem
268:5866, 1993.
  8. Hiemstra PS, Biewenga J, Gorter A, et al: Activation of complement
by human serum IgA, secretory IgA and IgA1 fragments. Mol Immu-
nol 25:527, 1988.
  9. Froelich CJ, Hanna WL, Poirier GG, et al: Granzyme B/perforin-medi-
ated apoptosis of Jurkat cells results in cleavage of poly(ADP-ribose)
polymerase to the 89-kDa apoptotic fragment and less abundant
64-kDa fragment. Biochem Biophys Res Commun 227:658, 1996.
10. Ghetie V, Ward ES: FcRn: The MHC class I-related receptor that is
more than an IgG transporter. Immunol Today 18:592, 1997.
11. Takizawa T, Anderson CL, Robinson JM: A novel Fc gamma R-
defined, IgG-containing organelle in placental endothelium. J Immu-
nol 175:2331, 2005.
12. Aroeti B, Casanova J, Okamoto C, et al: Polymeric immunoglobulin
receptor. Int Rev Cytol 137B:157, 1992.
13. Burrows PD, Cooper MD: IgA deficiency. Adv Immunol 65:245,
1997.
14. Stewart WW, Kerr MA: The specificity of the human neutrophil IgA
receptor (Fc alpha R) determined by measurement of chemilumi-
nescence induced by serum or secretory IgA1 or IgA2. Immunology
71:328, 1990.
15. Carsetti R, Kohler G, Lamers MC: A role for immunoglobulin
D: Interference with tolerance induction. Eur J Immunol 23:168,
1993.
16. Nitschke L, Kosco MH, Kohler G, et al: Immunoglobulin D-defi-
cient mice can mount normal immune responses to thymus-inde-
pendent and -dependent antigens. Proc Natl Acad Sci U S A
90:1887, 1993.
197
17. Roes J, Rajewsky K: Immunoglobulin D (IgD)-deficient mice reveal
an auxiliary receptor function for IgD in antigen-mediated recruit-
ment of B cells. J Exp Med 177:45, 1993.
18. Janeway JA, Travers P, Walport M, et al: The structure of a typical
antibody molecule. In Immunobiology, 5th ed. New York, Garland,
2001, p 95.
19. Tonegawa S: Somatic generation of antibody diversity. Nature
302:575, 1983.
20. Duchosal MA: B-cell development and differentiation. Semin Hema-
tol 34:2, 1997.
21. Akira S, Okazaki K, Sakano H: Two pairs of recombination signals
are sufficient to cause immunoglobulin V-(D)-J joining. Science
238:1134, 1987.
22. Chu G: Role of the Ku autoantigen in V(D)J recombination and
double-strand break repair. Curr Top Microbiol Immunol 217:113,
1996.
23. Wiesendanger M, Scharff MD, Edelmann W: Somatic hypermuta-
tion, transcription, and DNA mismatch repair. Cell 94:415, 1998.
24. Diamond B, Scharff MD: Somatic mutation of the T15 heavy
chain gives rise to an antibody with autoantibody specificity. Proc
Natl Acad Sci U S A 81:5841, 1984.
25. Kikuchi K, Kondo M: Developmental switch of mouse hematopoietic
stem cells from fetal to adult type occurs in bone marrow after birth.
Proc Natl Acad Sci U S A 103:17852, 2006.
26. Boumsell L, Bernard A, Lepage V, et al: Some chronic lymphocytic
leukemia cells bearing surface immunoglobulins share determinants
with T cells. Eur J Immunol 8:900, 1978.
27. Tedder TF, Inaoki M, Sato S: The CD19-CD21 complex regulates
signal transduction thresholds governing humoral immunity and
autoimmunity. Immunity 6:107, 1997.
28. Montecino-Rodriguez E, Leathers H, Dorshkind K: Identification of
a B-1 B cell-specified progenitor. Nat Immunol 7:293, 2006.
29. Picker LJ, Siegelman MH: Lymphoid tissues and organs. In Paul WE
(ed): Fundamental Immunology. Philadelphia, Lippincott-Raven,
1999, p 479.
30. Dorshkind K: Regulation of hemopoiesis by bone marrow stromal
cells and their products. Annu Rev Immunol 8:111, 1990.
31. Kincade PW: B lymphopoiesis: Global factors, local control. Proc
Natl Acad Sci U S A 91:2888, 1994.
32. O’Riordan M, Grosschedl R: Transcriptional regulation of early B-
lymphocyte differentiation. Immunol Rev 175:94, 2000.
33. Melchers F, Haasner D, Grawunder U, et al: Roles of IgH and L
chains and of surrogate H and L chains in the development of cells of
the B lymphocyte lineage. Annu Rev Immunol 12:209, 1994.
34. Papavasiliou F, Jankovic M, Nussenzweig MC: Surrogate or conven-
tional light chains are required for membrane immunoglobulin mu to
activate the precursor B cell transition. J Exp Med 184:2025, 1996.
35. Fang W, Weintraub BC, Dunlap B, et al: Self-reactive B lymphocytes
overexpressing Bcl-xL escape negative selection and are tolerized by
clonal anergy and receptor editing. Immunity 9:35, 1998.
36. Gross JA, Dillon SR, Mudri S, et al: TACI-Ig neutralizes molecules
critical for B cell development and autoimmune disease: Impaired B
cell maturation in mice lacking BLyS. Immunity 15:289, 2001.
37. Carsetti R, Rosado MM, Wardemann H: Peripheral development of
B cells in mouse and man. Immunol Rev 197:179, 2004.
38. Sprent J: Immunological memory. Curr Opin Immunol 9:37, 1997.
39. Tarlinton D: B-cell memory: Are subsets necessary? Nat Rev Immu-
nol 6:785, 2006.
40. Zandvoort A, Timens W: The dual function of the splenic marginal
zone: Essential for initiation of anti-TI-2 responses but also vital in
the general first-line defense against blood-borne antigens. Clin Exp
Immunol 130:4, 2002.
41. Alugupalli KR, Leong JM, Woodland RT, et al: B1b lymphocytes
confer T cell-independent long-lasting immunity. Immunity 21:379,
2004.
42. Shaffer AL, Lin KI, Kuo TC, et al: Blimp-1 orchestrates plasma
cell differentiation by extinguishing the mature B cell gene expres-
sion program. Immunity 17:51, 2002.
43. Merville P, Dechanet J, Desmouliere A, et al: Bcl-2+ tonsillar plasma
cells are rescued from apoptosis by bone marrow fibroblasts. J Exp
Med 183:227, 1996.
44. Slifka MK, Ahmed R: Long-lived plasma cells: A mechanism for
maintaining persistent antibody production. Curr Opin Immunol
10:252, 1998.
 198 DIAMOND  |  B Cells
45. Kim CH: The greater chemotactic network for lymphocyte traffick-
ing: Chemokines and beyond. Curr Opin Hematol 12:298, 2005.
46. Muller G, Lipp M: Concerted action of the chemokine and lympho-
toxin system in secondary lymphoid-organ development. Curr Opin
Immunol 15:217, 2003.
47. Cherukuri A, Dykstra M, Pierce SK: Floating the raft hypothesis:
Lipid rafts play a role in immune cell activation. Immunity 14:657,
2001.
48. Kishihara K, Penninger J, Wallace VA, et al: Normal B lymphocyte
development but impaired T cell maturation in CD45-exon 6 pro-
tein tyrosine phosphatase-deficient mice. Cell 74:143, 1993.
49. Birkeland ML, Monroe JG: Biochemistry of antigen receptor signal-
ing in mature and developing B lymphocytes. Crit Rev Immunol
17:353, 1997.
50. Carter RH, Fearon DT: CD19: Lowering the threshold for antigen
receptor stimulation of B lymphocytes. Science 256:105, 1992.
51. Haas KM, Hasegawa M, Steeber DA, et al: Complement receptors
CD21/35 link innate and protective immunity during Streptococcus
pneumoniae infection by regulating IgG3 antibody responses. Immu-
nity 17:713, 2002.
52. Kurosaki T: Molecular mechanisms in B cell antigen receptor signal-
ing. Curr Opin Immunol 9:309, 1997.
53. Sato S, Jansen PJ, Tedder TF: CD19 and CD22 expression recipro-
cally regulates tyrosine phosphorylation of Vav protein during B lym-
phocyte signaling. Proc Natl Acad Sci U S A 94:13158, 1997.
54. Cyster JG, Goodnow CC: Tuning antigen receptor signaling by
CD22: Integrating cues from antigens and the microenvironment.
Immunity 6:509, 1997.
55. Sato S, Miller AS, Inaoki M, et al: CD22 is both a positive and nega-
tive regulator of B lymphocyte antigen receptor signal transduction:
Altered signaling in CD22-deficient mice. Immunity 6:551, 1996.
56. Chan VW, Meng F, Soriano P, et al: Characterization of the B lym-
phocyte populations in Lyn-deficient mice and the role of Lyn in sig-
nal initiation and down-regulation. Immunity 7:69, 1997.
57. Coggeshall KM: Inhibitory signaling by B cell Fc gamma RIIb. Curr
Opin Immunol 10:306, 1998.
58. Hippen KL, Buhl AM, D’Ambrosio D, et al: Fc gammaRIIB1 inhibi-
tion of BCR-mediated phosphoinositide hydrolysis and Ca2+ mobiliza-
tion is integrated by CD19 dephosphorylation. Immunity 7:49, 1997.
59. Bolland S, Ravetch JV: Spontaneous autoimmune disease in
Fc(gamma)RIIB-deficient mice results from strain-specific epistasis.
Immunity 13:277, 2000.
60. Bikah G, Carey J, Ciallella JR, et al: CD5-mediated negative regula-
tion of antigen receptor-induced growth signals in B-1 B cells. Sci-
ence 274:1996, 1906.
61. Neel BG: Role of phosphatases in lymphocyte activation. Curr Opin
Immunol 9:405, 1997.
62. Pan C, Baumgarth N, Parnes JR: CD72-deficient mice reveal nonre-
dundant roles of CD72 in B cell development and activation. Immu-
nity 11:495, 1999.
63. Ujike A, Takeda K, Nakamura A, et al: Impaired dendritic cell matu-
ration and increased T(H)2 responses in PIR-B(-/-) mice. Nat Immu-
nol 3:542, 2002.
64. Nishimura H, Nose M, Hiai H, et al: Development of lupus-like auto-
immune diseases by disruption of the PD-1 gene encoding an ITIM
motif-carrying immunoreceptor. Immunity 11:141, 1999.
65. Westhoff CM, Whittier A, Kathol S, et al: DNA-binding antibodies
from viable motheaten mutant mice: Implications for B cell toler-
ance. J Immunol 159:3024, 1997.
66. Qu CK, Yu WM, Azzarelli B, et al: Biased suppression of hematopoi-
esis and multiple developmental defects in chimeric mice containing
Shp-2 mutant cells. Mol Cell Biol 18:6075, 1998.
67. Qu CK, Nguyen S, Chen J, et al: Requirement of Shp-2 tyrosine
phosphatase in lymphoid and hematopoietic cell development.
Blood 97:911, 2001.
68. Helgason CD, Kalberer CP, Damen JE, et al: A dual role for Src
homology 2 domain-containing inositol-5-phosphatase (SHIP) in
immunity: Aberrant development and enhanced function of B lym-
phocytes in ship -/- mice. J Exp Med 191:781, 2000.
69. Karnell FG, Brezski RJ, King LB, et al: Membrane cholesterol con-
tent accounts for developmental differences in surface B cell receptor
compartmentalization and signaling. J Biol Chem 280:25621, 2005.
70. Harnett MM, Katz E, Ford CA: Differential signalling during B-cell
maturation. Immunol Lett 98:33, 2005.
71. Abbas AK, Murphy KM, Sher A: Functional diversity of helper T
lymphocytes. Nature 383:787, 1996.
72. van Kooten C, Banchereau J: Functions of CD40 on B cells, dendritic
cells and other cells. Curr Opin Immunol 9:330, 1997.
73. Smith KG, Hewitson TD, Nossal GJ, et al: The phenotype and fate of
the antibody-forming cells of the splenic foci. Eur J Immunol 26:444,
1996.
74. Liu YJ, Arpin C: Germinal center development. Immunol Rev
156:111, 1997.
75. Revy P, Muto T, Levy Y, et al: Activation-induced cytidine deami-
nase (AID) deficiency causes the autosomal recessive form of the
hyper-IgM syndrome (HIGM2). Cell 102:565, 2000.
76. Muramatsu M, Kinoshita K, Fagarasan S, et al: Class switch recombi-
nation and hypermutation require activation-induced cytidine deam-
inase (AID), a potential RNA editing enzyme. Cell 102:553, 2000.
77. Betz AG, Neuberger MS, Milstein C: Discriminating intrinsic and
antigen-selected mutational hotspots in immunoglobulin V genes.
Immunol Today 14:405, 1993.
78. Tew JG, Wu J, Qin D, et al: Follicular dendritic cells and presenta-
tion of antigen and costimulatory signals to B cells. Immunol Rev
156:39, 1997.
79. Song H, Cerny J: Functional heterogeneity of marginal zone B cells
revealed by their ability to generate both early antibody-forming cells
and germinal centers with hypermutation and memory in response to
a T-dependent antigen. J Exp Med 198:2003, 1923.
80. Radic MZ, Zouali M: Receptor editing, immune diversification, and
self-tolerance. Immunity 5:505, 1996.
81. Gay D, Saunders T, Camper S, et al: Receptor editing: An
approach by autoreactive B cells to escape tolerance. J Exp Med
177:999, 1993.
82. Tiegs SL, Russell DM, Nemazee D: Receptor editing in self-
­reactive bone marrow B cells. J Exp Med 177:1009, 1993.
83. Chen C, Radic MZ, Erikson J, et al: Deletion and editing of B cells
that express antibodies to DNA. J Immunol 152:1994, 1970.
84. Bertrand FE, Golub R, Wu GE: V(H) gene replacement occurs in
the spleen and bone marrow of non-autoimmune quasi-monoclonal
mice. Eur J Immunol 28:3362, 1998.
85. Goodnow CC, Crosbie J, Adelstein S, et al: Altered immunoglobulin
expression and functional silencing of self-reactive B lymphocytes in
transgenic mice. Nature 334:676, 1988.
86. Cyster JG, Hartley SB, Goodnow CC: Competition for follicular
niches excludes self-reactive cells from the recirculating B-cell rep-
ertoire. Nature 371:389, 1994.
87. Cooke MP, Heath AW, Shokat KM, et al: Immunoglobulin signal
transduction guides the specificity of B cell-T cell interactions and is
blocked in tolerant self-reactive B cells. J Exp Med 179:425, 1994.
88. Hartley SB, Goodnow CC: Censoring of self-reactive B cells with
a range of receptor affinities in transgenic mice expressing heavy
chains for a lysozyme specific antibody. Int Immunol 6:1417, 1994.
89. Ashkenazi A, Dixit VM: Death receptors: Signaling and modulation.
Science 281:1305, 1998.
90. Schattner EJ, Elkon KB, Yoo DH, et al: CD40 ligation induces Apo-
1/Fas expression on human B lymphocytes and facilitates apoptosis
through the Apo-1/Fas pathway. J Exp Med 182:1557, 1995.
91. Garrone P, Neidhardt EM, Garcia E, et al: Fas ligation induces apoptosis
of CD40-activated human B lymphocytes. J Exp Med 182:1265, 1995.
92. Elkon KB, Marshak-Rothstein A: B cells in systemic autoimmune
disease: Recent insights from Fas-deficient mice and men. Curr Opin
Immunol 8:852, 1996.
93. Knudson CM, Korsmeyer SJ: Bcl-2 and Bax function independently
to regulate cell death. Nat Genet 16:358, 1997.
94. Veis DJ, Sorenson CM, Shutter JR, et al: Bcl-2-deficient mice dem-
onstrate fulminant lymphoid apoptosis, polycystic kidneys, and
hypopigmented hair. Cell 75:229, 1993.
95. Strasser A, Whittingham S, Vaux DL, et al: Enforced BCL2 expres-
sion in B-lymphoid cells prolongs antibody responses and elicits
autoimmune disease. Proc Natl Acad Sci U S A 88:8661, 1991.
96. Kilmon MA, Rutan JA, Clarke SH, et al: Low-affinity, Smith anti-
gen-specific B cells are tolerized by dendritic cells and macrophages.
J Immunol 175:37, 2005.
97. Ye YL, Chuang YH, Chiang BL: In vitro and in vivo functional anal-
ysis of CD5+ and CD5– B cells of autoimmune NZB x NZW F1 mice.
Clin Exp Immunol 106:253, 1996.
98. Murakami M, Honjo T: Involvement of B-1 cells in mucosal immu-
nity and autoimmunity. Immunol Today 16:534, 1995.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
99. Marion TN, Bothwell AL, Briles DE, et al: IgG anti-DNA autoanti-
bodies within an individual autoimmune mouse are the products of
clonal selection. J Immunol 142:4269, 1989.
100. Shlomchik M, Mascelli M, et al: Anti-DNA antibodies from auto-
immune mice arise by clonal expansion and somatic mutation.
J Exp Med 171:265, 1990.
101. Davies JM: Molecular mimicry: Can epitope mimicry induce autoim-
mune disease? Immunol Cell Biol 75:113, 1997.
102. Cunningham MW: Bacterial antigen mimicry. In Bona C, Simino-
vitch K, Zanetti M, et al (eds): Molecular Pathology of Autoimmune
Diseases. Chur Switzerland, Harwood Academic, 1993, p 245.
103. Zhang H, Kaur I, Niesel DW, et al: Lipoprotein from Yersinia entero-
colitica contains epitopes that cross-react with the human thyrotropin
receptor. J Immunol 158:1997, 1976.
104. El-Roiey A, Sela O, Isenberg DA, et al: The sera of patients with
Klebsiella infections contain a common anti-DNA idiotype (16/6)
and anti-polynucleotide activity. Clin Exp Immunol 67:507, 1987.
105. Putterman C, Limpanasithikul W, Edelman M, et al: The double
edged sword of the immune response: Mutational analysis of a murine
anti-pneumococcal, anti-DNA antibody. J Clin Invest 97:2251,
1996.
106. Ray SK, Putterman C, Diamond B: Pathogenic autoantibodies are
routinely generated during the response to foreign antigen: A para-
digm for autoimmune disease. Proc Natl Acad Sci U S A 93:2019,
1996.
107. McCluskey J, Farris AD, Keech CL, et al: Determinant spreading:
Lessons from animal models and human disease. Immunol Rev
164:209, 1998.
108. Daikh DI, Finck BK, Linsley PS, et al: Long-term inhibition of
murine lupus by brief simultaneous blockade of the B7/CD28 and
CD40/gp39 costimulation pathways. J Immunol 159:3104, 1997.
109. Rathmell JC, Fournier S, Weintraub BC, et al: Repression of B7.2
on self-reactive B cells is essential to prevent proliferation and allow
Fas-mediated deletion by CD4+ T cells. J Exp Med 188:651, 1998.
110. Koshy M, Berger D, Crow MK: Increased expression of CD40 ligand
on systemic lupus erythematosus lymphocytes. J Clin Invest 98:826,
1996.
199
111. Desai-Mehta A, Lu L, Ramsey-Goldman R, et al: Hyperexpression of
CD40 ligand by B and T cells in human lupus and its role in patho-
genic autoantibody production. J Clin Invest 97:2063, 1996.
112. Vinuesa CG, Cook MC, Angelucci C, et al: A RING-type ubiquitin
ligase family member required to repress follicular helper T cells and
autoimmunity. Nature 435:452, 2005.
113. Fanzo JC, Yang W, Jang SY, et al: Loss of IRF-4-binding protein leads
to the spontaneous development of systemic autoimmunity. J Clin
Invest 116:703, 2006.
114. Christensen SR, Shupe J, Nickerson K, et al: Toll-like receptor 7 and
TLR9 dictate autoantibody specificity and have opposing inflam-
matory and regulatory roles in a murine model of lupus. Immunity
25:417, 2006.
115. Berland R, Fernandez L, Kari E, et al: Toll-like receptor 7-dependent
loss of B cell tolerance in pathogenic autoantibody knockin mice.
Immunity 25:429, 2006.
116. Leadbetter EA, Rifkin IR, Hohlbaum AM, et al: Chromatin-IgG
complexes activate B cells by dual engagement of IgM and Toll-like
receptors. Nature 416:603, 2002.
117. Mackay F, Woodcock SA, Lawton P, et al: Mice transgenic for BAFF
develop lymphocytic disorders along with autoimmune manifesta-
tions. J Exp Med 190:1697, 1999.
118. Groom J, Kalled SL, Cutler AH, et al: Association of BAFF/BLyS
overexpression and altered B cell differentiation with Sjogren’s syn-
drome. J Clin Invest 109:59, 2002.
119. Harris DP, Haynes L, Sayles PC, et al: Reciprocal regulation of polar-
ized cytokine production by effector B and T cells. Nat Immunol
1:475, 2000.
120. Douglas RS, Woo EY, Capocasale RJ, et al: Altered response to and
production of TGF-beta by B cells from autoimmune NZB mice. Cell
Immunol 179:126, 1997.
121. Inaoki M, Sato S, Weintraub BC, et al: CD19-regulated signaling
thresholds control peripheral tolerance and autoantibody production
in B lymphocytes. J Exp Med 186:1923, 1997.
 11
KEY POINTS
Fibroblasts provide the structural basis of organ composition
and function but are more than just structure-building cells.
Fibroblasts are very sensitive to environmental changes and
actively regulate the composition and cellular dynamics of
connective tissues, including internal organs and membranes
such as the synovium.
Under disease conditions such as inflammation, fibroblasts
are critical switches that regulate the response to tissue injury,
contribute to the resolution or chronicity of the organ-specific
pathology, and determine the consequences of disease.
In rheumatoid arthritis, fibroblasts are a key part of the local
immune system and, through the integration of signals from
different sources, contribute to both disease initiation and
perpetuation.
While responding to environmental stimuli, rheumatoid
Synovial fibroblasts undergo fundamental changes that result
in their stable activation, which is maintained even in the
absence of continuous stimulation by inflammatory triggers.
Fibroblast-like synoviocytes are prominently involved in the
accumulation and pathologic differentiation of inflammatory
cells in the diseased synovium of rheumatoid arthritis.
PHYSIOLOGIC CHARACTERISTICS
AND FUNCTIONS OF FIBROBLASTS
Fibroblasts are ubiquitous cells of mesenchymal origin that
are present in virtually every body compartment. They are
the primary resident cells of all connective ­ tissues and, as
such, provide the structural basis of organ composition
and function. Fibroblasts are involved ­ prominently in the
remodeling of the extracellular matrix (ECM), have barrier
functions, and play an important role in tissue repair. How-
ever, it has become increasingly clear that fibroblasts are far
more than just structural cells that provide the meshwork
in which organ-specific cells reside and into which organ
function is embedded.1 Rather, fibroblasts react very specifi-
cally to environmental triggers, including soluble mediators,
ECM components, and chemical stimuli such as oxygen
tension and pH, and they actively regulate the composition
Fibroblasts
and Fibroblast-like
Synoviocytes
Thomas Pap  • Steffen Gay
and cellular dynamics of their respective tissues (Fig. 11-1).
Consequently, fibroblasts in different regions of the body
have distinct functions.
PRODUCTION OF EXTRACELLULAR MATRIX
COMPONENTS
Ensuring the homeostasis of the ECM is one of the primary
functions of fibroblasts. Fibroblasts produce a number of
ECM molecules, including collagens, fibronectin, proteo-
glycans, and others, and are critical in the assembly of these
molecules into a three-dimensional network. The types
of ECM molecules produced by individual populations of
fibroblasts differ from tissue to tissue, reflecting the diver-
sity of fibroblasts in different organs. For example, dermal
fibroblasts produce significant amounts of type VII collagen,
which holds together the epidermal and dermal layers in the
skin. Fibroblasts in other organs, such as the lung and kid-
ney, produce mainly interstitial collagens (particularly types
I and III), and specific features of fibroblasts from other ori-
gins, such as the gingiva, have been reported.2
In the synovial membrane, fibroblasts also have a barrier
function, in that they provide the joint cavity and the adja-
cent cartilage with lubricating molecules such as hyaluronic
acid and with plasma-derived nutrients. One peculiarity of the
synovial membrane is that it lacks a basement membrane and,
thus, the classic architecture of epithelium. As a consequence,
the most superficial layer of the synovium, the intimal lining
layer, must assume these functions. Recent data demonstrate
that although cellular contacts between the fibroblast-like
synoviocytes also lack tight junctions and desmosomes, the
recently discovered adhesion ­molecule cadherin-11 mediates
a strong homophilic adhesion between synoviocytes and is
largely responsible for their organization into tissue.3
Production of ECM components and cross-linking these
components into a specific three-dimensional structure consti-
tute a very active process in which fibroblasts have to migrate
to sites of tissue injury or remodeling and interact with ECM
molecules through specific surface receptors. Through such
receptors, fibroblasts must sense changes in both the struc-
ture and the cellular composition of connective tissues. They
respond dynamically by adjusting the production of ECM com-
ponents and cross-linking them into the appropriate matrix.
ATTACHMENT TO AND INTERACTION
WITH THE EXTRACELLULAR MATRIX
In the context of attachment to and interaction with the
ECM, integrins are important. Integrins are a complex
­family of adhesion molecules that contain heterodimers of
α and β chains. So far, at least 16 different α chains and 8
201
 202 PAP  |  Fibroblasts and Fibroblast-like Synoviocytes
Cell-cell interactions Chemical stimuli
(inflammatory cells, (e.g., pH oxygen
endothelial cells) tension)
Soluble mediators Extracellular matrix
(e.g., cytokines, (e.g., collagen,
growth factors) fibronectin)
Fibroblasts
Organ-specific reaction
(production and resorption of extracellular matrix,
production of soluble mediators, expression
of surface adhesion molecules)
Figure 11-1  Fibroblast stimulation signals. Fibroblasts are very
s­ ensitive cells that integrate signals from soluble mediators, cell-cell in-
teractions, chemical stimuli, and the extracellular matrix into a coordi-
nated, organ-specific response. This response includes remodeling of the
extracellular matrix, the secretion of soluble mediators, and the expres-
sion of surface molecules for cell-cell and cell-matrix interactions.
different β chains have been described that can combine
into at least 24 different integrins. For the attachment of
fibroblasts to the ECM components of connective tissue and
cartilage, β1 integrins are especially important. α1β1, α2β1
Integrins are the main adhesion molecules responsible for
the attachment of fibroblasts to collagen, but some data sug-
gest that the recently described α10β1 and α11β1 integrins
might also be involved. Other β1 integrins, such as α4β1
and α5β1 integrins, mediate the attachment of fibroblasts
to fibronectin and its spliced variants; in addition, αv inte-
grins are responsible for the attachment to vitronectin. The
engagement of integrin receptors on the surface of fibroblasts
results in the formation of focal adhesion complexes. These
focal adhesion complexes activate intracellular signaling
cascades that regulate the transcription of genes, thereby
controlling cell proliferation and survival, the secretion of
certain cytokines and chemokines, and matrix deposition
and resorption (Fig. 11-2).
Among the signaling molecules that transmit sig-
nals from the integrins to the cell interior, focal adhesion
kinase (FAK) plays a central role.4 FAK, a tyrosine kinase,
is recruited into newly established focal contacts and, in
turn, recruits other adapter proteins such as p130Cas and
Grb2. This leads to the activation of phosphatidylinositide
3-kinase (PI3K) and Src-kinase and promotes the initiation
of a variety of signaling cascades, such as the Raf-MEK-
ERK pathway. It is important to note, however, that these
signaling pathways can also be activated through FAK-
independent signaling events, such as through growth
factors. The exact mechanisms by which different signals
cooperate to mediate a specific response of fibroblasts and
how this translates into distinct pathologies are not yet fully
defined.
The picture is further complicated by the fact that
in addition to integrins, other cell surface molecules are
involved in the attachment of fibroblasts to ECM com-
ponents and in their response to growth factors. Among
these, transmembrane heparan sulfate proteoglycans have
generated special interest recently. Syndecans, a family of
such transmembrane heparan sulfate proteoglycans, possess
Paxillin
Integrin
receptor
Alpha Beta
subunit subunit
RAS
p130Cas
SHC SOS
FAK Raf
FYN Grb2
Src
P13K MEK
Actin
stress ERK
fibers Akt
Transcription
factors
Figure 11-2  Integrin signaling in fibroblasts. The engagement of
­integrins on the cell surface of fibroblasts results in the initiation of
signaling cascades that result in changes in (1) cell motility through
­reorganization of the cytoskeleton, (2) cell survival (e.g., through
­activation of the Akt-NFκB pathway), and (3) the production of matrix
molecules, matrix-degrading enzymes, and soluble mediators through
the activation of mitogen-activated protein kinases.
highly conserved cytoplasmic and transmembrane domains
but have highly variable extracellular domains. The extra-
cellular domains of syndecans contain different numbers of
glycosaminoglycan side chains through which they interact
with factors released during tissue injury, such as growth
factors and interleukins (ILs), but also with many ECM
components and adhesion molecules5 (Fig. 11-3). Syn-
decans are expressed on fibroblasts in a tissue-specific and
development-dependent manner. Data from syndecan knock-
out mice indicate that syndecan-4 is particularly important
for the fibroblast response to tissue injury. Syndecan-4–
­deficient mice show no gross abnormalities and are viable.
Under certain stress conditions, however, these mice show
differences compared with wild-type controls. They exhibit
alterations in wound healing, and the response of synde-
can-4–deficient fibroblasts to fibronectin attachment is sig-
nificantly altered.6 Also, syndecan-4–deficient fibroblasts are
compromised in their ability to differentiate into α-smooth
muscle actin–expressing myofibroblasts. The exact mecha-
nisms by which syndecans are involved in the functions of
different fibroblast populations remain to be determined, but
it appears that syndecans are important cell surface receptors
that, owing to their unique properties, can integrate signals
from ECM components and soluble ­factors.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
ECM-molecules
Growth factors
Heparan sulfate
attachment sites
Cell adhesion
domain (NXIP)
Shedding sites for
plasmin and thrombin
Transmembrane domain
C1
Conserved (C) and
V variable (V)
intracellular domains
C2
Figure 11-3  Structure of syndecans. Syndecans are transmembrane
heparan sulfate proteoglycans, some of which (e.g., syndecan-4) are
expressed prominently on mesenchymal cells, particularly fibroblasts.
Through their heparan sulfate side chains, they are able to bind not only
matrix components such as fibronectin but also growth factors. Their
intracellular part contains two highly conserved regions (C1 and C2), as
well as one variable domain (v). Activation of syndecans results in both
the engagement of focal contacts and the initiation of intracellular sig-
naling cascades that are not yet fully understood.
DEGRADATION OF EXTRACELLULAR MATRIX
BY FIBROBLASTS
In addition to being responsible for the deposition of ECM
molecules and their assembly, fibroblasts are involved in the
destruction and removal of the ECM. To this end, fibroblasts
produce a great variety of matrix-degrading enzymes, includ-
ing matrix metalloproteinases (MMPs) and cathepsins, that
can cleave ECM components. A detailed description of the
enzymes responsible for the degradation of cartilage- and bone-
associated matrix components is provided in Chapter 7. How-
ever, it is important to note that fibroblasts constitute a major
source of these enzymes and thus are prominently involved in
the degradation of ECM, particularly under disease conditions.
With the exception of MMP-2 and the MT-MMPs, which
are constitutively expressed by fibroblasts, MMP expression
is regulated by extracellular signals via transcriptional acti-
vation in fibroblasts. Three major groups of inducers can be
differentiated: proinflammatory cytokines, growth factors,
and matrix molecules. Among the cytokines, IL-1 is per-
haps the most potent inducer of a variety of MMPs, includ-
ing MMP-1, -3, -8, -13, and -14. Fibroblast growth factor
(FGF) and platelet-derived growth factor (PDGF) are also
known inducers of MMPs in fibroblasts, as they potenti-
ate the effect of IL-1 on MMP expression. Matrix proteins
(collagen, fibronectin) and especially their degradation
­products activate MMP expression in fibroblasts, providing
the possibility for site-specific MMP activation in regions of
matrix breakdown.7 Interestingly, studies have shown that
­microparticles derived from immune cells can directly stim-
ulate the expression of both inflammatory cytokines and
­disease-relevant MMPs in fibroblast-like synoviocytes.8
In fibroblasts, several pathways mediate signaling for the
transcriptional activation of MMPs. The activator protein-1
(AP-1) binding site is present in the promoter region of all
203
MMPs (except MMP-2), ­suggesting a central role of Jun/Fos
transcription factor binding. Indeed, there is ample experi-
mental evidence that all three mitogen-activated protein
kinase MAPK families—extracellular signal-regulated kinase
(ERK), c-Jun N-terminal kinase (JNK), and p38 kinase—
integrate extracellular signals upstream from jun/fos and are
involved in the regulation of MMP expression. Especially, the
induction of MMP-1, -9, and -13 is ­mediated through MAPK
signaling.9-11 Aside from AP-1, the promoter regions of some
MMPs contain nuclear factor κB (NFκB),9-11 STAT,12 and Ets13
binding sites. Activation of these transcription factors occurs
during the induction of MMP-1, -3, and -13, which are also
thought to be essential for the joint damage in rheumatoid
arthritis (RA). Activation of the various MAPK and tran-
scription factors, none of which are tissue-specific signaling
molecules, occurs in distinct subcompartments of the rheu-
matoid joint, thus determining a particular pattern of MMP
expression in the synovium.14
HETEROGENEITY OF FIBROBLASTS
Although fibroblasts share a number of characteristic
­features, as outlined earlier, they are far from being a uni-
form cell lineage. Rather, fibroblasts differ substantially
from one tissue to another. These differences become obvi-
ous when their growth and proliferation are investigated
together with the distinct profiles of the ECM components
they produce.
There are also differences in the production of cytokines,
chemokines, and growth factors by fibroblasts from differ-
ent organs. These differences can be explained by the dis-
tinct embryonic origins of fibroblasts in various body sites,
as well as by the specific tissue environment in the adult
organism. More importantly, however, there are significant
differences between fibroblasts within tissues. Heterogene-
ity of fibroblasts has been shown in the skin and the lung,
as well as in the synovial membrane, where fibroblasts in
the most superficial lining differ substantially from those in
the deeper layers (also called the sublining). Under disease
conditions such as RA, the heterogeneity of fibroblasts is an
important factor in the pathogenesis of disease; fibroblasts of
the most superficial layer of the rheumatoid synovium play a
key role in the progressive destruction of articular cartilage
and bone, thus contributing to disease progression.
FIBROBLASTS IN RHEUMATIC DISEASES
As outlined earlier, fibroblasts play a central role in ­ensuring
physiologic tissue homeostasis. Under disease conditions
such as inflammation, the role of fibroblasts becomes even
more central: they are critical switches that regulate the
response to tissue injury. Thus, fibroblasts contribute to the
resolution or perpetuation of organ-specific pathology and
largely determine the consequences of disease. In addition to
the production and resorption of matrix components, fibro-
blasts are involved in angiogenesis and regulate the accumu-
lation of inflammatory cells. To this end, fibroblasts secrete
chemokines and mediate the subsequent recruitment of
inflammatory cells to sites of tissue injury. In addition, they
can inhibit programmed cell death in inflammatory cells15
and modulate the activity and behavior of many cell lin-
eages through direct cell-to-cell interactions16 (Fig. 11-4).
 204 PAP  |  Fibroblasts and Fibroblast-like Synoviocytes
Production and resorption
Disease-specific of extracellular matrix
activation
Accumulation of
inflammatory cells
Fibroblasts
Neoangiogenesis
Figure 11-4  Role of fibroblasts in disease. Under disease conditions,
fibroblasts becomes important triggers that can, for instance, mediate
the switch from acute resolving to chronic persisting inflammation. The
role of fibroblasts in inflammatory diseases is not restricted to the pro-
duction of extracellular matrix; it also includes the recruitment and ac-
cumulation of inflammatory cells as well as neoangiogenesis.
Elucidation of the mechanisms by which fibroblasts
c­ ontribute to the perpetuation of inflammatory disease
and the progression of rheumatic disorders has significantly
changed our view of these cells.17 Initially, fibroblasts were
thought to be more or less passively responding cells that,
upon stimulation by environmental triggers, reacted by
changing the deposition or resorption of matrix. This view
has been replaced by the realization that these cells are cen-
tral components of organ-specific homeostasis and pathol-
ogy. It is understood that fibroblasts are a key part of the
immune system and integrate signals from different sources
into a coordinated tissue response.18 In this context, fibro-
blasts themselves may undergo fundamental changes while
responding to environmental stimuli. This is obvious, based
on observations that, during wound healing and under
fibrotic conditions, fibroblast-like cells are transformed into
myofibroblasts, which are distinct from fibroblasts in terms
of both their phenotype and their behavior.19 Such fibrotic
transformation of fibroblasts is also characteristic of systemic
sclerosis, a generalized fibrotic disorder that affects the skin
and various internal organs such as the lungs, heart, and
gastrointestinal tract. The overproduction of ECM compo-
nents, particularly type I, III, VI, and VII collagen, by skin
fibroblasts is a hallmark of the disease and is closely linked
to the disease-specific activation of these fibroblasts. This
pattern of activation includes not only a distinct profile
of ECM overproduction but also altered responses to both
inflammatory mediators and immune cells.20
Several lines of evidence suggest that in chronic,
­destructive arthritides such as RA, there is also stable
activation of fibroblast-like cells of the synovial inti-
mal lining, and this activation is maintained even in the
absence of continuous stimulation by inflammatory trig-
gers.21 This process appears to be distinct from that seen
in fibrotic disorders, and fibroblasts that have undergone
this ­activation show certain features of tumor cells, such as
anchorage-independent growth, firm attachment to ECM
molecules of the cartilage, alterations in their response
to apoptotic stimuli, and invasiveness toward articular
cartilage and bone (see later). This activation process is
sometimes referred to as tumor-like transformation. The
underlying mechanisms for this transition are not entirely
clear. However, there is ­evidence that the chronic ­exposure
of fibroblast-like cells in the synovial lining to the combina-
tion of inflammatory ­ cytokines, growth factors, and ECM
components, together with less clearly defined environmen-
tal triggers such as reactive oxygen or nitrogen hypoxia22
and possibly bacterial or viral products23 and microparticles,8
results in the imprinting or stable activation of these cells
into an aggressive phenotype. As a consequence of this sta-
ble activation, the disease process is perpetuated and might
even progress when inflammation is ameliorated or con-
trolled through anti-inflammatory treatment.
FIBROBLAST-LIKE SYNOVIOCYTES
IN RHEUMATOID ARTHRITIS
About one third of the cells in the most superficial lin-
ing layer of the rheumatoid synovium appear to originate
from resident synovial cells, which have a fibroblast-like
appearance and lack specific surface markers. They have
been called type B synoviocytes or fibroblast-like syno­
viocytes and can be identified by antibodies recognizing
prolyl-4-hydroxylase24 or antibodies against Thy-1/CD90.25
In addition to infiltration with inflammatory cells, the
increased number of these fibroblast-like synoviocytes con-
tributes significantly to many aspects of synovial pathol-
ogy—namely, hyperplasia (especially that of the lining
layer), joint destruction, and perpetuation of chronic
inflammation.17,26 It is important to note that fibroblasts
in the rheumatoid synovium differ significantly from nor-
mal fibroblast-like synoviocytes and, as part of a complex
cellular network, contribute to joint destruction by direct
mechanisms as well as through interaction with neighbor-
ing cells.27 In RA, fibroblast-like synoviocytes show features
of stable cellular activation, which provides the basis for
both their direct and indirect effects on joint destruction.
STABLE ACTIVATION OF FIBROBLASTS
IN THE RHEUMATOID SYNOVIUM
The hypothesis that activated fibroblast-like synoviocytes
are involved in rheumatoid joint destruction is based on
observations that date back to the 1970s.28 By analyzing
large numbers of synovial specimens from RA patients, it
was found that invasion of cartilage and subchondral bone
by synovial lining cells did not require the presence of
inflammatory cells. Moreover, fibroblast-like ­ synoviocytes
from RA patients exhibit considerable morphologic altera-
tions, including an abundant cytoplasm; a dense, rough
endoplasmic reticulum; and large, pale nuclei with sev-
eral prominent nucleoli21,28 (Fig. 11-5). Based on these
­morphologic ­analyses, studies in mice with severe combined
­immunodeficiency (SCID) were performed to investigate
the “transformed-appearing” phenotype of rheumatoid
fibroblasts. In this model, RA fibroblast-like synoviocytes
were coimplanted with normal human cartilage into SCID
mice (Fig. 11-6). Because of their defective immune systems,
these mice did not reject the implants, allowing investiga-
tors to study the aggressive behavior of RA fibroblasts in
the absence of human inflammatory cells and their factors.
It was shown that rheumatoid fibroblasts in this model not
only exhibited an invasive phenotype but also, and even
more intriguingly, were able to maintain this phenotype over
­prolonged periods in the absence of ­continuous stimulation
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
Figure 11-5  Phase contrast picture of rheumatoid arthritis fibroblast-
like synoviocytes.
by an ­inflammatory environment. A number of groups set
out to characterize the specific phenotype of fibroblast-like
synoviocytes in RA, and interest focused mainly on the char-
acteristics of these cells at the cellular and molecular levels,
as well as on the mechanisms of activation with respect to
their destructive properties. The results have shown that
the stable activation of RA fibroblast-like synoviocytes is
accompanied by alterations in a variety of proto-oncogenes
and tumor ­suppressors and ultimately results in the activation
of adhesion molecules, the production of matrix-degrading
enzymes, and changes in the susceptibility to programmed
cell death (Fig. 11-7).
ALTERED EXPRESSION OF PROTO-ONCOGENES
Expression of proto-oncogenes and transcriptional factors in
fibroblast-like synoviocytes is a major indication of the acti-
vated nature of these cells.29 A number of oncogenes such as
c-fos, ras, raf, sis, myb, and myc have been detected at ele-
vated levels in RA patients and are predominantly expressed
by synovial cells attaching cartilage and bone.29 Binding
sites for early-response genes such as egr-1 can be identified
in the promoters of the oncogenes sis and ras, and about
70% of RA patients exhibit high expression of Ras and Myc
proteins in synovial lining cells.30 The cysteine proteinase
cathepsin L, which is the major ras-induced protein in ras-
transformed murine NIH 3T3 cells, was detected in 50% of
RA cases, predominantly in synovial cells.31 Interestingly,
cathepsin L was colocalized with ras and myc. Some of these
proto-oncogenes appear to be directly involved in the regu-
lation of different MMPs. Gelatinases (MMP-2 and MMP-9)
and MT1-MMP are likely regulated by growth factors that
mediate their effects through the ras proto-oncogene, and
c-Ras plays a critical role in the increased expression and
proteolytic activation of MMPs in fibroblasts.32,33 Recently,
gene transfer with dominant negative (dn) mutants of Raf-1
and c-Myc demonstrated the relevance of the Ras-Raf-
MAPK pathways for the activation and invasive behavior of
205
Rheumatoid fibroblasts Normal human
or articular cartilage
control fibroblasts
Invasion No invasion
Figure 11-6  Severe combined immunodeficiency (SCID) mouse
model of rheumatoid cartilage destruction. Rheumatoid arthritis fibro-
blast-like synoviocytes are implanted along with normal human cartilage
into SCID mice. These mice do not reject the implants, allowing investiga-
tors to study the aggressive behavior of rheumatoid arthritis fibroblasts
in the absence of human inflammatory cells and their factors for at least
60 days. Rheumatoid fibroblasts (left) exhibit an invasive phenotype
and maintain this phenotype over prolonged periods in the absence of
­continuous stimulation by an inflammatory environment; in ­ contrast,
there is no such invasion with normal or osteoarthritic fibroblasts (right).
This experiment can be taken as evidence of the stable activation of
rheumatoid fibroblasts. At the same time, this model can be used to
study therapeutic effects on the fibroblasts.
­rheumatoid fibroblast-like synoviocytes.34 The clear effect of
dn-Raf-1 and dn-c-Myc on the invasiveness of rheumatoid
fibroblast-like synoviocytes in the SCID mouse model is in
line with the concept that the activation of relevant signal-
ing pathways is maintained in RA fibroblast-like synovio-
cytes in the absence of human inflammatory cells. Taken
together, different data suggest that the pathologic expres-
sion of proto-oncogenes constitutes an important step lead-
ing to the expression of matrix-degrading enzymes in RA
and progressive joint destruction.
Alterations in different tumor suppressor genes may
explain some features of fibroblast activation and sur-
vival in RA. For instance, somatic mutations of the tumor
suppressor gene p53 have been described in rheumatoid
synoviocytes.35 Based on additional evidence that some
of these mutations can exert dominant negative effects,
it has been hypothesized that the accumulation of p53
mutations may contribute to the activation of rheumatoid
fibroblast-like synoviocytes. Although mutations in the
p53 gene appear to show great variability, such mutations
may nonetheless constitute one mechanism that imprints
the aggressive behavior into RA fibroblast-like synovio-
cytes. This notion is supported by data showing that
inhibition of p53 in normal fibroblast-like synoviocytes
results in an invasive, RA-like phenotype.36 RA-specific
­expression of p53 in the synovial tissue in both the early
and late stages of RA has been demonstrated.37 Hence,
the local environment in the rheumatoid joint might
cause altered expression and function of p53 in a subset of
­rheumatoid fibroblasts, which in turn contributes to the
stable ­activation of these cells.38
 206 PAP  |  Fibroblasts and Fibroblast-like Synoviocytes
Proto-oncogenes
tumor suppressors
β1-integrins
syndecans
MMPs
cathepsins
Attachment
to cartilage
Matrix degradation
Figure 11-7  Invasive phenotype of rheumatoid arthritis fibroblast-like synoviocytes. As a result of the stable activation of rheumatoid arthritis
fibroblasts, these cells show three distinct features: increased expression of adhesion molecules, leading to attachment to the articular cartilage; up-
regulated production of matrix-degrading enzymes such as matrix metalloproteinases and cathepsins; and alterations in programmed cell death, which
contribute to synovial hyperplasia.
Aggressive RA fibroblast-like ­synoviocytes lack the expres-
sion of mRNA for the tumor suppressor phosphatase and
­tensin homolog (PTEN). PTEN is a tyrosine phosphatase
that shows homology to the cytoskeletal proteins tensin and
auxillin. Mutations in PTEN have been described in differ-
ent malignancies and are associated with their invasiveness
and metastatic properties.39,40 In RA, no mutations of PTEN
have been found, but rheumatoid synovium shows a distinct
pattern of PTEN expression, with negligible staining in the
lining but very strong expression in the sublining.41 These data
suggest that the lack of PTEN expression is an intrinsic feature
of rheumatoid fibroblast-like synoviocytes,41 which is of inter-
est because PTEN is involved in the regulation of FAK,42 and
some data indicate that PTEN is an essential mediator of FasL-
mediated apoptosis through the PI3K–Akt–NFκB pathway.43
Interesting data have come from studies of the transcrip-
tional factor NFκB in RA fibroblast-like synoviocytes. NFκB
is a dimeric (p65, p50), regulatory, DNA-binding protein
that interacts with a number of different signal cascades.
Several studies have demonstrated that NFκB is highly
activated in the synovial membrane of RA patients and
constitutes a key integrating factor for intracellular as well
as cytokine-mediated activation pathways.44 Thus, NFκB
is an important molecule that mediates the resistance of
fibroblasts against apoptosis45,46 and regulates inflammatory
cytokines,45,47 adhesion molecules,45,48 and matrix-degrad-
ing enzymes.45,49 The activation of NFκB in RA fibroblast-
like synoviocytes appears to constitute an important link
among synovial inflammation, hyperplasia, and matrix
­degeneration.44,45
Fas
FLIP
SUMO-1
BcI-2
Stable
activation
Altered
apoptosis
RESISTANCE AGAINST APOPTOSIS
Synovial fibroblasts from patients with RA are activated but
do not proliferate faster than those from patients with osteo-
arthritis. Using thymidine incorporation, only 1% to 5% of
synovial cells have been found to proliferate,50 and immu-
nohistochemistry for specific proliferation markers such as
Ki-67 revealed a very low number of positive cells.51 Also,
only 1% of fibroblast-like cells expressing proto-oncogenes
such as jun-B and c-fos were positive for Ki-67, indicating
that the majority of RA fibroblast-like synoviocytes do not
show accelerated proliferation.52 In contrast, more recent
data provide growing evidence of changes in the apoptotic
pathways in RA synovium, particularly within the lining
layer (for a review, see reference 53). When examined by
ultrastructural methods, less than 1% of lining cells exhibit
morphologic features of apoptosis.54
Mitochondrial Pathways of Apoptosis
Generally, apoptosis can be induced by intrinsic mito-
chondrial pathways or triggered through cell surface death
receptors.53,55 Members of the Bcl family have been identi-
fied as important regulators of mitochondrial pathways of
apoptosis. Specifically, Bcl-2 exerts strong antiapoptotic
effects and contributes to the pathogenesis of experimental
arthritis.56 In situ analysis has shown an increased presence
of Bcl-2 in human RA fibroblast-like synoviocytes,57 and
enhanced expression of Bcl-2 in rheumatoid fibroblast-like
synoviocytes correlates with synovial lining thickening and
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
­inflammation.58 Of interest, stimulation of RA fibroblast-like
synoviocytes with IL-15 suppresses Bcl-2 and Bcl-x(L)
mRNA.59 Apoptosis can be increased when the autocrine
stimulation of fibroblast-like synoviocytes with IL-15 is
inhibited.
Pathways of Receptor-Mediated Apoptosis
In addition to changes in the mitochondrial pathway of
apoptosis, there is evidence that fibroblast-like synoviocytes
are resistant to receptor-induced apoptosis. These receptors
include members of the tumor necrosis factor (TNF) recep-
tor family, specifically, TNF receptor 1 (TNFR1), TNF-
related apoptosis-inducing ligand (TRAIL) receptors 1 and
2, and Fas receptor. They all possess conserved intracellular
motifs, termed death domains, that, upon receptor activa-
tion, induce the formation of a signaling complex that initi-
ates the apoptotic cascade.
Several factors are involved in this resistance of fibroblast-
like synoviocytes against death receptor–induced cell death,
but the ultimate mechanisms are incompletely understood.
It is currently estimated that only about 15% of synovial
fibroblasts are susceptible to Fas.21 It has been conjectured
that the extensive resistance to Fas-induced apoptosis
despite the surface expression of Fas is conveyed by inflam-
matory cytokines present in the synovial fluid, such as TNF-α
and transforming growth factor-β (TGF-β), but also by sol-
uble Fas, elevated levels of which have been found in the
synovial fluid of patients with RA.60 However, this resistance
against Fas-induced apoptosis is maintained in vitro, indicat-
ing that intrinsic factors are also responsible for this feature.
Some evidence points to a role for the PI3K-Akt pathway in
Fas resistance. The antiapoptotic FLICE inhibitory protein
(Flip) is also elevated in the rheumatoid synovium, espe-
cially in the lining layer,61 and it correlates with low levels
of apoptosis in early RA.62 It is induced by TNF-α63 and pre-
vents the interaction of caspase-8 with the Fas-associated
death domain adapter protein. Its downregulation sensitizes
rheumatoid fibroblasts to Fas-induced apoptosis.64
Another molecule that modulates downstream mecha-
nisms of Fas is SUMO-1. It is a small ubiquitin-like protein
that, in contrast to ubiquitination, does not lead to the deg-
radation of proteins. Rather, SUMOylation results in altered
binding of modified proteins to subsequent substrates and
significantly affects the signaling of SUMOylated proteins.65
SUMO-1 also interacts with the Fas- and TNFR1-associated
death domain and protects against Fas- and TNFR1-induced
apoptosis.66 In the rheumatoid synovium, there is marked
expression of SUMO-1, predominantly in fibroblasts of the
lining layer and at sites of cartilage invasion. Normal synovial
tissues and synovial tissues of osteoarthritis patients do not
show prominent expression of SUMO-1.67 Intriguingly, rheu-
matoid fibroblasts maintain the high expression of SUMO-1
when analyzed in the SCID mouse model, and recent data
suggest that SUMO-1 is indeed involved functionally in
rheumatoid fibroblasts’ resistance to Fas-induced apoptosis.
Tumor Necrosis Factor-α
Although TNF-α can activate the apoptotic pathway through
caspase-3 and caspase-8, it can also initiate pathways diverg-
ing downstream of the TNF receptor–binding protein TNF
207
Receptor-Associated Death Domain (TRADD) and leading
to the activation of NFκB,68 which in TNF Receptor-Asso-
ciated Death Domain (turn promotes cell survival and the
production of proinflammatory factors.69 In RA fibroblasts,
TNF-α inhibits apoptosis and induces cell death only after
the inhibition of NFκB.70 TNF-α has been demonstrated to
rapidly and potently activate Akt kinase via PI3K, and inhi-
bition of PI3K strongly potentiates TNF-α–induced apopto-
sis.71 Apparently, however, not all the antiapoptotic effects
of TNF-α through the Akt pathway depend on NFκB.50
According to some studies, TNF-α interferes with Fas-medi-
ated apoptosis at physiologic levels and induces apoptosis
only at 100-fold higher levels. Of note, stimulation of rheu-
matoid fibroblast cell cultures with TNF-α also significantly
increases the production of soluble Fas.72 In this context,
it was recently shown that the tissue inhibitor of metallo-
proteinase-3 (TIMP-3) can sensitize rheumatoid fibroblast
to Fas ligand–induced apoptosis when expressed through
adenoviral gene transfer.72 In addition, adenoviral delivery
of TIMP-3 completely reverses the apoptosis-inhibiting
effects of TNF-α in these cells. These findings provide a link
between matrix degradation and rheumatoid fibroblasts’
resistance to apoptosis and indicate that overexpression of
TIMP-3 in such cells may have beneficial effects by both
inhibiting matrix degradation and facilitating cell death.
Taken together, these data suggest that apoptosis-
­suppressing signals outweigh apoptosis-inducting signals,
which can cause an imbalance of pro- and antiapoptotic path-
ways in synoviocytes. This may lead to an extended life span
of synovial lining cells and result in a prolonged expression of
matrix-degrading enzymes at sites of joint destruction.73
ATTACHMENT
The attachment of fibroblast-like synoviocytes to joint carti-
lage is one of the most prominent features of joint destruction.
This process appears to be pivotal for inflammatory arthritis.
Integrins
Integrins, which are key adhesion molecules for fibroblasts
(discussed earlier), are of interest not only because of their
function as receptor molecules but also because of their
interaction with several signaling pathways and cellular
proto-oncogenes.74 For instance, the expression of early
cell-cycle genes such as c-fos and c-myc is also ­ stimulated
by integrin-mediated cell adhesion, and gene ­ expression
driven by the fos promoter strongly synergizes with integrin-
mediated adhesion.75 Studies have demonstrated that, apart
from being expressed on lymphocytes, several β1 integrins
are highly expressed on fibroblast-like synoviocytes.76,77 The
binding of fibroblast-like synoviocytes to ECM is inhib-
ited, at least in part, by anti–ß1 integrin antibodies, with
the blocking efficacy being significantly higher in rheuma-
toid than in normal fibroblast-like synoviocytes.76 Of note
is that different integrins function as fibronectin receptors,
which is why the fibronectin-rich environment of the car-
tilage surface might facilitate the adhesion of fibroblast-like
synoviocytes to cartilage. In this context, it is important
to determine how the loss of ­cartilage components during
disease progression affects the adhesion of fibroblast-like
sy­noviocytes to the articular cartilage.
 208 PAP  |  Fibroblasts and Fibroblast-like Synoviocytes
Integrins not only facilitate the attachment of cells to
the ECM but also mediate intracellular signaling pathways.
Integrin signals are crucial for the growth of a number of
cell types. Specifically, rheumatoid fibroblasts require inte-
grin cosignaling for proliferation upon PDGF binding.78
Integrins contribute to Ras-dependent pathways79 involving
ERK, JNK, and Akt and modulate the activation of MAPKs
in response to growth factors.80 Importantly, the activation
of ß1 integrins induces the expression of proteases con-
tributing to ECM degradation,81 and their inhibition by
antibodies reduces the invasive capacity of fibroblast-like
synoviocytes.82
Vascular Adhesion Molecule 1
Vascular adhesion molecule 1 (VCAM-1; CD106) is a
member of the immunoglobulin gene superfamily and may
contain either six or seven immunoglobulin domains of
the H type.83 Several studies have demonstrated increased
VCAM-1 expression in inflamed synovium compared with
normal and osteoarthritic synovial tissues. Although some
data suggest that macrophage-like cells are the major source
of VCAM-1 in RA synovial tissue,84 most studies reveal
high expression of VCAM-1 mainly in fibroblast-like syn-
oviocytes. Moreover, different data suggest that VCAM-1
is particularly upregulated in the subpopulation of activated
lining fibroblasts.85-87 Increased expression of VCAM-1 is
associated with cell and subsequently articular cartilage
invasion.85 Studies in the SCID mouse model revealed
increased expression of VCAM-1 in RA fibroblast-like
synoviocytes, even in the absence of human inflammatory
cells.88 In addition to mediating the attachment to cartilage,
VCAM-1 produced by fibroblast-like synoviocytes might
contribute to T cell anergy,89 B cell pseudoemperipolesis,16
and angiogenesis.90
MATRIX DEGRADATION
Progressive joint destruction distinguishes RA from other
inflammatory joint diseases and is mediated by the con-
certed action of various proteinases. The most prominent of
these are MMPs and cathepsins,91,92 and several reports have
implicated MMPs in joint destruction (for a more compre-
hensive review of MMPs in RA, see reference 91).
Expression of Matrix Metalloproteinases
MMP-1 is found in the synovial membranes of all RA
patients but is present in the synovial samples of only about
55% to 80% of trauma patients.93 Synovial lining cells
produce MMP-1 in the diseased synovium, and MMP-1 is
released from these cells immediately after production (for a
review, see reference 94). As a result, expression of MMP-1
in the synovial fluid correlates with the degree of synovial
inflammation.95 However, serum concentrations of MMP-1
do not appear to reflect the levels in the synovial fluid; there-
fore, measuring serum MMP-1 has not been established as a
marker for disease activity.
MMP-3, or stromelysin, has a key role in joint ­destruction
because it not only degrades matrix molecules but also acti-
vates other pro-MMPs into their active forms. MMP-3 is
produced abundantly by ­ fibroblast-like ­ synoviocytes when
stimulated with ­ macrophage-­conditioned medium,96 and
fibroblast-like synoviocytes in the lining layer are a major
source of MMP-3 in synovium.97 Synovial fluids from
patients with RA contain about 100-fold higher concentra-
tions of MMP-3 than control samples,98 and increased lev-
els of MMP-3 have been found in the sera of patients with
RA.99 These increased serum levels correlate with systemic
inflammation at clinical and serologic levels,100-102 but it is
unclear whether levels of circulating MMP-3 reflect radio-
graphic damage. No correlation between serum MMP-3
levels and radiographic or functional scores was found,101
and there were no differences in the serum levels of MMP-3
in long-standing RA patients with low versus high erosion
scores.102 In contrast, other data indicate that serum MMP-3
levels predict joint damage at early stages of the disease.103
MMP-13, or collagenase-3, is also expressed at the
mRNA104 and protein levels,105 especially in the lining layer
of inflammatory synovium. Because of this localization,
along with its substrate specificity for collagen type II and
its relative resistance to known MMP inhibitors, MMP-13
is thought to play an important role in joint destruction.
Of interest, expression of MMP-13 correlates with elevated
levels of systemic inflammation markers,106 but studies of
osteoarthritis clearly demonstrated that the expression
of MMP-13 is not specific for RA. Rather, it appears that
MMP-13 is closely associated with the degeneration of car-
tilage in several pathologies.
MT-MMPs are also abundantly expressed in cells that
aggressively destroy cartilage and bone in RA.107 MT1-MMP
is produced constitutively by fibroblast-like synoviocytes,
and elevated levels have been found in RA. This is impor-
tant because MT1-MMP degrades ECM components as
well as activates other disease-relevant MMPs such as
MMP-2 and MMP-13. In a recent study that compared the
expression of MT-MMPs in RA, MT1-MMP was found to
be of particular relevance to RA.107 In this analysis, the
expression of MT3-MMP mRNA was seen in fibroblasts
and some macrophages, particularly in the lining layer;
however, the expression of MT2- and MT4-MMP was
characterized by scattered staining of only a few CD68–
fibroblasts.
Regulation of Matrix Metalloproteinases
The expression of MMPs in synovial cells is regulated by
several extracellular signals, including inflammatory cyto-
kines, growth factors, and molecules of the ECM. Among
the inflammatory cytokines, IL-1 and TNF-α are impor-
tant inducers of MMPs. IL-1 induces the expression of a
variety of MMPs, including MMP-1, -3, -8, -13, and -14.91
However, as demonstrated in several studies using anti-
gen-induced arthritis in animals, IL-1 not only enhances
the production of MMPs but also suppresses the synthe-
sis of ­proteoglycans.108 In some of these studies, anti–IL-1
treatment ­normalized chondrocyte synthetic function and
reduced the activation of MMPs.109 These data support
observations that overexpression of the IL-1 ­receptor antag-
onist using retroviral gene transfer significantly reduces
perichondrocytic matrix degradation in the SCID mouse
model.110 However, the mechanisms by which ­ cytokines
such as IL-1 induce MMPs are variable and depend on the
cell type.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
Other macrophage-derived inflammatory cytokines such
as TNF-α have also been shown to amplify the destructive
process by stimulating the expression of some MMPs. For
example, TNF-α can stimulate the production of MMP-1
in cultured synovial cells.111,112 However, animal models of
arthritis indicate a difference in the relative importance
of TNF-α and IL-1 with respect to inflammation and joint
destruction. Whereas TNF-α appears to be responsible
primarily for the extent of synovitis, IL-1 seems to have a
greater impact on the destruction of cartilage.113
Other cytokines that regulate the expression of MMP
in the RA synovium include IL-17 (MMP-1 and -9)114
and TGF-β (MMP-13).115 Growth factors such as FGF and
PDGF are also potent inducers of MMPs and potentiate
the effect of IL-1.116 MMP expression can also be induced
by matrix proteins such as collagen and fibronectin; their
degradation products in particular can activate the expres-
sion of MMPs in chondrocytes and fibroblasts, which sug-
gests the possibility of specific activation of MMPs at sites
of matrix degradation.7,117 As a consequence, the synthesis
of matrix-degrading enzymes in inflamed joints is regulated
not only by inflammatory cytokines and growth factors but
also by cleavage products of the destroyed matrix itself in an
amplifying fashion.
A close correlation between the expression of MMP-1,
-3, and -10 with the invasive growth of RA fibroblast-like
synoviocytes has been reported,118 but the specific contri-
bution of individual MMPs to matrix degradation is only
partly understood. Using gene transfer of ribozymes to
MMP-1, specific inhibition of MMP-1 significantly reduced
the production of this enzyme in RA fibroblast-like sy-
noviocytes and inhibited the cells’ invasiveness in the
SCID mouse model.119 In a similar study, gene transfer of
antisense RNA expression constructs against MT1-MMP, a
membrane-anchored MMP, also inhibited the invasiveness
of RA fibroblast-like synoviocytes. These techniques might
clarify the role of individual MMPs in joint destruction and
permit the development of drugs that selectively target dis-
ease-relevant MMPs.
Tissue Inhibitors of Metalloproteinase
MMP activity is normally balanced by the naturally
­occurring tissue inhibitors of metalloproteinase such as
TIMP-1 and TIMP-2. They interact with MMP-1 and
MMP-3 and are synthesized and secreted by chondrocytes,
fibroblast-like synoviocytes, and endothelial cells.120-122 In
situ hybridization studies demonstrated striking amounts
of TIMP-1 mRNA in the synovial lining of patients with
RA.120 However, the molar ratio of MMP to TIMP, rather
than the absolute level of TIMP, is crucial for joint destruc-
tion. In RA, the amount of MMPs produced far outweighs
the TIMPs produced, allowing destruction to take place.
This notion has been supported by data demonstrating that
the overexpression of TIMP-1 and TIMP-3 by gene trans-
fer may result in a marked reduction in the invasiveness of
RA fibroblast-like synoviocytes.123 Of interest, in addition to
inhibiting the degradation of ECM, TIMP-3 has a number
of distinctive features; for example, TIMP-3 prevents the
shedding of cell membrane proteins such as TNF receptor124
and IL-6 receptor125 and of TNF-α converting enzyme.126
Another ­interesting feature of TIMP-3 is its ability to induce
209
­apoptosis in ­ different cell types.127-130 It can also sensitize
fibroblast-like synoviocytes to Fas ligand–induced apoptosis
when expressed through adenoviral gene transfer.131
Cathepsins
Cathepsins are another major group of proteases involved
in joint destruction.92 They are classified by their catalytic
mechanism and cleave cartilage types II, IX, and XI, as
well as proteoglycans. Expression of the cysteine proteases
cathepsin B and L is increased in RA synovium, especially
at sites of cartilage invasion.31,132,133 As with MMPs, cathep-
sin production is induced by proto-oncogenes. Transfection
of fibroblasts with the ras proto-oncogene leads to cellular
transformation and the induction of cathepsin L.134 Several
studies have also shown that inflammatory cytokines such as
IL-1 and TNF-α can stimulate the production of cathepsins
B and L by fibroblast-like synoviocyte-like cells.135,136 In this
context, gene transfer of ribozymes to cathepsin L can sig-
nificantly inhibit the fibroblast-mediated cartilage degrada-
tion both in vitro and in vivo.137
Cathepsin K is a cysteine proteinase that plays an
important role in osteoclast-mediated bone resorption. In
addition, cathepsin K expression by RA fibroblast-like sy-
noviocytes and macrophages has been reported, especially
at the site of synovial invasion into articular bone, suggest-
ing that it participates in bone destruction.138
INTERACTIONS BETWEEN FIBROBLAST-LIKE
SYNOVIOCYTES AND INFLAMMATORY CELLS
The accumulation of inflammatory and immune cells,
­particularly macrophages, B cells, and T cells, is one of the
hallmarks of many forms of chronic arthritis. These inflam-
matory cells release a variety of cytokines that stimulate
neighboring cells and contribute to the specific environ-
ment in the rheumatoid joint. Cytokines derived from
­inflammatory cells, such as TNF, IL-1, interferon-γ, and
­others, contribute to the aggressive behavior of fibroblast-like
synoviocytes in diseases such as RA.
It is now evident that in addition to the effect of inflam-
matory cells on fibroblast-like synoviocytes, these resident
fibroblast-like synoviocytes contribute significantly to the
accumulation and survival of inflammatory cells. RA fibro-
blasts interact with and regulate important functions of T
cells, B cells, and macrophages. They are sentinels that,
upon contact with bacterial products such as lipopolysac-
charide and poorly defined components of necrotic cells
through Toll-like receptors, produce chemokines to recruit
inflammatory cells to the site of inflammation.23
Recruitment of T Cells
Interactions between fibroblasts and T cells appear to be
important for the specific composition of the inflamed
synovium. There is now agreement that activated fibroblasts
contribute to impaired apoptosis and anergy of synovial
lymphocytes.15
It is mainly through the regulation of local cytokine
­levels that fibroblasts play a key role in modulating the
transition from acute resolving inflammation to a persistent
immune response accompanied by chronic inflammation.17
 210 PAP  |  Fibroblasts and Fibroblast-like Synoviocytes
Several inflammatory cytokines have been implicated in the
interaction of rheumatoid fibroblasts and T cells. Prominent
examples include IL-8,139 IL-16,140,141 and IL-7 and IL-15,142
both activators of T cells. In addition, stromal cell–derived
factor 1,143 which stimulates the migration and inhibits the
activation-induced apoptosis of T cells, and particularly
IL-6, a highly abundant cytokine in inflammatory syno-
vial fluid that supports antigen-independent inflammation,
have been associated with maintenance of the inflammatory
milieu. Monocyte chemoattractant proteins have also been
implicated in diseases characterized by monocyte-rich infil-
trates, including RA.8,23,144
IL-16 levels in the synovial fluid correlate with the che-
motactic activity toward CD4+ T cells; these levels are high-
est in early RA.140 IL-16 expression is strongly induced in
fibroblasts by IL-1ß,145 a key inflammatory cytokine. Most
interestingly, IL-16 is induced by immunoglobulin G, tar-
geting the insulin-like growth factor-1 receptor in RA but
not in osteoarthritis. This mechanism may provide a link
between B cell activity and T cell infiltration and poten-
tially identify a novel therapeutic target.141
Interactions between rheumatoid fibroblast-like synovio-
cytes and T cells are important determinants of the cellular
composition of inflamed synovium. Thus, human thymo-
cytes and mitogen-activated peripheral blood T cells bind to
RA synoviocytes, whereas fresh peripheral blood T cells do
not exhibit this behavior. Of note, antibodies against CD2
and synovial cell lymphocyte function-associated antigen-3
inhibit this binding.146
Interaction of Fibroblast-Like Synoviocytes
and B Cells
Fibroblast-like synoviocytes have been implicated in the
direct attraction and accumulation of B lymphocytes in
inflamed joints. For instance, rheumatoid fibroblasts can act
as follicular dendritic cells and bind to B cells.147 This func-
tion is intrinsic to RA fibroblasts and quite specific for these
cells when compared with non-RA fibroblast-like synovio-
cytes. Also, B cells cocultured with rheumatoid fibroblasts
show reduced apoptosis and an increase in mitochondrial
apoptosis inhibitors such as Bcl-X(L).148 Fibroblasts promote
the survival of B cells mainly through the induction of Bcl-
X(L) expression and block their apoptosis through VLA-4
(CD49d/CD29)–VCAM-1 interactions. Other studies have
shown that B cells cocultured with fibroblast-like synovio-
cytes are protected from cell death in a cell contact– and
VCAM-1– dependent mechanism.149
Interaction of Fibroblast-Like Synoviocytes and
Synovial Macrophages
A variety of data support the notion that fibroblasts play a
significant role in the activation of macrophages and their
differentiation into multinucleated, bone-resorbing cells.
Specifically, the osteoclast differentiating factor known as
the receptor activator of NFκB (RANKL) has been identi-
fied as a major factor promoting osteoclastogenesis in RA
synovium. Synoviocytes produce large amounts of RANKL
in vivo, and these high levels of RANKL correlate with
the ability of the fibroblasts to generate osteoclasts from
PBMCs in vitro.150 These data suggest that fibroblast-like
Macrophages
IL-15
IL-18 MCP-1
RANKL
Lymphocytes
IFNγ
SDF-1α IL-2
VCAM-1
IL-15
RANKL
IL-16
TNFα
IL-1
IL-15
RANKL
RANKL
Osteoclasts Fibroblasts
Figure 11-8  Fibroblasts contribute to the accumulation and differen-
tiation of inflammatory cells in the rheumatoid arthritis synovium. Fibro-
blasts are part of a cellular network in the diseased synovial membrane.
As such, they are stimulated by inflammatory cells and, in turn, contribute
actively to the accumulation and differentiation of these cells.
s­ ynoviocytes contribute to ECM degradation not only
directly through the release of matrix-degrading enzymes
but also indirectly through the differentiation and activa-
tion of osteoclasts. The cooperation of synovial macrophages
and fibroblasts is also mediated by direct cell-cell interac-
tions through the ligation of CD55 on FLS and CD97 on
macrophages.151 Interestingly, these interactions take place
predominantly in the synovial lining, which mediates the
progressive destruction of cartilage and bone (Fig. 11-8).
SUMMARY
Fibroblasts are structural mesenchymal cells that form the
cellular infrastructure for most internal organs as well as for
bordering membranes such as the synovial membrane. They
are prominently involved in the deposition and resorption
of the ECM and thus are responsible for maintaining tis-
sue homeostasis. However, fibroblasts are far more than just
passively responding cells that build the “backbone” for
organ-specific function. Rather, they are very sensitive to
environmental changes. They react in a very specific manner
to a variety of stimuli and are capable of actively influencing
not only the composition of the ECM but also the cellular
composition of tissues and barrier membranes. Under dis-
ease conditions such as inflammation, fibroblasts, as a criti-
cal part of the organ-specific immune system, are involved
in the progression of organ damage as well as in the switch
from acute resolving to chronic persisting ­inflammation.
This notion is particularly true for fibroblast-like
­synoviocytes, which play a critical role in the pathogenesis
of RA. In addition to contributing to the recruitment of
inflammatory cells to the joint, they modulate the behav-
ior of these cells and are, in turn, regulated by the newly
recruited cells. More important, fibroblast-like synoviocytes
are crucial components in the hyperplastic lining layer and
in cartilage destruction. Further, rheumatoid fibroblasts
retain many of their specific characteristics, including the
ability to invade and degrade cartilage in environments
devoid of ­inflammatory cells.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
REFERENCES
  1. Smith RS, Smith TJ, Blieden TM, et al: Fibroblasts as sentinel cells:
Synthesis of chemokines and regulation of inflammation. Am J
Pathol 151:317-322, 1997.
  2. Lekic PC, Pender N, McCulloch CA: Is fibroblast heterogeneity rel-
evant to the health, diseases, and treatments of periodontal tissues?
Crit Rev Oral Biol Med 8:253-268, 1997.
  3. Valencia X, Higgins JM, Kiener HP, et al: Cadherin-11 provides
specific cellular adhesion between fibroblast-like synoviocytes. J
Exp Med 200:1673-1679, 2004.
  4. Mitra SK, Hanson DA, Schlaepfer DD: Focal adhesion kinase: In
command and control of cell motility. Nat Rev Mol Cell Biol 6:
56-68, 2005.
  5. Echtermeyer F, Baciu PC, Saoncella S, et al: Syndecan-4 core protein
is sufficient for the assembly of focal adhesions and actin stress fibers.
J Cell Sci 112:3433-3441, 1999.
  6. Echtermeyer F, Streit M, Wilcox-Adelman S, et al: Delayed wound
repair and impaired angiogenesis in mice lacking syndecan-4. J Clin
Invest 107:R9-R14, 2001.
  7. Loeser RF, Forsyth CB, Samarel AM, et al: Fibronectin fragment
­activation of proline-rich tyrosine kinase PYK2 mediates integrin
signals regulating collagenase-3 expression by human chondro-
cytes through a protein kinase C-dependent pathway. J Biol Chem
278:24577-24585, 2003.
  8. Distler JH, Jungel A, Huber LC, et al: The induction of matrix
­metalloproteinase and cytokine expression in synovial fibroblasts
stimulated with immune cell microparticles. Proc Natl Acad Sci U S A
102:2892-2897, 2005.
  9. Brauchle M, Gluck D, Di Padova F, et al: Independent role of p38
and ERK1/2 mitogen-activated kinases in the upregulation of matrix
metalloproteinase-1. Exp Cell Res 258:135-144, 2000.
10. Barchowsky A, Frleta D, Vincenti MP: Integration of the NF-kappaB
and mitogen-activated protein kinase/AP-1 pathways at the
­collagenase-1 promoter: Divergence of IL-1 and TNF-dependent
signal transduction in rabbit primary synovial fibroblasts. Cytokine
12:1469-1479, 2000.
­ 11. Mengshol JA, Vincenti MP, Coon CI, et al: Interleukin-1 induction
of collagenase 3 (matrix metalloproteinase 13) gene expression in
chondrocytes requires p38, c-Jun N-terminal kinase, and nuclear
­factor kappa B: Differential regulation of collagenase 1 and collage-
nase 3. Arthritis Rheum 43:801-811, 2000.
­ 12. Li WQ, Dehnade F, Zafarullah M: Oncostatin M-induced matrix
metalloproteinase and tissue inhibitor of metalloproteinase-3 gene
expression in chondrocytes requires Janus kinase/STAT signaling
pathway. J Immunol 166:3491-3498, 2001.
­ 13. Westermarck J, Seth A, Kahari VM: Differential regulation
of ­ interstitial collagenase (MMP-1) gene expression by ETS
­transcription factors. Oncogene 14:2651-2660, 1997.
­ 14. Redlich K, Kiener HP, Schett G, et al: Overexpression of ­transcription
factor Ets-1 in rheumatoid arthritis synovial membrane: Regulation
of expression and activation by interleukin-1 and tumor necrosis
­factor alpha. Arthritis Rheum 44:266-274, 2001.
­ 15. Salmon M, Scheel Toellner D, Huissoon AP, et al: Inhibition of T
cell apoptosis in the rheumatoid synovium. J Clin Invest 99:439-446,
1997.
­ 16. Burger JA, Zvaifler NJ, Tsukada N, et al: Fibroblast-like synoviocytes
support B-cell pseudoemperipolesis via a stromal cell-derived ­factor-1-
and CD106 (VCAM-1)-dependent mechanism. J Clin Invest
107:305-315, 2001.
­ 17. Buckley CD, Pilling D, Lord JM, et al: Fibroblasts regulate the
switch from acute resolving to chronic persistent inflammation.
Trends Immunol 22:199-204, 2001.
­ 18. Buckley CD: Why does chronic inflammatory joint disease persist?
Clin Med 3:361-366, 2003.
­ 19. Kissin EY, Merkel PA, Lafyatis R: Myofibroblasts and hyalinized
­collagen as markers of skin disease in systemic sclerosis. Arthritis
Rheum 54:3655-3660, 2006.
­ 20. Distler O, Pap T, Kowal-Bielecka O, et al: Overexpression of ­monocyte
chemoattractant protein 1 in systemic sclerosis: Role of platelet-derived
growth factor and effects on monocyte chemotaxis and collagen synthe-
sis. Arthritis Rheum 44:2665-2678, 2001.
­ 21. Pap T, Muller-Ladner U, Gay RE, et al: Fibroblast biology: Role
of synovial fibroblasts in the pathogenesis of rheumatoid arthritis.
Arthritis Res 2:361-367, 2000.
211
­ 22. Distler JH, Wenger RH, Gassmann M, et al: Physiologic responses
to hypoxia and implications for hypoxia-inducible factors in the
­pathogenesis of rheumatoid arthritis. Arthritis Rheum 50:10-23,
2004.
­ 23. Pierer M, Rethage J, Seibl R, et al: Chemokine secretion of rheuma-
toid arthritis synovial fibroblasts stimulated by Toll-like receptor 2
ligands. J Immunol 172:1256-1265, 2004.
­ 24. Hoyhtya M, Myllyla R, Piuva J, et al: Monoclonal antibodies to
human prolyl 4-hydroxylase. Eur J Biochem 141:472-482, 1984.
­ 25. Zimmermann T, Kunisch E, Pfeiffer R, et al: Isolation and
­characterization of rheumatoid arthritis synovial fibroblasts from
­primary culture—primary culture cells markedly differ from fourth-
passage cells. Arthritis Res 3:72-76, 2001.
­ 26. Qu Z, Garcia CH, O’Rourke LM, et al: Local proliferation of
­fibroblast-like synoviocytes contributes to synovial hyperplasia:
Results of ­ proliferating cell nuclear antigen/cyclin, c-myc, and
­nucleolar organizer region staining. Arthritis Rheum 37:212-220,
1994 (see comments).
­ 27. Firestein GS: Evolving concepts of rheumatoid arthritis. Nature
423:356-361, 2003.
­ 28. Fassbender HG: Histomorphological basis of articular cartilage
destruction in rheumatoid arthritis. Coll Relat Res 3:141-155, 1983.
­ 29. Muller-Ladner U, Gay RE, Gay S: Activation of synoviocytes. Curr
Opin Rheumatol 12:186-194, 2000.
­ 30. Trabandt A, Aicher WK, Gay RE, et al: Spontaneous expression of
immediately-early response genes c-fos and egr-1 in collagenase-pro-
ducing rheumatoid synovial fibroblasts. Rheumatol Int 12:53-59, 1992.
­ 31. Trabandt A, Aicher WK, Gay RE, et al: Expression of the
­collagenolytic and Ras-induced cysteine proteinase cathepsin L and
proliferation-associated oncogenes in synovial cells of MRL/I mice
and patients with rheumatoid arthritis. Matrix 10:349-361, 1990.
­ 32. Gum R, Wang H, Lengyel E, et al: Regulation of 92 kDa type IV
collagenase expression by the jun aminoterminal kinase- and the
extracellular signal-regulated kinase-dependent signaling cascades.
Oncogene 14:1481-1493, 1997.
­ 33. Korzus E, Nagase H, Rydell R, et al: The mitogen-activated ­protein
kinase and JAK-STAT signaling pathways are required for an oncostatin
M-responsive element-mediated activation of matrix metalloprotein-
ase 1 gene expression. J Biol Chem 272:1188-1196, 1997.
­ 34. Pap T, Nawrath M, Heinrich J, et al: Cooperation of Ras- and
c-Myc-dependent pathways in regulating the growth and ­invasiveness
of synovial fibroblasts in rheumatoid arthritis. Arthritis Rheum
50:2794-2802, 2004.
­ 35. Firestein GS, Echeverri F, Yeo M, et al: Somatic mutations in the p53
tumor suppressor gene in rheumatoid arthritis synovium. Proc Natl
Acad Sci U S A 94:10895-10900, 1997.
­ 36. Pap T, Aupperle KR, Gay S, et al: Invasiveness of synovial fibroblasts
is regulated by p53 in the SCID mouse in vivo model of cartilage
invasion. Arthritis Rheum 44:676-681, 2001.
­ 37. Seemayer CA, Kuchen S, Neidhart M, et al: p53 in rheumatoid
arthritis synovial fibroblasts at sites of invasion. Ann Rheum Dis
62:1139-1144, 2003.
­ 38. Muller-Ladner U, Nishioka K: p53 in rheumatoid arthritis: Friend or
foe? Arthritis Res 2:175-178, 2000.
­ 39. Li J, Yen C, Liaw D, et al: PTEN, a putative protein tyrosine
­phosphatase gene mutated in human brain, breast. prostate cancer.
Science 275:1943-1947, 1997 (see comments).
­ 40. Steck PA, Pershouse MA, Jasser SA, et al: Identification of a
­candidate tumour suppressor gene, MMAC1, at chromosome
10q23.3 that is mutated in multiple advanced cancers. Nat Genet
15:356-362, 1997.
­ 41. Pap T, Franz JK, Hummel KM, et al: Activation of synovial ­fibroblasts
in rheumatoid arthritis: Lack of expression of the tumour suppressor
PTEN at sites of invasive growth and destruction. Arthritis Res 2:
59-64, 2000.
­ 42. Tamura M, Gu J, Danen EH, et al: PTEN interactions with
focal adhesion kinase and suppression of the extracellular
matrix-dependent phosphatidylinositol 3-kinase/Akt cell survival
pathway. J Biol Chem 274:20693-20703, 1999.
­ 43. Di Cristofano A, Kotsi P, Peng YF, et al: Impaired Fas response and
autoimmunity in PTEN+/– mice. Science 285:2122-2125, 1999.
­ 44. Miagkov AV, Kovalenko DV, Brown CE, et al: NF-kappaB ­activation
provides the potential link between inflammation and hyperplasia
in the arthritic joint. Proc Natl Acad Sci U S A 95:13859-13864,
1998.
 212 PAP  |  Fibroblasts and Fibroblast-like Synoviocytes
­ 45. Georganas C, Liu H, Perlman H, et al: Regulation of IL-6 and IL-8
expression in rheumatoid arthritis synovial fibroblasts: The ­dominant
role for NF-kappa B but not C/EBP beta or c-jun. J Immunol
165:7199-7206, 2000.
­ 46. Makarov SS: NF-kappaB in rheumatoid arthritis: A pivotal ­regulator
of inflammation, hyperplasia, and tissue destruction. Arthritis Res
3:200-206, 2001.
­ 47. Karin M, Lin A: NF-kappaB at the crossroads of life and death. Nat
Immunol 3:221-227, 2002.
­ 48. Li P, Sanz I, O’Keefe RJ, et al: NF-kappa B regulates VCAM-
1 expression on fibroblast-like synoviocytes. J Immunol 164:
5990-5997, 2000.
­ 49. Vincenti MP, Coon CI, Brinckerhoff CE: Nuclear factor kappaB/p50
activates an element in the distal matrix metalloproteinase 1 ­promoter
in interleukin-1beta-stimulated synovial fibroblasts. Arthritis Rheum
41:1987-1994, 1998.
­ 50. Nykanen P, Bergroth V, Raunio P, et al: Phenotypic characterization
of 3H-thymidine incorporating cells in rheumatoid arthritis synovial
membrane. Rheumatol Int 6:269-271, 1986.
­ 51. Petrow P, Theis B, Eckard A, et al: Determination of ­ proliferating
cells at sites of cartilage invasion in patients with rheumatoid
­arthritis. Arthritis Rheum 40(Suppl):S251, 1997.
52. Kinne RW, Palombo Kinne E, Emmrich F: Activation of synovial
fibroblasts in rheumatoid arthritis. Ann Rheum Dis 54:501-504,
1995.
­ 53. Baier A, Meineckel I, Gay S, et al: Apoptosis in rheumatoid arthri-
tis. Curr Opin Rheumatol 15:274-279, 2003.
­ 54. Matsumoto S, Muller Ladner U, Gay RE, et al: Multistage ­apoptosis
and Fas antigen expression of synovial fibroblasts derived from
patients with rheumatoid arthritis. J Rheumatol 23:1345-1352,
1996.
­ 55. Pope RM: Apoptosis as a therapeutic tool in rheumatoid arthritis.
Nat Rev Immunol 2:527-535, 2002.
­ 56. Perlman H, Liu H, Georganas C, et al: Differential expression ­pattern
of the antiapoptotic proteins, Bcl-2 and FLIP, in experimental
­arthritis. Arthritis Rheum 44:2899-2908, 2001.
­ 57. Matsumoto S, Muller Ladner U, Gay RE, et al: Ultrastructural
­demonstration of apoptosis, Fas and Bcl-2 expression of rheumatoid
synovial fibroblasts. J Rheumatol 23:1345-1352, 1996.
­ 58. Perlman H, Georganas C, Pagliari LJ, et al: Bcl-2 expression in
­synovial fibroblasts is essential for maintaining mitochondrial
homeostasis and cell viability. J Immunol 164:5227-5235, 2000.
­ 59. Kurowska M, Rudnicka W, Kontny E, et al: Fibroblast-like
­synoviocytes from rheumatoid arthritis patients express functional
IL-15 receptor complex: Endogenous IL-15 in autocrine fashion
enhances cell proliferation and expression of Bcl-x(L) and Bcl- 2.
J Immunol 169:1760-1767, 2002.
­ 60. Hasunuma T, Kayagaki N, Asahara H, et al: Accumulation of soluble
Fas in inflamed joints of patients with rheumatoid arthritis. Arthritis
Rheum 40:80-86, 1997.
­ 61. Perlman H, Pagliari LJ, Liu H, et al: Rheumatoid arthritis ­synovial
macrophages express the Fas-associated death domain-like ­interleukin-
1beta-converting enzyme-inhibitory protein and are refractory to Fas-
mediated apoptosis. Arthritis Rheum 44:21-30, 2001.
­ 62. Catrina AI, Ulfgren AK, Lindblad S, et al: Low levels of apoptosis
and high FLIP expression in early rheumatoid arthritis synovium.
Ann Rheum Dis 61:934-936, 2002.
­ 63. Schedel J, Gay RE, Kuenzler P, et al: FLICE-inhibitory protein
expression in synovial fibroblasts and at sites of cartilage and bone
erosion in rheumatoid arthritis. Arthritis Rheum 46:1512-1518,
2002.
­64. Palao G, Santiago B, Galindo M, et al: Down-regulation of FLIP
sensitizes rheumatoid synovial fibroblasts to Fas-mediated apoptosis.
Arthritis Rheum 50:2803-2810, 2004.
­ 65. Melchior F: SUMO—nonclassical ubiquitin. Annu Rev Cell Dev
Biol 16:591-626, 2000.
­ 66. Okura T, Gong L, Kamitani T, et al: Protection against Fas/APO-1-
and tumor necrosis factor-mediated cell death by a novel protein,
sentrin. J Immunol 157:4277-4281, 1996.
­ 67. Franz JK, Pap T, Hummel KM, et al: Expression of sentrin, a novel
antiapoptotic molecule, at sites of synovial invasion in rheumatoid
arthritis. Arthritis Rheum 43:599-607, 2000.
­ 68. Hsu H, Shu HB, Pan MG, et al: TRADD-TRAF2 and TRADD-FADD
interactions define two distinct TNF receptor 1 signal transduction
pathways. Cell 84:299-308, 1996.
­ 69. Mountz JD, Hsu HC, Matsuki Y, et al: Apoptosis and rheumatoid
arthritis: Past, present, and future directions. Curr Rheumatol Rep
3:70-78, 2001.
­ 70. Liu H, Pope RM: The role of apoptosis in rheumatoid arthritis. Curr
Opin Pharmacol 3:317-322, 2003.
­ 71. Reddy SA, Huang JH, Liao WS: Phosphatidylinositol 3-kinase
as a mediator of TNF-induced NF-kappa B activation. J Immunol
164:1355-1363, 2000.
­ 72. Drynda A, Quax PH, Neumann M, et al: Gene transfer of tissue
inhibitor of metalloproteinases-3 reverses the inhibitory effects of
TNF-alpha on Fas-induced apoptosis in rheumatoid arthritis synovial
fibroblasts. J Immunol 174:6524-6531, 2005.
­ 73. Pap T, Cinski A, Baier A, et al: Modulation of pathways regulating
both the invasiveness and apoptosis in rheumatoid arthritis synovial
fibroblasts. Joint Bone Spine 70:477-479, 2003.
­ 74. Schwartz MA: Integrins, oncogenes, and anchorage independence.
J Cell Biol 139:575-578, 1997.
­ 75. Dike LE, Ingber DE: Integrin-dependent induction of early growth
response genes in capillary endothelial cells. J Cell Sci 109:
2855-2863, 1996.
­ 76. Rinaldi N, Schwarz EM, Weis D, et al: Increased expression of integ-
rins on fibroblast-like synoviocytes from rheumatoid arthritis in vitro
correlates with enhanced binding to extracellular matrix proteins.
Ann Rheum Dis 56:45-51, 1997.
­ 77. Ishikawa H, Hirata S, Andoh Y, et al: An immunohistochemical and
immunoelectron microscopic study of adhesion molecules in synovial
pannus formation in rheumatoid arthritis. Rheumatol Int 16:53-60,
1996.
­ 78. Sarkissian M, Lafyatis R: Integrin engagement regulates ­proliferation
and collagenase expression of rheumatoid synovial fibroblasts.
J Immunol 162:1772-1779, 1999.
­ 79. Lin TH, Chen Q, Howe A, et al: Cell anchorage permits efficient
signal transduction between ras and its downstream kinases. J Biol
Chem 272:8849-8852, 1997.
­ 80. Inoue H, Yamashita A, Hakura A: Adhesion-dependency of serum-
induced p42/p44 MAP kinase activation is released by retroviral
oncogenes. Virology 225:223-226, 1996.
­ 81. Ravanti L, Heino J, Lopez-Otin C, et al: Induction of collagenase-3
(MMP-13) expression in human skin fibroblasts by three-­dimensional
collagen is mediated by p38 mitogen-activated protein kinase. J Biol
Chem 274:2446-2455, 1999.
­ 82. Wang AZ, Wang JC, Fisher GW, et al: Interleukin-1beta-stimulated
invasion of articular cartilage by rheumatoid synovial f­ibroblasts
is inhibited by antibodies to specific integrin receptors and by
­collagenase inhibitors. Arthritis Rheum 40:1298-1307, 1997.
­ 83. Osborn L, Hession C, Tizard R, et al: Direct expression cloning of
vascular cell adhesion molecule 1, a cytokine-induced endothelial
protein that binds to lymphocytes. Cell 59:1203-1211, 1989.
­ 84. Higashiyama H, Saito I, Hayashi Y, et al: In situ hybridization study
of vascular cell adhesion molecule-1 messenger RNA expression in
rheumatoid synovium. J Autoimmun 8:947-957, 1995.
­ 85. Kriegsmann J, Keyszer GM, Geiler T, et al: Expression of vascular cell
adhesion molecule-1 mRNA and protein in rheumatoid synovium
demonstrated by in situ hybridization and immunohistochemistry.
Lab Invest 72:209-214, 1995.
­ 86. Morales DJ, Wayner E, Elices MJ, et al: Alpha 4/beta 1 integrin
(VLA-4) ligands in arthritis: Vascular cell adhesion molecule-1
expression in synovium and on fibroblast-like synoviocytes.
J ­Immunol 149:1424-1431, 1992.
­ 87. Matsuyama T, Kitani A: The role of VCAM-1 molecule in the
­pathogenesis of rheumatoid synovitis. Hum Cell 9:187-192, 1996.
­ 88. Muller-Ladner U, Kriegsmann J, Franklin BN, et al: Synovial
­fibroblasts of patients with rheumatoid arthritis attach to and
invade normal human cartilage when engrafted into SCID mice.
Am J Pathol 149:1607-1615, 1996.
­ 89. Kitani A, Nakashima N, Izumihara T, et al: Soluble VCAM-1
induces chemotaxis of Jurkat and synovial fluid T cells bearing high
affinity very late antigen-4. J Immunol 161:4931-4938, 1998.
­ 90. Koch AE, Halloran MM, Haskell CJ, et al: Angiogenesis mediated
by soluble forms of E-selectin and vascular cell adhesion molecule-1.
Nature 376:517-519, 1995.
­ 91. Pap T, Schett G, Gay S: Matrix metalloproteinases. In Smolen J,
Lipsky J (eds): Targeted therapies in Rheumatology. London, Martin
Dunitz, 483-497, 2002.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
­ 92. Muller-Ladner U, Gay RE, Gay S: Cysteine proteinases in arthri-
tis and inflammation. Perspect Drug Discovery Design 6:87-98,
1996.
­ 93. Konttinen YT, Ainola M, Valleala H, et al: Analysis of 16 ­different
matrix metalloproteinases (MMP-1 to MMP-20) in the synovial
membrane: Different profiles in trauma and rheumatoid arthritis.
Ann Rheum Dis 58:691-697, 1999.
­ 94. Sorsa T, Konttinen YT, Lindy O, et al: Collagenase in synovitis of
rheumatoid arthritis. Semin Arthritis Rheum 22:44-53m 1992.
­ 95. Maeda S, Sawai T, Uzuki M, et al: Determination of interstitial colla-
genase (MMP-1) in patients with rheumatoid arthritis. Ann Rheum
Dis 54:970-975, 1995.
­ 96. Okada Y, Takeuchi N, Tomita K, et al: Immunolocalization of matrix
metalloproteinase 3 (stromelysin) in rheumatoid synovioblasts (B cells):
Correlation with rheumatoid arthritis. Ann Rheum Dis 48:645-653,
1989.
­ 97. Tetlow LC, Lees M, Ogata Y, et al: Differential expression of
­gelatinase B (MMP-9) and stromelysin-1 (MMP- 3) by rheumatoid
synovial cells in vitro and in vivo. Rheumatol Int 13:53-59, 1993.
­ 98. Beekman B, van El B, Drijfhout JW, et al: Highly increased levels
of active stromelysin in rheumatoid synovial fluid determined by a
selective fluorogenic assay. FEBS Lett 418:305-309, 1997.
­ 99. Taylor DJ, Cheung NT, Dawes PT: Increased serum proMMP-3
in inflammatory arthritides: A potential indicator of synovial
­inflammatory monokine activity. Ann Rheum Dis 53:768-772, 1994.
100. Yoshihara Y, Obata K, Fujimoto N, et al: Increased levels of
­stromelysin-1 and tissue inhibitor of metalloproteinases-1 in sera
from patients with rheumatoid arthritis. Arthritis Rheum 38:
969-975, 1995.
101. Manicourt DH, Fujimoto N, Obata K, et al: Levels of ­ circulating
­collagenase, stromelysin-1, and tissue inhibitor of matrix
­metalloproteinases 1 in patients with rheumatoid arthritis:
­Relationship to serum levels of antigenic keratan sulfate and systemic
parameters of inflammation. Arthritis Rheum 38:1031-1039, 1995.
102. So A, Chamot AM, Peclat V, et al: Serum MMP-3 in rheumatoid
arthritis: Correlation with systemic inflammation but not with
­erosive status. Rheumatology (Oxford) 38:407-410, 1999.
103. Yamanaka H, Matsuda Y, Tanaka M, et al: Serum matrix
­metalloproteinase 3 as a predictor of the degree of joint destruction
during the six months after measurement, in patients with early
­rheumatoid arthritis. Arthritis Rheum 43:852-858, 2000.
104. Petrow P, Hummel KM, Franz JK, et al: In-situ detection of MMP-13
mRNA in the synovial membrane and cartilage-pannus junction in
rheumatoid arthritis. Arthritis Rheum 40:S336, 1997.
105. Lindy O, Konttinen YT, Sorsa T, et al: Matrix-metalloproteinase 13
(collagenase 3) in human rheumatoid synovium. Arthritis Rheum
40:1391-1399, 1997.
106. Westhoff CS, Freudiger D, Petrow P, et al: Characterization of
­collagenase 3 (matrix metalloproteinase 13) messenger RNA
­expression in the synovial membrane and synovial fibroblasts of
patients with rheumatoid arthritis. Arthritis Rheum 42:1517-1527,
1999.
107. Pap T, Shigeyama Y, Kuchen S, et al: Differential expression pat-
tern of membrane-type matrix metalloproteinases in rheumatoid
arthritis. Arthritis Rheum 43:1226-1232, 2000.
108. van de Loo FA, Joosten LA, van Lent PL, et al: Role of ­interleukin-1,
tumor necrosis factor alpha, and interleukin-6 in cartilage proteo-
glycan metabolism and destruction: Effect of in situ blocking in
murine antigen- and zymosan-induced arthritis. Arthritis Rheum 38:
164-172, 1995.
109. van den Berg WB, Joosten LA, Helsen M, et al: Amelioration
of ­ established murine collagen-induced arthritis with anti-IL-1
­treatment. Clin Exp Immunol 95:237-243, 1994.
110. Muller-Ladner U, Roberts CR, Franklin BN, et al: Human IL-1Ra
gene transfer into human synovial fibroblasts is chondroprotective.
J Immunol 158:3492-3498, 1997.
111. Dayer JM, Beutler B, Cerami A: Cachectin/tumor necrosis factor stim-
ulates collagenase and prostaglandin E2 production by human syno-
vial cells and dermal fibroblasts. J Exp Med 162:2163-2168, 1985.
112. Brinckerhoff CE, Auble DT: Regulation of collagenase gene ­expression
in synovial fibroblasts. Ann N Y Acad Sci 580:355-374, 1990.
113. Kuiper S, Joosten LA, Bendele AM, et al: Different roles of TNFα
and IL-1 in murine streptococcal wall arthritis. Cytokine 10:690-702,
1998.
213
114. Jovanovic DV, Martel-Pelletier J, Di Battista JA, et al: ­Stimulation
of 92-kd gelatinase (matrix metalloproteinase 9) production by
­interleukin-17 in human monocyte/macrophages: A possible role in
rheumatoid arthritis. Arthritis Rheum 43:1134-1144, 2000.
115. Ravanti L, Toriseva M, Penttinen R, et al: Expression of human
­collagenase-3 (MMP-13) by fetal skin fibroblasts is induced by
­transforming growth factor beta via p38 mitogen-activated protein
kinase. FASEB J 15:1098-1100, 2001.
116. Bond M, Fabunmi RP, Baker AH, et al: Synergistic upregulation of
metalloproteinase-9 by growth factors and inflammatory cytokines:
An absolute requirement for transcription factor NF-kappa B. FEBS
Lett 435:29-34, 1998.
117. Forsyth CB, Pulai J, Loeser RF: Fibronectin fragments and ­blocking
antibodies to alpha2beta1 and alpha5beta1 integrins stimulate
­mitogen-activated protein kinase signaling and increase ­collagenase 3
(matrix metalloproteinase 13) production by human articular chon-
drocytes. Arthritis Rheum 46:2368-2376, 2002.
118. Tolboom TC, Pieterman E, van der Laan WH, et al: Invasive
­properties of fibroblast-like synoviocytes: Correlation with growth
characteristics and expression of MMP-1, MMP-3, and MMP-10.
Ann Rheum Dis 61:975-980, 2002.
119. Rutkauskaite E, Zacharias W, Schedel J, et al: Ribozymes that inhibit
the production of matrix metalloproteinase 1 reduce the ­invasiveness
of rheumatoid arthritis synovial fibroblasts. Arthritis Rheum 50:
1448-1456, 2004.
120. Firestein GS, Paine MM: Stromelysin and tissue inhibitor of
­metalloproteinases gene expression in rheumatoid arthritis synovium.
Am J Pathol 140:1309-1314, 1992.
121. Clark IM, Powell LK, Ramsey S, et al: The measurement of
­collagenase, TIMP and collagenase-TIMP complex in synovial fluids
from patients with osteoarthritis and rheumatoid arthritis. Arthritis
Rheum 36:372-380, 1993.
122. DiBattista JA, Pelletier JP, Zafarullah M, et al: Coordinate regulation
of matrix metalloproteases and tissue inhibitor of metalloproteinase
expression in human synovial fibroblasts. J Rheumatol Suppl 43:
123-128, 1995.
123. van der Laan WH, Quax PH, Seemayer CA, et al: Cartilage
­degradation and invasion by rheumatoid synovial fibroblasts is
­inhibited by gene transfer of TIMP-1 and TIMP-3. Gene Ther
10:234-242, 2003.
124. Smith MR, Kung H, Durum SK, et al: TIMP-3 induces cell death
by stabilizing TNF-alpha receptors on the surface of human colon
carcinoma cells. Cytokine 9:770-780, 1997.
125. Hargreaves PG, Wang F, Antcliff J, et al: Human myeloma cells
shed the interleukin-6 receptor: Inhibition by tissue inhibitor of
­metalloproteinase-3 and a hydroxamate-based metalloproteinase
inhibitor. Br J Haematol 101:694-702, 1998.
126. Amour A, Slocombe PM, Webster A, et al: TNF-alpha ­ converting
enzyme (TACE) is inhibited by TIMP-3. FEBS Lett 435:39-44, 1998.
127. Ahonen M, Baker AH, Kahari VM: Adenovirus-mediated gene
delivery of tissue inhibitor of metalloproteinases-3 inhibits invasion
and induces apoptosis in melanoma cells. Cancer Res 58:2310-2315,
1998.
128. Baker AH, Zaltsman AB, George SJ, et al: Divergent effects of ­tissue
inhibitor of metalloproteinase-1, -2, or -3 overexpression on rat
­vascular smooth muscle cell invasion, proliferation, and death in vitro:
TIMP-3 promotes apoptosis. J Clin Invest 101:1478-1487, 1998.
129. Baker AH, George SJ, Zaltsman AB, et al: Inhibition of invasion and
induction of apoptotic cell death of cancer cell lines by ­overexpression
of TIMP-3. Br J Cancer 79:1347-1355, 1999.
130. Majid MA, Smith VA, Easty DL, et al: Adenovirus mediated gene
delivery of tissue inhibitor of metalloproteinases-3 induces death
in retinal pigment epithelial cells. Br J Ophthalmol 86:97-101,
2002.
131. Drynda A, Quax PH, Neumann M, et al: Gene transfer of tissue
inhibitor of metalloproteinases-3 reverses the inhibitory effects of
TNF-alpha on Fas-induced apoptosis in rheumatoid arthritis synovial
fibroblasts. J Immunol 174(10): 6524-6531, 2005.
132. Keyszer G, Redlich A, Haupl T, et al: Differential expression of
cathepsins B and L compared with matrix metalloproteinases and
their respective inhibitors in rheumatoid arthritis and osteoarthritis:
A parallel investigation by semiquantitative reverse transcriptase-
polymerase chain reaction and immunohistochemistry. Arthritis
Rheum 41:1378-1387, 1998.
 214 PAP  |  Fibroblasts and Fibroblast-like Synoviocytes
133. Keyszer GM, Heer AH, Kriegsmann J, et al: Comparative ­ analysis
of cathepsin L, cathepsin D, and collagenase messenger RNA
­expression in synovial tissues of patients with rheumatoid ­ arthritis
and ­ osteoarthritis, by in situ hybridization. Arthritis Rheum 38:
976-984, 1995.
134. Joseph L, Lapid S, Sukhatme V: The major ras induced protein in
NIH3T3 cells is cathepsin L. Nucleic Acids Res 15:3186, 1987.
135. Huet G, Flipo RM, Colin C, et al: Stimulation of the secretion of
latent cysteine proteinase activity by tumor necrosis factor alpha and
interleukin-1. Arthritis Rheum 36:772-780, 1993.
136. Lemaire R, Huet G, Zerimech F, et al: Selective induction of the
secretion of cathepsins B and L by cytokines in synovial fibroblast-
like cells. Br J Rheumatol 36:735-743, 1997.
137. Schedel J, Seemayer CA, Pap T, et al: Targeting cathepsin L (CL)
by specific ribozymes decreases CL protein synthesis and cartilage
destruction in rheumatoid arthritis. Gene Ther 11:1040-1047, 2004.
138. Hummel KM, Petrow PK, Franz JK, et al: Cysteine proteinase
­cathepsin K mRNA is expressed in synovium of patients with
­rheumatoid arthritis and is detected at sites of synovial bone
­destruction. J Rheumatol 25:1887-1894, 1998.
139. Min DJ, Cho ML, Lee SH, et al: Augmented production of
­chemokines by the interaction of type II collagen-reactive
T cells with ­ rheumatoid synovial fibroblasts. Arthritis Rheum 50:
1146-1155, 2004.
140. Franz JK, Kolb SA, Hummel KM, et al: Interleukin-16, pro-
duced by synovial fibroblasts, mediates chemoattraction for CD4+
T ­lymphocytes in rheumatoid arthritis. Eur J Immunol 28:2661-2671,
1998.
141. Pritchard J, Tsui S, Horst N, et al: Synovial fibroblasts from
patients with rheumatoid arthritis, like fibroblasts from Graves’
disease, express high levels of IL-16 when treated with Igs against
insulin-like growth factor-1 receptor. J Immunol 173:3564-3569,
2004.
142. McInnes IB, al Mughales J, Field M, et al: The role of interleukin-15
in T-cell migration and activation in rheumatoid arthritis. Nat Med
2:175-182, 1996.
143. Nanki T, Hayashida K, El Gabalawy HS, et al: Stromal cell-derived
factor-1-CXC chemokine receptor 4 interactions play a central role
in CD4+ T cell accumulation in rheumatoid arthritis synovium.
J Immunol 165:6590-6598, 2000.
144. Koch AE, Kunkel SL, Harlow LA, et al: Enhanced production of
monocyte chemoattractant protein-1 in rheumatoid arthritis. J Clin
Invest 90:772-779, 1992.
145. Sciaky D, Brazer W, Center DM, et al: Cultured human fibroblasts
express constitutive IL-16 mRNA: Cytokine induction of active
IL-16 protein synthesis through a caspase-3-dependent mechanism.
J Immunol 164:3806-3814, 2000.
146. Haynes BF, Grover BJ, Whichard LP, et al: Synovial microenvi-
ronment-T cell interactions: Human T cells bind to fibroblast-like
­synovial cells in vitro. Arthritis Rheum 31:947-955, 1988.
147. Lindhout E, van Eijk M, van Pel M, et al: Fibroblast-like
­synoviocytes from rheumatoid arthritis patients have intrinsic
properties of ­follicular dendritic cells. J Immunol 162:5949-5956,
1999.
148. Hayashida K, Shimaoka Y, Ochi T, et al: Rheumatoid arthritis
­sy­novial stromal cells inhibit apoptosis and up-regulate Bcl-xL
­expression by B cells in a CD49/CD29-CD106-dependent ­mechanism.
J Immunol 164:1110-1116, 2000.
149. Reparon-Schuijt CC, van Esch WJ, van Kooten C, et al: ­Regulation
of synovial B cell survival in rheumatoid arthritis by vascular
cell ­ adhesion molecule 1 (CD106) expressed on fibroblast-like
­synoviocytes. Arthritis Rheum 43:1115-1121, 2000.
150. Shigeyama Y, Pap T, Kunzler P, et al: Expression of osteoclast
­differentiation factor in rheumatoid arthritis. Arthritis Rheum
43:2523-2530, 2000.
151. Hamann J, Wishaupt JO, van Lier RA, et al: Expression of the
­activation antigen CD97 and its ligand CD55 in rheumatoid syno-
vial tissue. Arthritis Rheum 42:650-658, 1999.
 12
KEY POINTS
Neutrophils are myeloid-lineage cells that are characterized
by the presence of large quantities of granules containing
enzymes and other potentially toxic agents involved in host
defense.
Neutrophils are short-lived, terminally differentiated cells that
exist primarily in the bloodstream, where they participate in
host surveillance of foreign organisms.
Neutrophils function in acute inflammation and provide
an ­essential defense against acute bacterial infections;
abnormalities of neutrophil function are uncommon and
­ prior exposure to the particle. This chapter reviews the salient
impair ability to respond to life-threatening infections.
The main role of the neutrophil is to phagocytose foreign
­particles, primarily bacteria, and degrade them through
­activation of proteases, activation of other antibiotic
­molecules, and generation of toxic oxygen radicals.
Neutrophils play a role as a major inflammatory cell in many
rheumatic conditions and may be attracted into tissues by
noninfectious stimuli, such as complement activation and
lipid inflammatory mediators.
Eosinophils are myeloid-lineage cells that contain many
cytoplasmic granules containing proteins such as major
basic protein, eosinophil cationic protein, eosinophil-derived
­neurotoxin, and eosinophil peroxidase.
Eosinophils are thought to function in the defense of
­helminths and other parasites; however, evidence for such an
antihelminthic role is limited.
In contrast to neutrophils, eosinophils are not primarily
phagocytic cells, but are thought to discharge their granule
contents adjacent to the much larger organisms that may be
their targets.
Eosinophilia may be seen in many rheumatic diseases,
­including Churg-Strauss syndrome and less common
diseases such as eosinophilic fasciitis and the idiopathic
­hypereosinophilic syndromes.
Polymorphonuclear leukocytes constitute a family of hemato-
poietically derived cells that share the feature of a multilobed
nucleus. These leukocytes also share a property of possessing
highly developed populations of intracytoplasmic granules,
divisible into subsets and distinct between cell types. The
presence of these granules permits the further designation of
polymorphonuclear leukocytes as granulocytes. Based on the
histocytochemical staining properties of their respective gran-
ules, three classes of polymorphonuclear leukocytes have been
identified: neutrophils, eosinophils, and basophils. Neutrophil
(polymorphonuclear neutrophil) granules stain with neutral
dyes, the granules of eosinophils are most ­effectively stained
Neutrophils
and Eosinophils
Michael H. Pillinger  • 
Jose U. Scher  • 
Steven B. Abramson
with acidic dyes such as eosin, and basophil granules stain
with basic dyes. In a standard polychromatic Wright stain of a
peripheral blood smear, the cytoplasm of neutrophils, eosino-
phils, and basophils appears blue-pink (neutrophils), pink
(eosinophils), and blue (basophils). These classes of polymor-
phonuclear leukocytes differ with respect to not only appear-
ance, but also biochemistry and function. ­Polymorphonuclear
leukocytes constitute an important part of the organism’s sys-
tem of innate immunity: Their responses to foreign organisms
or antigens or both are preprogrammed and do not depend on
features of neutrophils and eosinophils.
NEUTROPHILS
Neutrophils are the body’s first line of defense against ­foreign
invaders and constitute the major cell type involved in acute and
some forms of chronic inflammatory disease. The importance
of neutrophils in bacterial defense is shown by patients who
have hereditary defects in neutrophil function and are prone
to repeated and often life-threatening infections. ­Neutrophils
are the most prevalent leukocyte in the bloodstream, typically
constituting greater than 50% of all bloodstream leukocytes.
During bacterial infection, the percentage of neutrophils may
increase to 80% or more. In contrast, tissue concentrations of
resting (inactive) neutrophils seem to be quite low. Neutrophils
may be thought of as surveillance cells—sweeping through
the bloodstream scanning for infections or other inflamma-
tory events. The capacity of neutrophils to destroy foreign
organisms is matched in some circumstances, however, by a
capacity for host tissue destruction.
NEUTROPHIL MYELOPOIESIS AND CLEARANCE
The neutrophil majority in the bloodstream is duplicated
in the marrow, where 60% of hematopoietic capacity may
be dedicated to neutrophil production. Daily, 1011 neutro-
phils may be released into the bloodstream. Neutrophil
development in the marrow takes about 14 days, originat-
ing with the hematopoietic stem cell. Stem cells fated to
become neutrophils first differentiate into myeloblasts,
which retain the capacity to develop into eosinophils, baso-
phils, and neutrophils. Subsequent differentiation leads to
the neutrophilic promyelocyte, a dedicated precursor of the
neutrophil, and proceeds through the stages of neutrophilic
myelocytes, metamyelocyte, band cell, and mature neutro-
phil. At the metamyelocyte stage, neutrophil mitosis ceases,
whereas neutrophil development and organization of gran-
ules ­ continues. Neutrophils are terminally differentiated;
they neither divide nor alter their gross phenotype after
their release from the marrow.
215
 216 Pillinger  |  Neutrophils and Eosinophils

A C

1.0 µm
B D 2.0 µm

Figure 12-1  Resting and stimulated neutrophil morphology. A and B, Transmission (A) and scanning (B) electron micrographs of resting neutrophils.
In A, note the multilobed nucleus and the rich population of granules. At least two granule populations of granules may be discerned: The larger, darker
granules represent the primary (azurophilic) granules, whereas the smaller, slightly paler granules are predominantly secondary (specific) granules and
may include a population of gelatinase granules (arrow indicates primary granule). In B, note the relatively smooth surface area with some membrane
surface irregularities. C and D, Transmission (C) and scanning (D) electron micrographs of neutrophils 1 minute after stimulation with zymosan. The
cellular diameter is enlarged, and the overall surface (plasma) membrane area is greatly increased. Most of the membrane contributing to the increased
surface area is supplied via the fusion of internal granule membranes with the plasma membrane. In C, this fusion is apparent as the depletion of
granules, leading to an appearance of empty vesicles (arrow indicates a partially depleted primary granule; clear circular areas represent fully depleted
vesicles whose membranes are fused to the plasma membrane). In D, this fusion is apparent as the increase in plasma surface membrane extensions,
known as lamellipodia.

Given the origin of neutrophils in a pluripotent stem cell,
and the precise phases of their development, the mechanisms
regulating neutrophil differentiation are of considerable inter-
est. Although incompletely understood, studies have empha-
sized the role of a particular complement of cytokines that
seem to direct the early cells toward neutrophil development.
Principal among the cytokines described to date are granu-
locyte colony-stimulating factor (G-CSF) and granulocyte-
macrophage colony-stimulating factor (GM-CSF).1
Neutrophils released from the marrow have a bloodstream
half-life of approximately 6 hours and a tissue half-life only
marginally longer. The high output and short half-life of neu-
trophils imply that neutrophil clearance mechanisms must
exist. When exposed to appropriate stimuli such as tumor
necrosis factor (TNF)-α and Fas (CD95) ligand, neutrophils
undergo apoptosis or programmed cell death.2,3 Senescent
or apoptotic bloodstream neutrophils are cleared largely by
liver and spleen macrophages (reticuloendothelial system).
It is a matter of speculation whether tissue neutrophils are
cleared primarily via local macrophages.
NEUTROPHIL MORPHOLOGY AND CONTENT
Neutrophil nuclei tend to have more lobes than nuclei of
other polymorphs, typically three to five (Fig. 12-1). The
multilobed nature of this nucleus reflects a condensation
of chromatin that suggests that neutrophils might be inca-
pable of transcription. It is now appreciated, however, that
­ eutrophils retain the capacity for constitutive and stimu-
n
lated protein synthesis, albeit at a limited rate.
Neutrophil granules identifiable by classic histochemical
staining comprise two classes (see Fig. 12-1). Neutrophil pri-
mary granules form first and, by virtue of their staining ten-
dencies, are also referred to as azurophilic granules. These
granules are oval or round and vary in size. They are similar,
and functionally equivalent, to the lysosomes of other cells.
In contrast, neutrophil secondary granules constitute a pop-
ulation unique to neutrophils, a fact reflected in the alterna-
tive nomenclature of specific granules often used to describe
these structures. Characteristic of ­ azurophilic granules is
the presence of myeloperoxidase, an enzyme that catalyzes
the formation of hypochlorous acid from chloride in the
presence of superoxide anion (O2−).4 The presence of large
amounts of this enzyme in azurophilic granules lends collec-
tions of neutrophils (pus) their typical greenish yellow color.
­Consistent with their role as lysosomes, azurophilic granules
also contain a variety of proteases and other enzymes, includ-
ing elastase, lysozyme, acid phosphatase, and cathepsins and
enzymes directed at nucleic acids and sugars. One particu-
larly important family of ­ proteinases found in neutrophils
are the matrix metalloproteinases (MMPs), including neu-
trophil collagenase-2 (MMP-8), gelatinase-B (MMP-9), and
stromelysin (MMP-3). In ­ contrast to ­ azurophilic granules,
specific granules possess an extensive array of membrane-
associated proteins, including cytochromes, ­ signaling mol-
ecules, and receptors. ­Specific granules ­constitute a reservoir
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
Table 12-1  Neutrophil Granule Contents
Secretory Vesicles
Relative size Smallest
Soluble components Plasma proteins
Membrane-associated FMLP receptor
components CD11b/CD18
Cytochrome b558
Alkaline phosphatase
Uroplasminogen activator
CD10, CD13, CD16, CD45
CR1
Decay accelerating factor
BPI, bactericidal/permeability-increasing protein; FMLP, formyl-methionyl-leucyl-phenylalanine; MMP, matrix metalloproteinase; TNF, tumor necrosis factor.
of proteins destined for topologically external surfaces of
phagocytic vacuoles themselves and the plasma membrane
(Table 12-1).4
Further study has confirmed the existence of two addi-
tional classes of vesicles. Gelatinase granules are almost
identical in size to specific granules and share some proteins
in common with them. As their name implies, however,
gelatinase granules are noteworthy for their high concen-
trations of gelatinase, a latent enzyme with the capacity for
tissue destruction.5 Secretory vesicles are smaller and lighter
than the other classes and do not seem to contain proteo-
lytic enzymes.6 Rather, they are noteworthy for an extensive
series of membrane-associated proteins, including receptors
otherwise identified with the plasma membrane.7 These and
other data suggest that the secretory vesicle is a reservoir of
neutrophil plasma membrane and membrane proteins.
Azurophilic and specific granules further contain anti-
microbial proteins and peptides that are a cornerstone of
innate immunity. A detailed description of the neutrophil’s
armamentarium against foreign invaders is beyond the scope
of this chapter, but a few whose mechanisms of action have
more recently been elucidated warrant mention. Elastase,
already mentioned previously, aids in the killing of gram-neg-
ative bacteria via degradation of bacterial outer membrane
protein A.8 Elastase-deficient mice are more susceptible to
infection with gram-negative (but not gram-positive) organ-
isms than wild-type mice. The defensins, normally located
in azurophilic granules, are found in mg/mL concentrations
in the phagocytic vacuole (see later) and render target cell
membranes permeable. Bactericidal/permeability-inducing
protein, also located in azurophilic granules, acts in concert
with the defensins; it potently neutralizes endotoxin and is
cytotoxic to gram-negative bacteria.9 Bactericidal/permeabil-
ity-inducing protein also enhances the activity of secretory
phospholipase A2, which has activity against gram-negative
and gram-positive bacteria. Lactoferrin (found in specific
granules) deprives microorganisms of iron and has antiviral
and antibacterial effects.
Neutrophil proteases play important roles beyond their
antimicrobial effects; they also may amplify or dampen the
217
Gelatinase Granules Specific Granules Azurophilic Granules
Intermediate Intermediate Largest
Gelatinase   Gelatinase Myeloperoxidase
Acetyltransferase MMP-3 Glucuronidase
MMP-8 Elastase
MMP-9 Lysozyme
Lactoferrin Proteinase 3
β2-microglobulin α1-antitrypsin
Defensins
Cathespsin
BP1
FMLP receptor FMLP receptor CD63, CD68
CD11b/CD18 CD11b/CD18
Deacylating enzyme Cytochrome b558
CD66, CD67
Fibronectin receptor
TNF-α receptor
innate and adaptive immune response. Lactoferrin released
during phagocytosis may inhibit proliferation of mixed lym-
phocyte cultures by decreasing release of interleukin (IL)-2,
TNF-α, and IL-1. Proteinase 3 has been found to augment
release of active TNF-α and IL-1 in monocyte/neutrophil
cocultures by releasing the membrane bound forms of these
cytokines.10 Gelatinase B has been shown to convert latent
IL-1 into its active form11 and to potentiate IL-8 activity by
truncating this chemoattractant and increasing its release,
amplifying neutrophilic influx.12 Neutrophil elastase also may
play a proinflammatory role by virtue of its ability to cleave
and disrupt phosphatidyl serine receptors on macrophages.
Apoptotic cells undergo membrane alterations that lead to
expression of phosphatidyl serine on their outer membrane
surface, and interaction of phosphatidyl serine with its recep-
tor leads to macrophage responses that downregulate inflam-
mation through the generation of transforming growth factor
(TGF)-β.13 By disrupting these interactions, the presence of
neutrophil elastase may permit inflammation to continue.14
NEUTROPHIL ACTIVATION AND FUNCTION
For bloodstream neutrophils to destroy foreign targets in
the periphery, they first must sense the presence of such tar-
gets at a distance. They must adhere to and pass through
the endothelium of postcapillary venules (diapedesis) and
migrate to the source of the signal (chemotaxis). Finally,
neutrophils must encounter a target, engulf it, and destroy
it. Collectively, these processes are referred to as neutrophil
activation. Because of the potential for tissue destruction,
neutrophil activation must be carefully regulated. The inter-
nal responses through which a cell translates an encounter
with a stimulus into a particular phenotypic response are
termed signal transduction (Fig. 12-2).15,16
Stimuli and Receptors
Classic chemoattractants include lipid mediators (e.g.,
leukotriene B4 [LTB4], platelet-activating factor) and pro-
teins/peptides (e.g., formylated peptides, the complement
 218 Pillinger  |  Neutrophils and Eosinophils

Immune complex

Growth
Endothelial factor Chemoattractant
ICAM Tyrosine kinase Serpentine seven
FcγRII CD11b CD18 receptor receptor

Ras α
Grb2 SOS γ
P P GTP GTP

Rac Rho β Heterotrimeric G protein

Raf

Membrane ruffling, Stress fiber

superoxide generation formation P13K PLC
MEK

DAG IP3

Translocation ERK cPLA2

PKC Ca++

ERK AA
AP-1 system Calmodulin
kinase
Transcriptional Adhesion, superoxide
regulation Nucleus generation

Figure 12-2  Signaling pathways in neutrophil activation. Engagement of Fc, growth factor, and chemoattractant receptors and adhesion molecules
initiate signaling pathways that result in proinflammatory neutrophil responses, including cytoskeletal and morphologic changes, activation of adhe-
sion molecules and the superoxide generating system (NADPH oxidase), and regulation of transcription. Some of the well-established pathways partici-
pating in these responses are illustrated (see text for details).

split product C5a, and IL-8). Chemoattractants in vivo are
formed at sites of inflammation—either produced at the site
by inflammatory cells, such as LTB4 or IL-8, or liberated
from already synthesized proteins, as in the case of C5a. The
ability of formylated peptides, such as N-formyl-methionyl-
leucyl-phenylalanine, to stimulate neutrophils probably
represents a particularly ancient arm of the innate immune
response because prokaryotic, but not eukaryotic, cells
synthesize proteins whose first amino acid is a formylated
methionine. Chemoattractants also have the capacity to
stimulate most other aspects of neutrophil activation. Their
individual potencies for particular responses may differ,17
however, suggesting that they may serve overlapping, but
distinct functions in neutrophil activation.
Bloodstream activation of neutrophils depends on the
presence of specific surface receptors. Chemoattractant
receptors belong to a class known as seven-transmembrane-
domain receptors, or “serpentine seven” receptors, com-
posed of a single protein chain whose seven hydrophobic
domains span the plasma membrane. Binding of a particular
chemoattractant occurs in a pocket on the cytoplasmic face,
close to or below the level of the plasma membrane.
Receptors for soluble ligands other than chemoattrac-
tants also have been identified on neutrophils, including
receptors for growth factors, colony-stimulating factors,
and cytokines.16 These receptors fall into several families
distinct from the ­ serpentine sevens. Growth factor recep-
tors are members of the protein tyrosine kinase receptor
family, in which ligand interaction with two identical or
related receptors brings them into proximity, causing their
phosphorylation and activation. Some nonchemoattractant
ligands do not directly activate neutrophils, but may modu-
late their function. Pretreatment of neutrophils with either
insulin or GM-CSF results in amplification of subsequent
neutrophil responses to chemoattractants, a process referred
to as priming.
G Proteins
Ligation of seven-transmembrane-domain receptors results in
the interaction of cytoplasmic elements of the receptor with a
class of effectors known as heterotrimeric guanosine triphos-
phate (GTP)–binding proteins (G proteins). G proteins are
composed of α, β, and γ subunits, and individual G protein
types are distinguished by their particular combination of sub-
units. In neutrophils, the predominant G proteins are of the
Gi family.15 G protein γ subunits are modified by the addition
of prenyl (polyisoprene) and carboxyterminal methyl groups,
which serve to anchor them to the plasma membrane. All
G proteins share the capacity, localized to their α subunits,
to bind GTP and subsequently to hydrolyze it to guano-
sine diphosphate (GDP). G proteins are active when GTP
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
bound, but inactive in the GDP-bound form. Engagement
of the appropriate seven-transmembrane-domain receptor
results in the binding of GTP on the α subunit. As a conse-
quence of GTP binding, heterotrimeric G proteins dissoci-
ate into α and β/γ components, each with specific effector
functions.
A monomeric class of low-molecular-weight (20 to
25 kD) GTP-binding proteins (LMW-GBPs) also has been
described. Because the first, prototypical LMW-GBP to
be described was the proto-oncogene ras, these also are
referred to as ras-related proteins. LMW-GBPs combine,
in one molecule, the prenyl and methyl modifications of
the G protein γ subunit with the GTP-binding capacity of
the α subunit. At least four families of LMW-GBP have
been described: the Ras family, whose members play roles
in cell growth and division; the Rho family, which func-
tions in cytoskeletal rearrangements18; and the Rab and Arf
families, which are crucial for vesicular and endomembrane
trafficking.19 All four classes of LMW-GBPs are represented
in neutrophils.
Second Messengers
Second messengers are small, diffusible molecules that are
generated in response to stimuli and transmit signals from
membrane receptors to downstream effector proteins. In
the classic model of neutrophil activation, engagement
of receptors results in the activation of phospholipase C,
which cleaves phosphoinositol triphosphate into diacyl­
glycerol and inositol 1,4,5-triphosphate. ­Diacylglycerol and
inositol 1,4,5-triphosphate mediate the influx of cytosolic
calcium and the activation of protein kinase C. Other phos-
pholipases present in the neutrophil include phospholipase
A2, which cleaves phosphatidylcholine or ethanolamine or
both and is responsible for the ­ generation of arachidonic
acid, and phospholipase D, which cleaves phosphatidyl-
choline into phosphatidic acid and choline.20 Although the
above-described second messengers all have been impli-
cated in neutrophil activation, other lipid ­ mediators may
have negative regulatory effects. Sphingosine and ceramide
each inhibits neutrophil phagocytosis.21
In addition to lipids, other organic and inorganic
messenger molecules have been characterized. Intracel-
lular concentrations of cyclic adenosine monophosphate
(cAMP), a classic second messenger, increase rapidly in
neutrophils exposed to stimuli and inhibitors. cAMP in
these settings is likely to provide a negative regulatory
(off) signal because direct exposure to cAMP inhibits
neutrophil responses, probably through the activation of
protein kinase A.17,22 In contrast, increases in cyclic gua-
nosine monophosphate have a modest enhancing effect
on some neutrophil responses. Nitric oxide (NO), an
important molecule in the regulation of host defense, also
is produced in neutrophils, albeit in low levels.23 Endog-
enously produced NO in neutrophils is likely to play an
important role in signal transduction; several studies have
documented the capacity of exogenously added NO to
exert a variety of effects, including inhibition of reduced
nicotinamide adenine dinucleotide phosphate (NADPH)
oxidase, actin polymerization, and chemotaxis (see later).
Excessive NO production has been implicated in many
rheumatic diseases.24
219
Kinases and Kinase Cascades
There has been explosive growth in understanding of how
kinases—proteins capable of enzymatically adding phos-
phate groups to target molecules—contribute to signaling
in myeloid and nonmyeloid cells. Nonetheless, it is likely
that studies to date have elucidated only a small portion of
the molecules involved. Protein kinase C (PKC), actually
a family of kinases, was among the first kinases implicated
in neutrophil activation and is activated in response to che-
moattractants. The ability of phorbol myristate acetate—a
synthetic activator of PKC—to stimulate neutrophil
responses, including adhesion and O2− generation, supports
a role for PKC in neutrophil activation.25 In addition, inhibi-
tors of PKC block stimulation of neutrophil functions.
The mitogen-activated protein kinases are a family of
serine threonine kinases including the ERK, p38, and Jun
kinase (JNK) families. In neutrophils, chemoattractants and
other stimuli are capable of activating p38, Jnk, and Erk, on
time courses consistent with neutrophil activation.26,27 A role
for Erk activation in signaling for neutrophil O2− has been
­proposed, but remains controversial; a role in neutrophil
adhesion and phagocytosis seems to be better established.17,28
Phosphatidylinositol 3-kinase is a set of related enzymes
that are found in abundance in neutrophils and ­ primarily
catalyze the phosphorylation, not of proteins, but of the 3-
position of phosphatidylinositol phospholipids. The main
active product of phosphatidylinositol 3-kinases seems to be
phosphatidylinositol triphosphate. Chemoattractants rap-
idly activate phosphatidylinositol 3-kinase in neutrophils,
where it seems to play a role in diverse neutrophil functions,
including O2− generation, adhesion, and degranulation.29
Phosphatidylinositol 3-kinase also may regulate neutrophil
survival and apoptosis.30
Neutrophil Adhesion
One of the earliest, crucial aspects of the inflammatory
response is the ability of bloodstream neutrophils to adhere
to vascular endothelium preparatory to movement into the
tissues (Fig. 12-3). Stimulated neutrophils also possess the
ability to adhere to each other, a process termed homotypic
aggregation, which may bring in vivo bloodstream neutro-
phils into proximity with neutrophils already adherent to
the vessel or concentrate them at a site of inflammation.
Extensive investigation has provided significant insight
into the mechanisms involved in neutrophil adhesion. Sev-
eral families of interacting adhesion molecules have been
shown to exist on neutrophils and endothelial cells, includ-
ing the selectins, integrins, intercellular adhesion molecules
(ICAMs), and sialylated glycoproteins.
The selectins consist of three related molecules (L-
selectin on leukocytes, E-selectin on endothelial cells, and
P-selectin on activated platelets and endothelial cells) shar-
ing a common structure of two or more complement regu-
latory domains, an epidermal growth factor–like domain,
and a lectin domain. Each binds to a sialylated glycopro-
tein on the surface of its interacting cell: E-selectin binds
to the sialyl Lewisx antigen on neutrophils, P-selectin binds
P-selectin glycoprotein-1 on neutrophils, and L-selectin
binds P-selectin glycoprotein-1 and GlyCAM-1 on the
endothelium. Selectin expression is largely constitutive,
 220 Pillinger  |  Neutrophils and Eosinophils
Rolling Tight Adhesion Diapedesis
Selectin
Selectin Endothelial
Integrin
(active) cell
Integrin Sialylated
(inactive) glycoprotein ICAM
Basement
membrane Cytokines Chemoattractants
Figure 12-3  Neutrophil adhesion to the vascular endothelium. Left,
Rolling. An unstimulated neutrophil adheres with low affinity to the un-
stimulated endothelium of a postcapillary venule, a process mediated
by the interaction of selectins (on neutrophil and endothelium) with
­sialylated glycoproteins and resulting in the rolling of neutrophils along
the vessel wall. Center, Tight adhesion. Exposure of the neutrophil to
chemoattractants results in activation of integrins (CD11a/CD18, CD11b/
CD18); exposure of endothelium to cytokines results in the expression
of ICAMs. These molecules interact, resulting in tight adhesion. Concur-
rently, selectins may be shed from the cell surfaces. Right, Diapedesis. A
neutrophil undergoes diapedesis, passing across the endothelium and
making its way through the basement membrane. Bloodstream neutro-
phils have the capacity to adhere to and move out of the vasculature in
response to tissue signals for inflammation.
but selectin/sialylated glycoprotein interactions are of low
affinity and transient. The result of these interactions is
that a pool of bloodstream neutrophils is, at any one time,
loosely marginated to the vascular surface and moving along
it slowly in a rolling, tumbleweed-like motion. Exposure of
neutrophils and endothelium to appropriate stimuli (e.g.,
adrenergic discharge, corticosteroids) leads to shedding of
selectins and neutrophil release (stress demargination), with
apparent increases in the peripheral neutrophil count.
The integrins are a large family of heterodimeric mol-
ecules generated by various combinations of α and β chains.
Similar to the selectins, they require divalent cations (Ca2+
or Mg2+ or both) to engage their ligands. Neutrophils express
three β2-type integrins, each constructed from a distinct α
subcomponent (CD11a, CD11b, or CD11c) and a common
β2 chain (CD18). Integrins use the ICAMs as their counter-
ligands. CD11b/CD18 (also called Mac-1 or CR3) binds to
fibrinogen, factor X, heparin, and the complement compo-
nent iC3b in addition to ICAM and is most strongly impli-
cated in neutrophil/endothelial and neutrophil/neutrophil
interactions. In contrast to the selectins, neutrophil CD11b/
CD18 is constitutively expressed, but inactive; stimulation
of neutrophils by chemoattractants and other agents results
in changes in the activation state of CD11b/CD18 and
increases its affinity for ICAMs and other ligands.31 Stimula-
tion of endothelium with cytokines such as IL-1 results in
increased expression of ICAM-1 and ICAM-2, providing a
coordinate mechanism for the regulation of adhesion. In con-
trast to selectin-mediated adhesion, integrin/ICAM interac-
tions are high affinity and persistent. Stimulation of rolling
neutrophils results in their tight adhesion to vessel walls
and constitutes the first committed step in the movement
of neutrophils into the tissues. ­Additionally, engagement of
integrins by their counterligands sends signals into the cell
(“outside in” signaling), regulating selective cell responses
such as ­cytoskeletal ­reorganization, oxidant ­production, and
Figure 12-4  Neutrophil diapedesis through the vascular endothelium.
A neutrophil passaging between, or through, one or more endothelial
cells is illustrated. The characteristic neutrophil multilobed nucleus and
multiple granule types are visible. The leading edge of the neutrophil,
passing through the endothelium, is relatively devoid of granule con-
tents, suggesting that it represents the formation of a specialized struc-
ture for diapedesis, including the formation of F-actin cytoskeleton.
degranulation. Outside-in signaling through CD11b/CD18
also coordinates with signaling through Fc receptor FcγRIII
(see later) to mediate phagocytosis of particles opsonized
by IgG and the complement component iC3b. Crosstalk
between neutrophils and endothelial cells also has been
shown to be a CD11b/CD18-dependent event: Cross-­linking
of CD18 on neutrophils leads to increased permeability of
endothelial cells, probably through the release of neutrophil
proteases.32
The function of CD11a/CD18 on neutrophils has been
controversial, but accumulating data indicate that this mol-
ecule may be necessary for neutrophil adhesion and emigra-
tion.33 The function of CD11c/CD18 is less clear, although
it may play a role in neutrophil phagocytic activity.34
Diapedesis and Chemotaxis
The mechanism by which neutrophils pass through the
endothelial barrier is unclear. One report suggests a model
in which neutrophils pass directly through pores generated
within the endothelial cells themselves, but neutrophils
alternatively may pass between endothelial cells by disrup-
tion of cell-cell junctions (Fig. 12-4).35 Diapedesis occurs via
homotypic interactions between adhesion molecules found
on neutrophils and endothelial cells known as ­ platelet-
­endothelial cell adhesion molecules (PECAMs). These
molecules are concentrated at endothelial cell junctions,
and antibodies that block PECAM inhibit transmigration
in vitro by limiting neutrophils to the apical surface of the
endothelium. Transmigrating neutrophils undergo upregula-
tion of α6β1, an integrin that mediates binding to laminin
(a key component of the perivascular basement membrane).
Antibodies to α6β1 generally block neutrophil transmi-
gration, but fail to do so in a PECAM knockout mouse,
­implicating α6β1/PECAM as crucial to the passage of neu-
trophils out of the vasculature.36 CD47, otherwise known as
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
integrin-associated protein,37 and CD99, expressed on neu-
trophils and endothelial junctions, also have been impli-
cated in neutrophil passage through the endothelium.38
When beyond the endothelium, most neutrophils pause
for a time before essaying the basement membrane (basal
lamina). Classic studies by Huber and Weiss39 suggest that
neutrophils pass through the basement membrane via active
disruption of its patency, without elucidation of known
proteases or oxygen radicals. The disruptions are rapidly
repaired by an unknown mechanism, probably involving
the endothelium.
Chemotaxis in the direction of a gradient is achieved by
the extension of membrane ruffles (lamellipodia), followed
by anchorage of the ruffles to the substrate and withdrawal
of the trailing edge of the cell in the direction of move-
ment. These changes are accomplished primarily through
rearrangement of the actin cytoskeleton. Actin is a 41-kD
protein that exists as a soluble, globular monomeric form (g-
actin) and as an insoluble linear polymer (f-actin). F-actin
may be assembled (extended) at one end (barbed end) and
disassembled at the other, under the control of regulatory
molecules. During chemotaxis, f-actin formation and exten-
sion is concentrated at the leading edge of the neutrophil,
permitting extension of the cell membrane (see Fig. 12-4).
Chemoattractant receptors also concentrate at the leading
edge, defining the cell’s directional response to the gradient
(headlight phenomenon). As the neutrophil moves along,
receptors that were formerly at the leading edge are swept to
the tail and internalized.40
Phagocytosis and Degranulation
Neutrophil phagocytosis of an encountered bacterium or
other particle requires direct contact. Neutrophils are gen-
erally poor at phagocytosing unmodified targets, particularly
encapsulated bacteria. Optimal phagocytosis depends on
opsonization (from the Greek, “to prepare for the table”),
the modification of a target via its decoration with immu-
noglobulin or complement components or both. Neutro-
phils express two families of receptors for the Fc portion
of complexed or aggregated IgG: low-affinity FcγRIIa and
high-affinity FcγRIIIb. During some infections, or after in
vitro stimulation with interferon or G-CSF, neutrophils
also express the high-affinity receptor FcγRI, which binds
­monomeric IgG.
FcγRIIa binds subclasses of IgG with varying efficiency
depending on a polymorphism at amino acid position 131.
Confusingly, the “low-responder” allotype (so named because
of its weak interaction with mouse IgG1) binds human IgG2
efficiently, whereas the “high-responder” allotype (which
binds mouse IgG1 efficiently) does not. FcγRIIIb polymor-
phisms of neutrophil antigens NA1 and NA2 also determine
binding to IgG subclasses. Individuals homozygous for the
NA2 allele have a lower capacity to mediate phagocytosis
than individuals homozygous for the NA1 allele. These dif-
ferences have important implications for rheumatic diseases
in which immune complexes play an important role (see
later).
Phagocytosis is an active process that involves exten-
sion of the neutrophil membrane (filopodia and lamello-
podia formation) and invagination of the neutrophil at the
locus of the target. Engagement of FcγR and complement
221
­receptors results in the activation of diverse signaling path-
ways. Elegant studies by Caron and Hall indicate that FcγR
and complement receptors play distinct roles in phagocyto-
sis40a. Although engagement of CR3 (CD11b/CD18) results
in actin stress fiber formation and invagination, engagement
of FcγRII results primarily in extension of membranes out
from and around the target. Signaling by these receptors
depends on the activation of distinct members of the Rho
family of LMW-GBPs. These observations remain to be spe-
cifically confirmed for neutrophils, however. On activation,
neutrophils degranulate, a term actually reflecting two dis-
tinct processes. Vesicles can fuse with the plasma membrane,
spilling their contents into the extracellular space (see Fig.
12-1), or they can fuse with the phagocytic vacuole to form
a phagolysosome. The former type of degranulation is regu-
lated differentially from the latter and favors mobilization
of lighter granules in response to stimuli (secretory vesicles
> gelatinase granules > specific granules > azurophilic gran-
ules). In the latter type of degranulation (phagolysosome
formation), fusion of azurophilic granules with the phago-
cytic vacuole results in the delivery of proteolytic enzymes,
myeloperoxidase, and antibacterial proteins to the site of
the ingested bacterium. Fusion of specific granules with the
phagocytic vacuole permits the delivery of collagenase and
the appropriate localization of cytochrome b558, a requisite
for NADPH oxidase (see later). Containment of potentially
toxic substrates within the phagolysosome keeps host tis-
sue damage and neutrophil autodestruction in check.41 As
discussed subsequently, however, in several of the rheumatic
diseases, neutrophilic activation plays an important role in
abetting inflammation and host tissue damage.
Reduced Nicotinamide Adenine Dinucleotide
Phosphate Oxidase System
In addition to the collection of proteases and other antibac-
terial proteins contained in their granules, neutrophils have
the capacity to kill bacteria through the generation of toxic
oxygen metabolites. This process, frequently referred to as
the respiratory burst, is extremely potent and requires tight
regulation to prevent neutrophil autodestruction. Studies in
cell-free systems have established the so-called minimal sys-
tem required for O2− generation—NADPH oxidase.42,43 The
central component of NADPH oxidase is cytochrome b558,
which is localized to the membranes of specific granules and
consists of two subunits: a 22-kD component (gp22phox, for
phagocyte oxidase) and a 91-kD component (gp91phox). This
cytochrome lacks independent activity, however. Three
cytosolic proteins also are required: a 47-kD and a 67-kD
component (p47phox and p67phox) and a LMW-GBP, p21rac. On
neutrophil stimulation, the p47phox and p67phox components
translocate to the membranes to form an active complex
with the cytochrome.44 Although p21rac also translocates in
response to stimuli, the significance of its translocation is
more controversial.45 A fifth protein, p40phox, also has been
reported to be associated with p47/p67 in the cytosol. Evi-
dence suggests that p40phox may regulate the oxidase system
in a positive and a negative manner (Fig. 12-5).46
When assembled and activated, the NADPH oxidase
transfers electrons from NADPH to generate O2−:
2O 2 + NADPH NADPH
 Oxidase
→ O 2 − + NADP + + H +
 222 Pillinger  |  Neutrophils and Eosinophils

a key role in the body’s defense against microorganisms. The

current view that oxidant production, via the production of
p22phox gp91phox
hypochlorous acid by myeloperoxidase, is the neutrophil’s
most powerful tool against microbes has been challenged,
however. Mice lacking either NADPH oxidase or elastase
and cathepsin G are susceptible to infection, implying that
GDP both arms of defense—oxidant production and protease-
p21rac mediated microbial destruction—are equally crucial. Super-
oxide production in phagocytic vacuoles causes the pH to
p67phox rise (secondary to the consumption of protons necessary to
p47phox
make H2O2), which causes an influx of K+. The resulting
increase in ionicity liberates cationic proteases from the
p40phox anionic proteoglycan matrix, freeing them to kill bacteria.
rho In this new model, oxidants are not primarily destructive
GDI Activation to microbes, but rather necessary to facilitate proteolytic
damage.47 In support of this model is the fact that myelo-
_
2O2 ï peroxidase deficiency is common (1:2000), yet surprisingly
benign.

p22phox gp91phox NEUTROPHIL PRODUCTION

p21rac 2O2
p67phox
p47phox +
GTP
NADPH
p40phox
NADP+ + H+
rho
GDI
Figure 12-5  Assembly of the neutrophil NADPH oxidase system. Top,
Basic components of the NADPH oxidase as they are distributed in a
resting state. The cytochrome-b558, composed of the two subunits
gp91phox and p22phox, are membrane associated, whereas p47phox,
p67phox, and the more recently identified p40phox exist as a complex
in the cytoplasm. p21rac in an inactive, GDP-bound form also resides in
the cytoplasm, in association with a chaperone (Rho-GDI) that sheaths
its hydrophobic tail to permit solubility. Bottom, Activation of the neu-
trophil leads to translocation of the cytosolic components of the oxidase
to the neutrophil membrane, where they form an active complex with
the cytochrome, resulting in the generation of oxygen. The potentially
damaging oxidase system is carefully regulated through the segregation
and assembly of its component parts.
A subsequent, spontaneous dismutase reaction rapidly
produces hydrogen peroxide:
O 2 − + 2H + → H 2 O 2 + O 2
Although O2− and H2O2 can kill organisms in vitro,
they are short-lived and probably do not account for most
of the bacterial killing capacity of the system under normal
circumstances. (Many bacteria possess catalase, an enzyme
that degrades H2O2.) Rather, the production of H2O2 within
the same space into which the myeloperoxidase has been
released permits the generation of large quantities of hypo-
chlorous acid (chlorine bleach), a powerful oxidant with
potent killing capacity. Hypochlorous acid may interact
further with proteins to form chloramines, less potent but
longer-lived oxidants. Neutrophil oxidant production plays
OF PROINFLAMMATORY
MEDIATORS
Arachidonic Acid Metabolites
The capacity of stimulated neutrophils to liberate arachi-
donic acid from membranes has implications for the propa-
gation of acute inflammation. Although arachidonic acid
itself has chemoattractant and neutrophil stimulatory prop-
erties,28,48,49 its metabolites are more crucial to regulation of
inflammation. Best recognized among these are the leuko­
trienes. Neutrophils have the capacity to produce LTB4,48
a highly potent lipid mediator for the chemoattraction of
other neutrophils. Intermediates of leukotriene production,
such as 5-hydroxyeicosatetraenoic acid, also are produced
by neutrophils and may have stimulatory properties.28,50
An alternative class of lipoxygenase products, the lipox-
ins, have been characterized.48 Synthesis of lipoxins requires
coordinated activity of neutrophil 5-lipoxygenase and a
related enzyme (either 12-lipoxygenase or 15-lipoxygenase)
in another cell type—either platelets or endothelial cells (Fig.
12-6).51 In contrast to leukotrienes, lipoxins inhibit neutro-
phil function and are anti-inflammatory,52 suggesting that the
assembly of a mixed population of inflammatory cells may trig-
ger the synthesis of anti-inflammatory molecules (resolvins),
contributing to the subsequent resolution of inflammation.
Aspirin has been shown to stimulate the generation of bio-
logically active epilipoxins, suggesting a previously unappre-
ciated mechanism for its anti-inflammatory action.53
The cyclooxygenase (COX) (endoperoxide synthase)
pathway is the other major pathway of arachidonic acid
metabolism. Arachidonic acid metabolized by COX is con-
verted into prostaglandin H,54 which undergoes further cell
type–specific conversion to a variety of other prostaglandins.
The prostaglandins of most relevance to inflammation are
those of the E series, particular prostaglandin E2. Prostaglan-
dins of the E series have numerous proinflammatory effects,
including increased vasodilation, vascular permeability, and
pain. The direct effects of prostaglandin E on neutrophils
seem to be inhibitory, probably through elevations of intra-
cellular cAMP.55 Although resting neutrophils exhibit little
COX activity, persistent neutrophil activation results in
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
12-LO
LXA4
AA 5-LO LTA4 12-LO
LXB4
12-LO
Neutrophil
Platelet
LXA4
AA 15-LO 15-HETE 5-LO
LXB4
Neutrophil
Epithelial cell
Figure 12-6  Generation of the anti-inflammatory lipoxins A4 and B4
­depends on the interaction between two different classes of inflam-
matory cells. Top, Lipoxin generation by neutrophils and platelets. Ara-
chidonic acid (AA) generated by activated neutrophils is converted by
neutrophil 5-lipoxygenase (5-LO) into leukotriene A4 (LTA4). LTA4 may
be converted by 12-LO in nearby platelets into lipoxin A4 (LXA4) and li-
poxin B4 (LXB4). Bottom, Lipoxin generation by epithelial cells and neu-
trophils. AA generated by epithelial cells may be converted by 15-LO into  cells/L. Autosomal dominant and autosomal recessive sub-
15-­hydroxyeicosatetraenoic acid (15-HETE). In the setting of inflamma-
tion, 5-LO from adjacent neutrophils subsequently may convert 15-HETE
into LXA4 and LXB4.
upregulation of COX-2,56 suggesting that neutrophils may
contribute prostaglandin E2 to the proinflammatory brew
and the downregulation of their own activity.
Cytokine Production
Although the relative amount of cytokine production by neu-
trophils is small, the large numbers of neutrophils present in
infected or inflammatory sites suggests that overall neutrophil
cytokine production may play a role in recruiting additional
neutrophils to the target area. Among the cytokines produced
by neutrophils are IL-1, IL-8, IL-12, TNF-α, TNF-β, MIP-1α,
oncostatin M, MCP-1, and TGF-β.57,58 TGF-β is a powerful neu-
trophil chemoattractant,59 and its recruitment of neutrophils
into an inflammatory space may lead to additional neutrophil
cytokine production, including further production of TGF-β.60
TGF-β also has potent anti-inflammatory effects,13 however,
suggesting that neutrophils also may participate in the reso-
lution of inflammation. Activated neutrophils also produce
an antagonist to IL-1, the IL-1 receptor antagonist.61,62 The
efficacy of recombinant IL-1 receptor antagonist (anakinra)
in the treatment of rheumatoid arthritis and in autoinflam-
matory diseases such as Still’s disease63 emphasizes its clinical
importance in downregulating synovial inflam­mation.
An intriguing mechanism has been defined through
which neutrophils might downregulate the action of protein
inflammatory mediators. Apoptotic neutrophils (present
during the resolving phase of inflammation) show increased
expression of the chemokine receptor CCR5 on their sur-
face, and this receptor can scavenge, and reduce the soluble
223
concentration of, chemokines such as CCL3 and CCL5.
These data emphasize again that neutrophils are not only
inflammatory cells, but also may play a direct role in the
subsequent resolution of inflammation.64
HERITABLE DISORDERS OF NEUTROPHIL
FUNCTION
A wide variety of acquired conditions result in neutrophil
dysfunction or depletion or both, including malignancies
(myeloid leukemias), metabolic abnormalities (diabetes),
and drugs (corticosteroids, chemotherapy). In addition, many
rare, congenital disorders of neutrophils have been identified
(Table 12-2). In general, patients with impaired neutrophil
function are prone to infection by bacteria (predominantly
Staphylococcus aureus, Pseudomonas species, Burkholderia) and
fungi (Aspergillus, Candida),65 but not viruses and parasites.
The major sites of infection include skin, mucous mem-
branes, and lungs, but any site may be affected, and spreading
abscesses are common. Most of these diseases are potentially
life-threatening in the absence of available effective therapy.
Diseases of Diminished Neutrophil Number
Severe congenital neutropenia (Kostmann’s syndrome)
results from marrow arrest of bone marrow myelopoiesis and
leads to neutrophil counts persistently less than 0.5 × 109
types have been identified.66 Patients are prone from early
infancy to severe bacterial infections, including omphali-
tis, pneumonia, otitis, gingivitis, and perirectal infections.
Because acute inflammation is lacking, infections tend to
spread extensively before coming to attention. Mortality
has been high. Therapy consists of antibiotics and long-
term therapy with recombinant human G-CSF, which may
help maintain normal or near-normal neutrophil counts.67
A milder form of neutropenia (benign congenital neutrope-
nia) with higher neutrophil counts and fewer infections also
has been observed. Another variant is cyclic neutropenia,
which causes transient, recurrent neutropenia on a 21-day
cycle. Studies suggest that defects in neutrophil elastase
affect neutrophil survival in the marrow and may be respon-
sible for severe congenital and cyclic neutropenia.68
Leukocyte Adhesion Deficiencies
Leukocyte adhesion deficiency type 1 (LAD1) results from
an autosomal recessive defect in production of the CD18
chain of β2 integrins. Consequently, neutrophil β2 integrins
fail to form,69 and bloodstream neutrophils are unable to
adhere firmly to vascular endothelium and to transmigrate to
sites of infection. Phagocytosis also is impaired. The clinical
picture is similar to that of the neutropenias, with recurrent
life-threatening infections. Peripheral neutrophil counts
are typically elevated, however, reflecting the inability of
cells to exit the vasculature. Complete LAD1 manifests in
infancy.70 LAD2 results from an autosomal recessive defect
in the glycosylation of sialyl Lewisx, the neutrophil counter-
ligand for endothelial selectins. Patients with LAD2 have
neutrophils that are unable to roll along the endothelium
and have symptoms similar to LAD1, but also may have
mental retardation, short stature, distinctive facies, and the
 224 Pillinger  |  Neutrophils and Eosinophils

Table 12-2  Heritable Disorders of Neutrophil Function

Disorder Defect Inheritance Presentation Therapy Typical Prognosis
Neutropenia
Severe congenital Maturation arrest   AR (HAX1 Bacterial infections RhG-CSF Improved with
­neutropenia (Kostmann’s (<0.5 × 109 PMN/L) ­mutations) ­(omphalitis, abscesses, ­treatment
syndrome) gingivitis, UITs)
Chronic benign (0.2-2 × 109 PMN/L) ? Mild infections None Good
neutropenia
Cyclic ­neutropenia Stem cell defect, elastase AD (ELA2 Infection during   RhG-CSF Improved with
gene deficiency (nadir ­mutations) nadirs ­treatment
every 21 days)
Adhesion Deficiency
Leukocyte adhesion Absent or abnormal AR Leukocytosis   Marrow Fair-poor
­deficiency type 1 CD18; deficiency in   recurrent infections   ­transplant
β2-­integrin chain of (skin mucous,
leukocyte ­adhesion ­membranes,  
molecules gastrointestinal  
tract)
Leukocyte adhesion Absent sialyl-Lewisx AR Neutrophilia; infection poor
­deficiency type 2 ­retardation, short
stature
Leukocyte adhesion Impaired activation of AR Leukocytosis; recurrent poor
­deficiency type 3 Rap1 GTPase infections; bleeding
tendency
Chemotaxis Deficiency
Hyper-lgE syndrome Chemotaxis defect AD eczema, recurrent Skin care; Good
­infections and elevated ­antibiotics
serum lgE levels
Granule Disorders
Chédiak-Higashi   Defective lysosomal   AR Albinism; infection Marrow Poor
syndrome trafficking regulator   transplant;
gene antibiotics
Specific granule   Abnormal/reduced specific AR? Infection of skin, mucous Fair-good
deficiency and azurophilic granules membranes, lungs
(lactoferrin deficiency)
Myeloperoxidase   Myeloperoxidase absent Variable   None Transfusion Excellent
deficiency (mostly AR) of HLA
identical
leukocytes
if severe
p 14 deficiency Defective endosomal Recessive Albinism; infection; None known ?
adapter protein gene short stature to date
Oxidase Defects
Chronic granulomatous gp91phox absent X-linked 50% Early childhood Interferon-γ Improved with
­disease (multiple types) p22phox absent AR 5% infections, especially ­treatment
p47phox absent AR 35% skin and mucous
p67phox absent AR 5% ­membranes, abscesses
AR, autosomal, recessive; PMN, polymorphonuclear neutrophil; RhG-CSF, recombinant human granulocyte colony-stimulating factor; UTIs, urinary tract
­infections.

Bombay (hh) blood type.70,71 An additional leukocyte adhe-
sion deficiency also has been identified (LAD3). Patients
with LAD3 have normal populations of leukocyte surface
integrins, but lack the ability to signal these molecules into
an active state. Because integrin activation in this condi-
tion also is deficient in platelets, patients with LAD3 are at
increased risk for infection and bleeding.72
Neutrophil Granule Defects
The best-known defect in neutrophil granule formation is
Chédiak-Higashi syndrome. Chédiak-Higashi syndrome is
an autosomal recessive disorder in which granule ­subtypes—
in neutrophils, but also in lymphocytes, melanocytes,
Schwann cells, and others—undergo disordered fusion,
resulting in giant, dysfunctional granules.70 The cause seems
to relate to a defect in the gene for lysosomal transport
protein (Lyst).73 Patients with Chédiak-Higashi syndrome
present with partial oculocutaneous albinism, neutropenia,
frequent infection, mild bleeding diathesis, and neurologic
abnormalities. Approximately 85% of patients who survive
childhood enter a so-called accelerated phase, a lymphoma-
like infiltration of lymphocytes and histiocytes throughout
the body, which is generally fatal. Other diseases of neutro-
phil granules have less ominous prognoses. A novel immu-
nodeficiency syndrome relating to lack of the endosomal
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
adapter protein p14 also has been described. Patients with
this syndrome have congenital neutropenia with structurally
abnormal neutrophil primary granules and abnormalities of
B cells, cytotoxic T cells, and melanocytes. In addition to
immunodeficiency, clinical findings include short stature
and partial albinism.74
Oxidase Deficiencies—Chronic Granulomatous
Disease
Chronic granulomatous disease resembles other diseases of
neutrophil dysfunction in that it results in severe, recurrent
infections of the skin and mucous membranes. Osteomyelitis
and intra-abdominal abscesses are common. In contrast to
the other diseases, infection in patients with chronic granu-
lomatous disease generally results in a delayed, but quantita-
tively normal neutrophil response. Because of incapacity to
kill organisms, however, the accumulation of neutrophils at
a site of infection generally results in granuloma formation,
rather than clearance of the target. Cutaneous infections
tend to show persistent drainage and scarring. The presence
of even partially responsive neutrophils results in a lower
frequency of sepsis in these patients relative to patients with
absolute neutropenia. Chronic granulomatous disease is
typically a disease of early childhood, although some milder
cases may be recognized later in life.70
Chronic granulomatous disease comprises a group of dis-
eases. In each, a genetic defect in a different component
of NADPH oxidase results in failure of neutrophils (and
other phagocytes) to generate O2−. Previously, treatment
for chronic granulomatous disease consisted of aggressive
antibiotic prophylaxis and therapy. The addition of long-
term therapy with recombinant human interferon-γ has
improved the outcome significantly for patients with this
disease, and pilot attempts at gene therapy have yielded
encouraging results.75
Neutrophils engaged in host defense entered into a novel
death program, resulting in the elucidation of neutrophil
extracellular traps, extracellular structures composed of
chromatin and granule proteins that bind and kill microor-
ganisms. This process depends on the generation of reactive
oxygen species, however, and patients with chronic granu-
lomatous disease lack the capacity to produce neutrophil
extracellular traps. The inability to generate neutrophil
extracellular traps may contribute to the immune deficiency
seen in chronic granulomatous disease.76,77
NEUTROPHIL RELEVANCE TO RHEUMATIC
DISEASE
Neutrophil-mediated Tissue Destruction
Despite sophisticated regulatory mechanisms, tissue ­destruction
by neutrophils is common. Several mechanisms may permit
the release of neutrophil proteases and oxygen radicals into the
extracellular milieu. First, necrosis or destruction of neutro-
phils, or both, may liberate cellular contents indiscriminately.
Second, studies have revealed that degranulation and O2− gen-
eration may begin before complete closure of the phagocytic
vacuole, releasing products either into the external environ-
ment or against a target surface. Neutrophils may destroy host
tissues that they have been misdirected to attack. Although
225
serum and joint fluid ­contain antiproteases and antioxidants,
the “protected space” between a neutrophil and a surface (e.g.,
cartilage) may exclude these factors. Release of hypochlorous
acid, myeloperoxidase, and proteinases into the extracellular
milieu may inactivate the protective compounds and act as an
“antiproteinase shield.”41
Neutrophil Fc Receptor Polymorphisms
Given that polymorphisms of FcγR determine phagocytic
capacity of IgG isotypes, it is not surprising that they deter-
mine susceptibility to diseases in which autoantibodies play
a key role. Phagocytes from individuals with one FcγRIIa
polymorphism (H131) allele are able to bind and phagocy-
tose IgG2; phagocytes from individuals with a different poly-
morphism (R131) cannot. In white European and African
American populations, patients with lupus nephritis have
a higher frequency of the FcγRIIa-R131 allele than control
groups; their relative inability to clear immune complexes
may make them more susceptible to renal disease.78-80 The
significance of different Fc receptor polymorphisms may
vary among rheumatologic diseases. Tse and colleagues81,82
found no association between FcγRIIa polymorphisms and
likelihood of developing antineutrophil cytoplasmic anti-
body (ANCA)–associated vasculitides, but they found an
overrepresentation of homozygosity for the FcγRIIIb NA1
allele among patients with antimyeloperoxidase antibodies.
Few studies have been published on Fc polymorphisms and
susceptibility or severity of rheumatoid arthritis. A more
recent study found no association between FcγRIIa type
and rheumatoid arthritis susceptibility, but did find a cor-
relation between patients with extra-articular disease and
the homozygous R/R 131 genotype.83 In contrast, FcγRIII
receptor expression might be increased in rheumatoid
arthritis.84
Gout
Gout may be the quintessential neutrophilic rheumatic
disease. Although the initiation of an acute gouty attack
seems to involve the phagocytosis of urate crystals by syno-
vial macrophages and the generation of cytokines such as
IL-1 and IL-8, the hallmark of acute gout is the presence
of enormous numbers of neutrophils (sometimes >100,000/
mm3) in the affected joint space. Urate crystals in the joint
are capable of nonspecifically binding immunoglobulin
and fixing complement by the classic and alternative path-
ways. C5a liberated from the complement fixation process
attracts neutrophils to the joint space, where they phagocy-
tose opsonized crystals via receptor-dependent mechanisms,
resulting in further activation of the neutrophil and pro-
duction of LTB4, IL-8, and other mediators. Activation of
neutrophils results in the ingress of additional neutrophils.
Neutrophils in the gouty joint may damage joint structures
through discharge of contents directly into the joint fluid
during crystal phagocytosis, or directly against cartilage dur-
ing attempted phagocytosis of urate crystals embedded in
or adherent to cartilage. In addition, interaction of phago-
cytosed urate crystals with lysosomal membranes results in
the dissolution of the latter, spilling lysosomal proteases
into the cytoplasm and, eventually, into the extracellular
space.85
 226 Pillinger  |  Neutrophils and Eosinophils
Rheumatoid Arthritis
Although the focus in rheumatoid arthritis in recent years
has been on the role of T cells, B cells, synovial fibroblasts,
and mononuclear cells, the first inflammatory cell type to
be appreciated in rheumatoid arthritis was the neutrophil.
Rheumatoid arthritis in the joint may be conceptualized as
a two-compartment inflammatory disease: In the synovium,
lymphocytes, fibroblasts, and macrophages predominate,
but the joint space contains neutrophils almost exclusively.
Although the numbers of neutrophils in the rheumatoid
joint tend to be less than those seen in gout, they are still
quite large, with 10 billion cells per day cycling through a
30-mL effusion. The classic model suggests that rheumatoid
factor–based immune complexes, produced in the pannus
and present in the joint space in high concentrations, can
fix complement and draw neutrophils into the joint space
in high numbers. Once there, in vitro studies have docu-
mented the ability of neutrophils to bind to cartilage surfaces
embedded with immune complexes and to damage them via
incomplete phagocytosis. No adequate in situ demonstration
of direct neutrophil attack on cartilage has been offered yet,
however. The fact that seronegative arthritides, such as pso-
riatic arthritis, lack rheumatoid factor, but nonetheless share
with rheumatoid arthritis the presence of synovial hyper-
plasia and a large neutrophilic infiltrate in the joint space
suggests that immune complex formation may be important,
but not absolutely required, for neutrophil influx. The ability
of rheumatoid synovial monocytic leukocytes to secrete IL-1
and IL-8 and other cytokines indicates that pannus itself may
play an important role in the attraction of neutrophils out of
the bloodstream and into the joint. Although few in num-
ber, neutrophils within the pannus have been documented
to concentrate at the pannus/cartilage border, suggesting a
possible role in pannus-driven marginal erosion.53
In addition to promoting joint destruction, neutrophils
in the rheumatoid joint may contribute to the propagation
of pannus and of rheumatoid inflammation. As noted ear-
lier, neutrophils themselves can produce proinflammatory
cytokines; expression of several such cytokines, including
oncostatin M, MIP-1α, IL-1β, and IL-8, is increased in rheu-
matoid neutrophils, especially from rheumatoid arthritis
synovial fluid.86 Injection of lysates of neutrophil granules
into joints in animal models produces a synovitis indistin-
guishable histologically from rheumatoid synovitis, an effect
that can be reproduced by injection with purified active or
inactive myeloperoxidase.87,88 Neutrophil proteins also may
regulate synovial proliferation through effects on other resi-
dent or immigrant cell populations. Neutrophil proteinase
3 may enhance the proinflammatory effects of monocytes,
by cleaving and releasing active IL-1 and TNF-α from the
surface of the latter. Neutrophil defensins enhance phago-
cytosis by macrophages and stimulate the activation and
degranulation of mast cells, an interesting observation in
light of a report that mice deficient in mast cells are resis-
tant to the development of erosive arthritis.89 Neutrophil
proteases also enhance the adherence of rheumatoid syno-
vial fibroblasts to articular cartilage,90 and neutrophils may
regulate synovial vascularization through the production
of vascular endothelial growth factor, leading to endothe-
lial proliferation.91 Finally, several studies have raised the
possibility that, under certain conditions of stimulation,
­ eutrophils can serve as antigen presenting cells. Neutro-
n
phils in rheumatoid arthritis synovial fluid synthesize and
express large amounts of class II major histocompatibility
complex.92 The importance of neutrophils to the rheuma-
toid process may be underscored by rheumatoid arthritis
animal models in which mice deficient in neutrophils are
resistant to the arthritic process.93,94
Vasculitis
Neutrophils may be identified, to a greater or lesser degree,
in the lesions of virtually all kinds of vasculitis. The mecha-
nisms of neutrophil accumulation may vary, however, with
different mechanisms predominating in different conditions.
The early observation that infusions of allospecies serum
produced acute inflammation in skin and joints (serum
sickness), together with the appreciation that subcutane-
ous rechallenge with previously administered antigen leads
to intense local inflammation (Arthus reaction), led to the
development of a model in which immune complex deposi-
tion in the blood vessels results in complement activation
and an influx of neutrophils to the affected site. Because
immune complex formation is a hallmark of many primary
rheumatic vasculitides (e.g., essential mixed cryoglobuli-
nemia, hypersensitivity vasculitis, Henoch-Schönlein pur-
pura), it is likely that immune complex deposition is crucial
to the genesis of these diseases. In several of these vasculi-
tides, neutrophil disruption and fragmentation—clasis—is a
prominent pathologic finding, leading to their designation
under the rubric leukocytoclastic vasculitis. In some rheumatic
diseases in which vasculitis is a secondary phenomenon,
such as rheumatoid arthritis and systemic lupus erythemato-
sus, the role of immune complex deposition also is implicit.
It also has been suggested that patients with lupus experi-
ence transient accumulations of neutrophils (leukoaggre-
gation) in small vessels of the lungs and other tissues, as a
result of complement activation within these vessels or in
the soluble phase.95
Upregulation of adhesion molecules on endothelial cells
or neutrophils themselves or both is an alternative mecha-
nism through which neutrophil accumulation in vessels may
be propagated. The Schwartzmann phenomenon, in which
reinjection of cellular material leads to vascular inflammation
via a cytokine-dependent, immune complex–independent
mechanism, is a model for this avenue to vasculitis. Adhe-
sion molecule upregulation may be particularly relevant to
vasculitides in which immune complex formation is not a
hallmark, such as giant cell (temporal) arteritis. Detailed
analyses of the inflammatory cells involved in giant cell
arteritis indicate the presence of T cells producing IL-1β and
IL-6 that may act on vascular endothelium.96 It is likely that
many rheumatic diseases employ immune complex–depen-
dent and immune complex–independent mechanisms in the
pathogenesis of neutrophil ingress into vascular structures.
In addition to the role of immune ­complexes, Belmont and
coworkers97 have shown the induction of adhesion molecules
in patients with systemic lupus erythematosus.
Several vasculitides are noteworthy for the presence,
in the serum of affected patients, of antibodies directed
at cytoplasmic components of neutrophils (ANCA).97
ANCA-positive vasculitides are discussed in detail in
Chapter 82.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
Neutrophilic Dermatoses and Familial Mediterranean
Fever
Sweet’s syndrome, named after the physician who first
described it in 1964, is characterized by fever; neutrophilia;
and painful erythematous papules, nodules, and plaques. It
can be subdivided into five groups: idiopathic, parainflam-
matory (associated with inflammatory bowel disease or
infection), paraneoplastic (most commonly in the setting of
leukemia), pregnancy related, and drug associated (usually
after treatment with G-CSF). Most important clinically,
it is a diagnosis of exclusion. Sweet’s syndrome frequently
appears after an upper respiratory tract infection and has a
propensity to involve the face, neck, and upper extremities.
When found on the legs, Sweet’s syndrome lesions can be
confused with erythema nodosum. Histopathology is char-
acterized by dense neutrophilic infiltrate in the superficial
dermis and edema of the dermal papillae and papillary der-
mis. Leukocytoclasia may suggest leukocytoclastic vasculitis,
although vascular damage is absent. It is typically accompa-
nied by peripheral neutrophilia. Treatment with systemic
corticosteroids usually induces a dramatic resolution of the
lesions and the systemic symptoms. Although the etiology
of the disease is unclear, many authors believe that Sweet’s
syndrome may represent a form of hypersensitivity reaction
to microbial or tumor antigens. Antibiotics do not influence
the course of the disease in most patients.
Pyoderma gangrenosum is characterized by painful ulcer-
ating cutaneous lesions over the lower extremities, usually
in patients with an underlying inflammatory illness. Inflam-
matory bowel disease, rheumatoid arthritis, and seronega-
tive arthritis are the most common associations, although an
association with malignancy also has been reported. Fifteen
percent of patients have a benign monoclonal gammopa-
thy, usually IgA. Similar to Sweet’s syndrome, pyoderma
gangrenosum is a diagnosis of exclusion; is characterized
on biopsy specimen by neutrophilic infiltrate; and usually
remits with systemic corticosteroids, although topical and
intralesional injections of corticosteroids also may be benefi-
cial. Other rare neutrophilic dermatoses include rheumatoid
neutrophilic dermatitis, described as symmetric erythema-
tous nodules on extensor surfaces of joints; bowel-associated
dermatosis-arthritis syndrome occurring after bowel bypass
surgery for obesity; and neutrophilic eccrine hidradenitis,
sometimes linked to acute myelogenous leukemia.
In familial Mediterranean fever (discussed in detail in
Chapter 113), patients experience episodic inflammatory
exacerbations, characterized by large influxes of neutrophils.
A defect in an anti-inflammatory protein, pyrin, seems to
permit the inappropriate development of inflammation.
Pyrin has been shown to be expressed exclusively in myeloid
cells, including neutrophils and eosinophils.98
Effects of Antirheumatic Agents on Neutrophil
Functions
Many antirheumatic therapies currently in use have been
documented to act at least partly at the level of the neu-
trophil. Nonsteroidal anti-inflammatory drugs (NSAIDs)
are the most frequently used class of antirheumatic agents.
By virtue of their ability to inhibit COX activity and
­prostaglandin production, moderate doses of NSAIDs have
227
diverse effects on inflammation, including inhibition of vas-
cular permeability and modulation of pain. At higher, clini-
cally anti-inflammatory concentrations, NSAIDs inhibit
chemoattractant-stimulated neutrophil CD11b/CD18-
dependent adhesion and degranulation and NADPH oxi-
dase activity.99-101 It is unlikely, however, that these effects
are due solely to COX inhibition because (1) as noted ear-
lier, neutrophils exhibit little COX activity under normal
circumstances, and (2) concentrations of NSAIDs required
to inhibit neutrophil function exceed the concentrations
required to inhibit COX. High-dose NSAIDs seem to have
other, pleiotropic effects on neutrophil signaling. Our labo-
ratory has shown the capacity of aspirin and the poor COX
inhibitor sodium salicylate to inhibit Erk activation in a
manner consistent with inhibition of adhesion, suggesting
that salicylates may have unique effects on inflammation.102
Similar to nonsteroidals, glucocorticoids exert potent
effects on neutrophils, including inhibition of neutrophil
phagocytic activity and adhesive function. The ability of
steroids to increase peripheral blood neutrophil counts
acutely—an effect known as demargination—may be
directly attributable to the release of neutrophils adherent
to vessel walls. In addition, glucocorticoids inhibit phos-
pholipase A2 and leukotriene and prostaglandin production.
Glucocorticoids also may regulate the expression of COX-2
and stimulate the release of lipocortins, compounds with
anti-inflammatory effects on neutrophils. Although the abil-
ity of glucocorticoids to interact with cytosolic receptors and
to regulate transcription is unlikely to be directly relevant
to neutrophil function, these longer term effects may have
indirect relevance in that steroids possess the capacity to
reduce production of IL-1 and other cytokines at inflamma-
tory sites.
Other anti-inflammatory/immunomodulatory agents also
have well-established effects on neutrophils. Methotrexate,
widely used in rheumatoid arthritis, has no direct neutrophil
effect, but is capable of producing indirect effects, probably
by virtue of its ability to stimulate the release of adenosine
from surrounding cells. Some data suggest that ­methotrexate-
induced adenosine release might inhibit phagocytosis, O2−
production and adhesion,103 and that treatment of patients
with methotrexate inhibits the capacity of neutrophils to
generate LTB4.104
Colchicine, a standard agent in the treatment of gout
and familial Mediterranean fever, inhibits microtubule for-
mation and has pleiotropic effects on neutrophils, including
inhibition of adhesion via decrements in selectin expres-
sion.105 Colchicine has been observed to stimulate the
expression of pyrin in neutrophils. Because pyrin deficien-
cies are implicated in familial Mediterranean fever, this
observation suggests a previously unappreciated mechanism
of action of colchicine in neutrophilic diseases.98
Sulfasalazine has been shown to inhibit neutrophil respon-
siveness to chemoattractants; to inhibit chemotaxis, degran-
ulation, and O2− production; to decrease LTB4 production;
and to scavenge oxygen metabolites. Similar to sulfasalazine,
gold salts may scavenge toxic metabolites. Gold salts, still in
use for rheumatoid arthritis in many parts of the world, also
decrease neutrophil collagenase activity and reduce E-selec-
tin expression on endothelium.106
The current era of biologic therapies has been ushered
in through the introduction of agents designed to block the
 228 Pillinger  |  Neutrophils and Eosinophils
effects of TNF-α or IL-1. As noted earlier, IL-1 and TNF-α
directly affect neutrophil function, including priming for
stimulus-induced responses such as O2− production, cartilage
destruction, and production of cytokines such as IL-8 and
LTB4. Nonetheless, studies examining the effects of anti-
TNF treatment on neutrophil function measured ex vivo
have not indicated extensive action. Treatment of patients
with etanercept107 or adalimumab108 induced no effect on
neutrophil ex vivo responses, including chemotaxis, phago-
cytosis, and superoxide generation (although CD69 levels
were reduced). Reduction of neutrophil populations in
rheumatoid arthritis joint effusions after anti-TNF therapy
is more likely due to alteration of the inflammatory environ-
ment, rather than to direct effects on the neutrophils them-
selves. Consistent with this suggestion, etanercept inhibited
neutrophil rolling and adhesion in vivo in a rat model of
uveitis.109
EOSINOPHILS
The eosinophil line shares many features with the other
families of polymorphonuclear granulocytes. In contrast
to the neutrophil, however, the eosinophil is primarily a
tissue-localized cell. Eosinophils are produced in numbers
smaller than neutrophils, and their half-life in the blood is
shorter (3 to 8 hours) owing to higher rates of diapedesis.
Normal bloodstream levels of eosinophils tend to be low—
typically less than 5% of blood leukocytes. When in the tis-
sues, eosinophils are more long-lived than their neutrophil
cousins, with estimates ranging from 2 to 14 days. Tissue
eosinophils are found in greatest concentrations in gastro-
intestinal mucosa, suggesting that they participate in barrier
rather than bloodstream surveillance.1,110
EOSINOPHIL DEVELOPMENT AND MORPHOLOGY
Similar to neutrophils, eosinophils follow a classic pattern
of granulocyte differentiation, passing through blast, promy-
elocyte, myelocyte, metamyelocyte, and band stages before
reaching maturity.1 Along the way, eosinophils succes-
sively acquire morphologically distinct classes of granules.
The factors required for eosinophil differentiation include
GM-CSF and IL-3, which also are required for neutrophil
differentiation and cannot account for eosinophil commit-
ment. An essential role for IL-5 in eosinophil development
also has been described, supported by the observation that
intravenous administration of IL-5 rapidly results in periph-
eral eosinophilia. IL-5 may not be completely eosinophil-
specific, however, because studies in animal models suggest
that it is also trophic for B cells. Similar to IL-5, IL-2 can
stimulate eosinophilia. The IL-2 effect seems to be mediated
via production of IL-5, however. CCL11 (eotaxin-1) also
may cause bone marrow release of mature eosinophils and
eosinophil precursors,111 via engagement of CCR3 receptors,
which are mainly expressed on eosinophils.112 Cooperation
between IL-5 and eotaxins, in particular eotaxin-1, seems
to be needed to induce tissue eosinophilia. Knockout mice
with targeted deletion of CCR3 show deficiency in gastro-
intestinal eosinophils.
When viewed under hematoxylin and eosin staining, the
eosinophil appears slightly larger than the neutrophil (12
to 17 μm). Its nucleus is typically bilobed. Most striking is
the presence of large, pink-staining granules. In addition,
lipid bodies occasionally may be seen—nonvesicular accu-
mulations of arachidonic acid and other lipids, presumably
liberated from plasma membrane. These are not unique,
however, and may be detected occasionally in neutrophils
as well.
Eosinophils contain at least three distinguishable classes
of granules (Table 12-3). Primary granules form first and are
analogous to the primary (azurophilic) granules of neutro-
phils. In contrast to neutrophils, eosinophil primary gran-
ules lack myeloperoxidase. Eosinophil primary granules are
most numerous in eosinophilic promyelocytes and persist
in smaller numbers in mature forms. As a result, eosino-
phil primary granules have been less thoroughly studied
than their neutrophil counterparts. In mature eosinophils,
a lysophospholipase that is present in large quantities (7%
to 10% of total eosinophil protein) has been tentatively
localized to primary granules, which when released extra-
cellularly precipitates into bipyramidal structures known as
Charcot-Leyden crystals.113 The deposition of these crystals
in tissues is frequently taken as evidence of present or past
­eosinophilia.
The large granules visible in mature eosinophils are spe-
cific granules, which form during the myelocyte stage. When
viewed under scanning electron microscopy, specific gran-
ules show a dense crystalline core surrounded by an inter-
mediate-density matrix. Because of their greater size and
number (>90% of overall granule population), eosinophil-
specific granules have yielded to isolation and immunocyto-
chemical examination, and their contents have been at least
partially evaluated. Among the contents probably localized
to the specific granules are lysosomal enzymes (acid and
neutral hydrolases, collagenase, cathepsin, and gelatinase),
lectins, and components of the oxidase system. Most distinct
is the presence of four highly basic proteins that lend the
granule its tinctorial properties. Major basic protein (MBP),
an 11,000-kD protein with an isoelectric point value of 11,
accounts for more than 50% of the total granule protein and
is the major, or possibly sole, component of the crystalline
core. Trace amounts of MBP (<0.1% of that found in eosino-
phils) also may be observed in basophils. Eosinophil cationic
protein (ECP), actually a heterogeneous group of several
related proteins (18 to 21 kD molecular weight), also is pres-
ent in large amounts (upto 10% on weight/weight basis).
Table 12-3  Eosinophil Granule Contents
Arylsulfatase Primary Specific
Granules Granules Granules
Relative Smallest Intermediate Largest
size
contents Arylsulfatase Lysophospholipase Major basic protein
Acid phospha- Eosinophil cationic
tase protein
Eosinophil-derived
neurotoxin
Eosinophil
peroxidase
Acid hydrolases
Neutral hydrolase
Collagenase
Cathepsin
Gelatinase
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
Eosinophil-derived neurotoxin (18 kD molecular weight),
the third of the basic granular proteins, is slightly less basic
(isoelectric point value of 8.9) than the aforementioned
proteins and is present in smaller quantities. In contrast
to MBP and ECP, which have likely roles in host defense,
eosinophil-derived neurotoxin is mainly recognized for its
function as a neurotoxin for myelinated neurons, the evolu-
tionary advantage of which is unclear. The Gordon phenom-
enon, in which injection of eosinophil-laden tissue into an
animal produces profound neurologic deficits, is likely due to
eosinophil-derived neurotoxin. Finally, eosinophil-specific
granules contain large quantities of eosinophil peroxidase,
an enzyme distinctly different from neutrophil myeloperoxi-
dase, but probably subsuming the same function of generat-
ing hypohalides for cell killing and the activation of latent
proteinases.114 ECP, EDW, and eosinophil peroxidase are
localized within the primary granule matrix region. A third
population of smaller eosinophilic granules has been iden-
tified by virtue of its acid phosphatase and arylsulfatase B
content and is present mainly in tissue eosinophils.
EOSINOPHIL ACTIVATION AND DISTRIBUTION
Similar to neutrophils, eosinophils undergo activation in
response to stimuli and are capable of adhesion, chemo-
taxis, phagocytosis, degranulation, and O2− generation.
Eosinophils respond to many of the same chemoattractant
stimuli as neutrophils, although with different sensitivities.
In addition, eosinophils respond to numerous stimuli that
do not affect neutrophils, including IL-3, IL-5, RANTES,
and MIP-1α. Whether the distribution of these factors is
sufficient to explain the tissue distribution of eosinophils
relative to other granulocytes is uncertain; however, eosino-
phils and mast cells secrete IL-3 and IL-5, suggesting their
capacity to attract additional eosinophils to sites of atopy.
Eosinophils not only express adhesion molecules identi-
fied on neutrophils—CD11a/CD18, CD11b/CD18, and L-
selectin—but also others, such as the α4β1 integrin VLA-4.
IL-4 and IL-13 induce the expression of the VLA-4 coun-
terligand, VCAM-1, via an eotaxin-1, STAT6-dependent
pathway.115 Oncostatin-M, an IL-6/gp130 family member,
also seems to upregulate VCAM-1 in an eotaxin-1, STAT6-
dependent manner and to play a role in eosinophil accumu-
lation in a mouse model.116
Eosinophils also differ from neutrophils in their reper-
toire of immunoglobulin receptors. Although eosinophils
possess IgG receptors, they are relatively sparse. Rather,
the predominant immunoglobulin receptors on the eosino-
phil surface are high affinity for IgA, consistent with the
eosinophil’s role in barrier defense.117 Although eosinophils
are activated by IgG and IgA, they are most potently acti-
vated by secretory IgA, probably owing to the presence of a
receptor unique for the secretory component.118 In contrast
to earlier teaching, expression of IgE receptors on eosino-
phils surfaces is minimal and most likely of little biologic
significance.119,120
NORMAL EOSINOPHIL FUNCTION
Although many studies have shown the capacity of eosino-
phils to phagocytose bacteria, others have indicated that
these cells are poor at phagocytosis, and it is likely that
229
antibacterial defense is not a primary eosinophil function.
Eosinophils most likely participate in host defense against
multicellular, helminthic parasites. Host eosinophilia in
response to parasitic, but not bacterial infection supports
such an interpretation. Although eosinophils can phago-
cytose small parasitic forms, more typically they attach, in
a polarized manner, to the surface of larger parasites, and
discharge their granular contents into the protected space
between the parasite and the eosinophil. Although O2− gen-
eration and proteinase release may play a role in this attack,
the specific granule-associated, basic proteins are probably
the major weapon in the eosinophil’s antiparasitic arma-
mentarium. In vitro studies have shown the capacities of
these proteins, particularly ECP and MBP, to kill protozoa.
Although ECP is about 10-fold more potent, the higher
concentration of MBP present in the granules has led to the
consensus that it is the dominant parasite toxin. Parasitic
killing by each of the proteins seems to depend on its capac-
ity to disrupt the plasma membrane; in the case of ECP,
membrane disruption occurs through the formation of pores
or channels.1 Eosinophils (similar to neutrophils) have been
reported to present antigen to T cells; it is not established
whether antigen presentation plays any role in the antipara-
sitic effect.121 Although epidemiologic and in vitro evidence
supports a role for eosinophils in parasitic defense, in vivo
confirmation of such a role is equivocal. In particular, sev-
eral studies indicate that eosinophil ablation (through IL-5
depletion) has no effect on the course of parasite infections
in mice.122,123 This might reflect redundancy of antiparasitic
defenses.
EOSINOPHIL RELEVANCE TO RHEUMATIC DISEASE
Asthma
Although perhaps not strictly a rheumatic disease, asthma
represents the most common inflammatory/autoimmune
disease in which eosinophils predominate. Although the
presence of blood and pulmonary eosinophilia in asthma
has been appreciated for a century, the role of eosino-
phils in the pathogenesis of this disease remains a subject
of intense study. Asthmatic eosinophilia is stimulated by
high levels of IL-5 and other cytokines. Eosinophils pos-
sess multiple mechanisms through which they can, at least
potentially, enhance the asthmatic response. Similar to
neutrophils, stimulated eosinophils synthesize LTA4 from
arachidonic acid. In contrast to neutrophils, however,
eosinophil metabolism of LTA4 leads to the production not
of LTB4, but of LTC4 and LTD4 (cysteinyl leukotrienes),
both potent bronchoconstrictors.124 Eosinophils them-
selves are exquisitely sensitive to the effects of cysteinyl
leukotrienes, which stimulate eosinophil adhesion, migra-
tion, and degranulation and the proliferation of eosinophil
progenitors.125-127 The cysteinyl leukotriene receptor antag-
onists (lukasts) are effective in the treatment of asthma
and have been shown to have direct effects on eosinophils
in vivo and in vitro, including reduction of eosinophil
transmigration and reduction of pulmonary and peripheral
eosinophilia.128-130 Lukasts seem to have beneficial effects
on other diseases characterized by eosinophilia, including
cystic fibrosis, eosinophilic gastroenteritis, and atrophic
dermatitis.131-133
 230 Pillinger  |  Neutrophils and Eosinophils
Platelet-activating factor also is produced by stimulated
eosinophils and has bronchoconstricting activities. Release
of specific granule proteins per se have multiple proasth-
matic effects, including (1) epithelial damage secondary to
membrane perturbations similar to those seen in parasites
and (2) activation of mast cells with subsequent histamine
and leukotriene production. MBP also may act specifically
as an antagonist of muscarinic M2 receptors, resulting in
enhanced vagal tone and increased bronchospasm.134
Eosinophils and Rheumatic Diseases
Although hypereosinophilia occasionally can be observed in
virtually all rheumatic diseases, it is relatively uncommon in
most, owing in part to the widespread use of corticosteroid
therapy.135 In Churg-Strauss vasculitis, hypereosinophilia
is the classic laboratory abnormality accompanying a con-
stellation of pulmonary and renal vasculitis and asthma. In
some cases, peripheral eosinophil counts in Churg-Strauss
vasculitis have been reported to exceed 50% of total leu-
kocytes. The cluster of asthma and eosinophilia accompa-
nying Churg-Strauss vasculitis suggests that this syndrome
may represent an atopic response to a foreign antigen. IgE
response varies, however, and asthma may precede the rest
of the disease by years. The presence of antimyeloperoxi-
dase antibody (perinuclear ANCA), an IgG class antibody,
is common, suggesting a broader autoimmune response.
Eosinophilia-myalgia syndrome was first observed in
1989 in New Mexico and was defined by the Centers for
Disease Control and Prevention for surveillance purposes
as peripheral eosinophilia and muscle pains unexplained by
other illnesses. Rash and skin edema are common findings.
Follow-up of cases over time revealed the frequent appear-
ance of fibrosing fasciitis, which in more severe disease
results in skin retraction, particularly over the veins, where
it gives rise to a train-track appearance. Intensive epide-
miologic investigation pinpointed the likely cause of the
epidemic to the consumption of l-tryptophan supplements
produced by a single manufacturer, probably owing to trace
contaminants. Discontinuation of the sale of the supple-
ment led to resolution of the epidemic, although sporadic,
tryptophan-independent cases continue to be reported.136
Eosinophilic fasciitis, a condition first described in 1975,
resembles eosinophilia-myalgia syndrome in that it involves
fasciitis and eosinophilia, but differs in that myalgias are
not a prominent feature, and organ involvement is unusual.
Clinically, the skin of patients with eosinophilic fasciitis
bears some resemblance to the skin of patients with sys-
temic sclerosis, but the distribution is typically on the distal
extremities with sparing of the hands and feet. An epidemic
similar to eosinophilia-myalgia syndrome was seen in Spain
in 1981, relating to the consumption of adulterated rapeseed
oil (toxic oil syndrome). Although in these syndromes it is
unclear whether the eosinophils act as mediators of fasciitis
or merely as reporters of exposure to an atopic antigen, the
capacity of eosinophils to produce TGF-β suggests a poten-
tial role for these cells in the generation of fibrous tissue.
The presence of eosinophils in the affected tissues varies,
however, with most of the infiltrates comprising other leu-
kocytes.137
A single study has shown indirect evidence for the pres-
ence of increased numbers of eosinophils (Charcot-Leyden
crystal deposition) in progressive systemic sclerosis. The
­relevance of this observation remains to be determined.
Primary Eosinophilic Syndromes
Idiopathic hypereosinophilic syndrome has been defined
as (1) persistent eosinophils numbering 1500/mm3 for
6 or more months (or until death); (2) absence of para-
sites, allergy, or other cause of eosinophilia; and (3) signs
and symptoms of organ involvement relating directly to
eosinophils or eosinophil accumulation. Morbidity is largely
from eosinophil tissue infiltration, and granuloma forma-
tion may occur.138 Idiopathic hypereosinophilic syndrome
has gained attention with the description of patients with a
genetic rearrangement, del4 (q12q12), that results in fusion
of the platelet-derived growth factor receptor-α (PDGFRα)
and Fip1-like 1 (FIP1L1) genes, generating a novel, con-
stitutively active tyrosine kinase responsible for the clonal
expansion of eosinophils. Targeted therapy with the selective
tyrosine kinase inhibitor imatinib has become an effective
tool in many cases of hypereosinophilic syndrome associ-
ated with the FIP1L1-PDGFRA fusion gene.139 A placebo-
controlled trial indicated that mepolizumab, an anti-IL-5
monoclonal antibody, also might offer benefit.140 To date,
corticosteroids remain the primary treatment for idiopathic
hypereosinophilic syndrome.
Eosinophilic esophagitis is a more recently recognized
entity defined by the accumulation of eosinophils in the
esophagus, which, in contrast to gastroesophageal reflux
disease, does not respond to therapy with proton-pump
inhibitors.141 Patients with eosinophilic esophagitis are pre-
dominantly young men and have high levels of eosinophils
in the esophageal mucosa, extensive hyperplasia, a high
rate of atopic disease, and normal pH monitoring com-
pared with patients with gastroesophageal reflux disease.
The prevalence seems to be increasing, notably among
whites from Western countries.142 Oral fluticasone propio-
nate and mepolizumab have been proven effective in initial
trials.143,144
Löffler’s syndrome is a self-limiting eosinophilic pneumo-
nitis with peripheral eosinophilia, presumably a hypersen-
sitivity reaction. Allergic bronchopulmonary aspergillosis
also represents a hypersensitivity reaction and may be indis-
tinguishable from Löffler’s syndrome. A novel eosinophilic
syndrome has been described, consisting of nodules, eosin-
ophilia, rheumatism, dermatitis, and swelling (NERDS);
because only a few cases have been reported to date, the
clinical identity of this illness awaits validation.145
Addison’s Disease
Addison’s disease is a disorder of adrenal failure resulting in
underproduction of steroid hormones. Addison’s disease fre-
quently is accompanied by peripheral eosinophilia. In con-
trast to increases in peripheral neutrophil counts, the ability
of glucocorticoids to reverse the eosinophilia of Addison’s
disease implicates that class of agents as a key regulator in
the downregulation of eosinophil number. Glucocoticoids
rapidly reduce eosinophil numbers in most hypereosino-
philic and nonhypereosinophilic patients, a fact enshrined
in the clinical maxim that detectable levels of eosinophils
in a patient on long-term glucocorticoid therapy may be
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
evidence of noncompliance with medication. Whether
glucocorticoids have their effect on eosinophil production,
release, or survival remains to be determined. Regardless of
the mechanism of their effect, the use of glucocoticoids to
reduce eosinophil count is an important strategy in reducing
morbidity from hypereosinophilic diseases.
Acknowledgments
The authors express their gratitude to Dr. Gerald Weissmann for gener-
ously sharing images for figures. The authors also thank Ms. Madeline Rios
for her excellent judgment and tireless editorial support.
REFERENCES
  1. Bainton DF: Developmental biology of neutrophils and eosinophils.
In Gallin JI, Snyderman R, Fearon DT, et al (eds): Inflammation:
Basic Principles and Clinical Correlates 3rd ed. Philadelphia, Lip-
pincott Williams & Wilkins, 1999, pp 13-34.
  2. Murray J, Barbara JA, Dunkley SA, et al: Regulation of neutrophil
apoptosis by tumor necrosis factor-alpha: Requirement for TNFR55
and TNFR75 for induction of apoptosis in vitro. Blood 90:2772-2783,
1997.
  3. Tortorella C, Piazzolla G, Spaccavento F, et al: Spontaneous and Fas-
induced apoptotic cell death in aged neutrophils. J Clin Immunol
18:321-329, 1998.
  4. Borregaard N, Lollike K, Kjeldsen L, et al: Human neutrophil
­granules and secretory vesicles. Eur J Haematol 51:187-198,
1993.
  5. Dewald B, Bretz U, Baggiolini M: Release of gelatinase from a novel
secretory compartment of human neutrophils. J Clin Invest 70:
518-525, 1982.
  6. Borregaard N, Miller LJ, Springer TA: Chemoattractant-regulated
mobilization of a novel intracellular compartment in human neutro-
phils. Science 237:1204-1206, 1987.
  7. Calafat J, Kuijpers TW, Janssen H, et al: Evidence for small intra-
cellular vesicles in human blood phagocytes containing cytochrome
b558 and the adhesion molecule CD11b/CD18. Blood 81:3122-3129,
1993.
  8. Belaaouaj A, Kim KS, Shapiro SD: Degradation of outer membrane
protein A in Escherichia coli killing by neutrophil elastase. Science
289:1185-1188, 2000.
  9. Elsbach P: The bactericidal/permeability-increasing protein (BPI) in
antibacterial host defense. J Leukoc Biol 64:14-18, 1998.
10. Coeshott C, Ohnemus C, Pilyavskaya A, et al: Converting enzyme-
independent release of tumor necrosis factor alpha and IL-1beta from
a stimulated human monocytic cell line in the presence of activated
neutrophils or purified proteinase 3. Proc Natl Acad Sci U S A
96:6261-6266, 1999.
11. Schonbeck U, Mach F, Libby P: Generation of biologically active
IL-1 beta by matrix metalloproteinases: A novel caspase-1-indepen-
dent pathway of IL-1 beta processing. J Immunol 161:3340-3346,
1998.
12. Van den Steen PE, Proost P, Wuyts A, et al: Neutrophil gelatinase
B potentiates interleukin-8 tenfold by aminoterminal processing,
whereas it degrades CTAP-III, PF-4, and GRO-alpha and leaves
RANTES and MCP-2 intact. Blood 96:2673-2681, 2000.
13. Huynh ML, Fadok VA, Henson PM: Phosphatidylserine-dependent
ingestion of apoptotic cells promotes TGF-beta1 secretion and the
resolution of inflammation. J Clin Invest 109:41-50, 2002.
14. Vandivier RW, Fadok VA, Hoffmann PR, et al: Elastase-mediated
phosphatidylserine receptor cleavage impairs apoptotic cell ­clearance
in cystic fibrosis and bronchiectasis. J Clin Invest 109:661-670,
2002.
15. Bokoch GM: Chemoattractant signaling and leukocyte activation.
Blood 86:1649-1660, 1995.
16. Kato T, Kitagawa S: Regulation of neutrophil functions by proinflam-
matory cytokines. Int J Hematol 84:205-209, 2006.
17. Pillinger MH, Feoktistov AS, Capodici C, et al: Mitogen-activated
protein kinase in neutrophils and enucleate neutrophil cytoplasts:
Evidence for regulation of cell-cell adhesion. J Biol Chem 271:
12049-12056, 1996.
231
18. Hall A: Rho GTPases and the control of cell behaviour. Biochem Soc
Trans 33(Pt 5):891-895, 2005.
19. Kawasaki M, Nakayama K, Wakatsuki S: Membrane recruitment of
effector proteins by Arf and Rab GTPases. Curr Opin Struct Biol
15:681-689, 2005.
20. Morgan CP, Sengelov H, Whatmore J, et al: ADP-ribosylation-­factor-
regulated phospholipase D activity localizes to secretory vesicles and
mobilizes to the plasma membrane following N-formylmethionyl-
leucyl-phenylalanine stimulation of human neutrophils. Biochem J
325(Pt 3):581-585, 1997.
21. Raeder EM, Mansfield PJ, Hinkovska-Galcheva V, et al: Sphingosine
blocks human polymorphonuclear leukocyte phagocytosis through
inhibition of mitogen-activated protein kinase activation. Blood
93:686-693, 1999.
22. Smolen JE, Stoehr SJ, Kuczynski B: Cyclic AMP inhibits secretion
from electroporated human neutrophils. J Leukoc Biol 49:172-179,
1991.
23. Amin AR, Attur M, Vyas P, et al: Expression of nitric oxide ­synthase
in human peripheral blood mononuclear cells and neutrophils.
J Inflamm 47:190-205, 1995.
24. Clancy RM, Amin AR, Abramson SB: The role of nitric oxide in
inflammation and immunity. Arthritis Rheum 41:1141-1151, 1998.
25. Nauseef WM, Volpp BD, McCormick S, et al: Assembly of the
­neutrophil respiratory burst oxidase: protein kinase C promotes cyto-
skeletal and membrane association of cytosolic oxidase components.
J Biol Chem 266:5911-5917, 1991.
26. Nick JA, Avdi NJ, Young SK, et al: Selective activation and
­functional significance of p38alpha mitogen-activated protein kinase
in lipopolysaccharide-stimulated neutrophils. J Clin Invest 103:
851-858, 1999.
27. Avdi NJ, Nick JA, Whitlock BB, et al: Tumor necrosis factor-alpha
activation of the c-Jun N-terminal kinase pathway in human neutro-
phils: Integrin involvement in a pathway leading from cytoplasmic
tyrosine kinases apoptosis. J Biol Chem 276:2189-2199, 2001.
28. Capodici C, Pillinger MH, Han G, et al: Integrin-dependent homo-
typic adhesion of neutrophils: arachidonic acid activates Raf-1/Mek/
Erk via a 5-lipoxygenase- dependent pathway. J Clin Invest 102:
165-175, 1998.
29. Capodici C, Hanft S, Feoktistov M, et al: Phosphatidylinositol
3-kinase mediates chemoattractant-stimulated, CD11b/CD18-
­dependent cell-cell adhesion of human neutrophils: Evidence for an
ERK-independent pathway. J Immunol 160:1901-1909, 1998.
30. Kilpatrick LE, Lee JY, Haines KM, et al: A role for PKC-delta
and PI 3-kinase in TNF-alpha-mediated antiapoptotic signaling in
the human neutrophil. Am J Physiol Cell Physiol 283:C48-C57,
2002.
31. Philips MR, Buyon JP, Winchester R, et al: Up-regulation of the
iC3b receptor (CR3) is neither necessary nor sufficient to promote
­neutrophil aggregation. J Clin Invest 82:495-501, 1988.
32. Gautam N, Herwald H, Hedqvist P, et al: Signaling via beta(2)
­integrins triggers neutrophil-dependent alteration in endothelial
­barrier function. J Exp Med 191:1829-1839, 2000.
33. Ding ZM, Babensee JE, Simon SI, et al: Relative contribution of
LFA-1 and Mac-1 to neutrophil adhesion and migration. J Immunol
163:5029-5038, 1999.
34. Mayadas TN, Cullere X: Neutrophil beta2 integrins: moderators of
life or death decisions. Trends Immunol 26:388-395, 2005.
35. Feng D, Nagy JA, Pyne K, et al: Neutrophils emigrate from venules
by a transendothelial cell pathway in response to FMLP. J Exp Med
187:903-915, 1998.
36. Dangerfield J, Larbi KY, Huang MT, et al: PECAM-1 (CD31)
homophilic interaction up-regulates alpha6beta1 on transmigrated
­neutrophils in vivo and plays a functional role in the ability of alpha6
integrins to mediate leukocyte migration through the perivascular
basement membrane. J Exp Med 196:1201-1211, 2002.
37. Liu Y, Merlin D, Burst SL, et al: The role of CD47 in neutrophil
transmigration: increased rate of migration correlates with increased
cell surface expression of CD47. J Biol Chem 276:40156-40166,
2001.
38. Lou O, Alcaide P, Luscinskas FW, et al: CD99 is a key mediator
of the transendothelial migration of neutrophils. J Immunol 178:
1136-1143, 2007.
39. Huber AR, Weiss SJ: Disruption of the subendothelial basement
membrane during neutrophil diapedesis in an in vitro construct of a
blood vessel wall. J Clin Invest 83:1122-1136, 1989.
 232 Pillinger  |  Neutrophils and Eosinophils
40. Hallett MB: Controlling the molecular motor of neutrophil chemo-
taxis. Bioessays 19:615-621, 1997.
40a. Caron E, Hall A: Identification of two distinct mechanisms of phago-
cytosis controlled by different RhoGTPases, Science 282(5394):1717-
1721, 1998.
41. Weiss SJ: Tissue destruction by neutrophils. N Engl J Med
320:365-376, 1989.
42. DeLeo FR, Quinn MT: Assembly of the phagocyte NADPH oxidase:
Molecular interaction of oxidase proteins. J Leukoc Biol 60:677-691,
1996.
43. Nauseef WM: Cytosolic oxidase factors in the NADPH-dependent
oxidase of human neutrophils. Eur J Haematol 51:301-308, 1993.
44. Clark RA, Volpp BD, Leidal KG, et al: Two cytosolic components
of the human neutrophil respiratory burst oxidase translocate to the
plasma membrane during cell activation. J Clin Invest 85:714-721,
1990.
45. Philips MR, Feoktistov A, Pillinger MH, et al: Translocation of
p21rac2 from cytosol to plasma membrane is neither necessary nor
sufficient for neutrophil NADPH oxidase activity. J Biol Chem
270:11514-11521, 1995.
46. Matute JD, Arias AA, Dinauer MC, et al: p40phox: The last NADPH
oxidase subunit. Blood Cells Mol Dis 35:291-302, 2005.
47. Reeves EP, Lu H, Jacobs HL, et al: Killing activity of neutrophils
is mediated through activation of proteases by K+ flux. Nature 416:
291-297, 2002.
48. Samuelsson B, Dahlen SE, Lindgren JA, et al: Leukotrienes and
lipoxins: Structures, biosynthesis, and biological effects. Science
237:1171-1176, 1987.
49. Abramson SB, Leszczynska-Piziak J, Weissmann G: Arachidonic acid
as a second messenger: Interactions with a GTP-binding protein of
human neutrophils. J Immunol 147:231-236, 1991.
50. O’Flaherty JT, Rossi AG: 5-hydroxyicosatetraenoate stimulates
neutrophils by a stereospecific, G protein-linked mechanism. J Biol
Chem 268:14708-14714, 1993.
51. Chiang N, Arita M, Serhan CN: Anti-inflammatory circuitry:
Lipoxin, aspirin-triggered lipoxins and their receptor ALX. Prosta-
glandins Leukot Essent Fatty Acids 73:163-177, 2005.
52. Serhan CN: Lipoxins and aspirin-triggered 15-epi-lipoxins are the
first lipid mediators of endogenous anti-inflammation and resolution.
Prostaglandins Leukot Essent Fatty Acids 73:141-162, 2005.
53. Papayianni A, Serhan CN, Phillips ML, et al: Transcellular biosyn-
thesis of lipoxin A4 during adhesion of platelets and neutrophils
in experimental immune complex glomerulonephritis. Kidney Int
47:1295-1302, 1995.
54. Hamberg M, Svensson J, Samuelsson B: Prostaglandin endoperox-
ides: A new concept concerning the mode of action and release of
prostaglandins. Proc Natl Acad Sci U S A 71:3824-3828, 1974.
55. Pillinger MH, Philips MR, Feoktistov A, et al: Crosstalk in
­signal transduction via EP receptors: Prostaglandin E1 inhibits
­chemoattractant-induced mitogen-activated protein kinase activity in
human neutrophils. Adv Prostaglandin Thromboxane Leukot Res 23:
311-316, 1995.
56. Maloney CG, Kutchera WA, Albertine KH, et al: Inflammatory
­agonists induce cyclooxygenase type 2 expression by human neutro-
phils. J Immunol 160:1402-1410, 1998.
57. Lord PC, Wilmoth LM, Mizel SB, et al: Expression of interleukin-1
alpha and beta genes by human blood polymorphonuclear leukocytes.
J Clin Invest 87:1312-1321, 1991.
58. Scapini P, Lapinet-Vera JA, Gasperini S, et al: The neutrophil as a
cellular source of chemokines. Immunol Rev 177:195-203, 2000.
59. Reibman J, Meixler S, Lee TC, et al: Transforming growth factor
beta 1, a potent chemoattractant for human neutrophils, bypasses
classic signal-transduction pathways. Proc Natl Acad Sci U S A 88:
6805-6809, 1991.
60. Fava RA, Olsen NJ, Postlethwaite AE, et al: Transforming growth
factor beta 1 (TGF-beta 1) induced neutrophil recruitment to syno-
vial tissues: Implications for TGF-beta-driven synovial inflammation
and hyperplasia. J Exp Med 173:1121-1132, 1991.
61. McColl SR, Paquin R, Menard C, et al: Human neutrophils produce
high levels of the interleukin 1 receptor antagonist in response to
granulocyte/macrophage colony-stimulating factor and tumor necro-
sis factor alpha. J Exp Med 176:593-598, 1992.
62. Malyak M, Swaney RE, Arend WP: Levels of synovial fluid interleu-
kin-1 receptor antagonist in rheumatoid arthritis and other arthropa-
thies: Potential contribution from synovial fluid neutrophils. Arthritis
Rheum 36:781-789, 1993.
63. Fitzgerald AA, Leclercq SA, Yan A, et al: Rapid responses to anakinra
in patients with refractory adult-onset Still’s disease. Arthritis Rheum
52:1794-1803, 2005.
64. Ariel A, Fredman G, Sun YP, et al: Apoptotic neutrophils and
T cells sequester chemokines during immune response resolution
through modulation of CCR5 expression. Nat Immunol 7:1209-
1216, 2006.
65. Rosenzweig SD, Holland SM: Phagocyte immunodeficiencies and
their infections. J Allergy Clin Immunol 113:620-626, 2004.
66. Skokowa J, Germeshausen M, Zeidler C, et al: Severe congenital
neutropenia: Inheritance and pathophysiology. Curr Opin Hematol
14:22-28, 2007.
67. Dale DC, Cottle TE, Fier CJ, et al: Severe chronic neutropenia:
Treatment and follow-up of patients in the Severe Chronic Neutro-
penia International Registry. Am J Hematol 72:82-93, 2003.
68. Horwitz MS, Duan Z, Korkmaz B, et al: Neutrophil elastase in cyclic
and severe congenital neutropenia. Blood 109:1817-1824, 2007.
69. Anderson DC, Springer TA: Leukocyte adhesion deficiency: An
inherited defect in the Mac-1, LFA-1, and p150,95 glycoproteins.
Annu Rev Med 38:175-194, 1987.
70. Holland SM, Gallin JI: Evaluation of the patient with recurrent
­bacterial infections. Annu Rev Med 49:185-199, 1998.
71. Etzioni A, Frydman M, Pollack S, et al: Brief report: Recurrent severe
infections caused by a novel leukocyte adhesion deficiency. N Engl
J Med 327:1789-1792, 1992.
72. Etzioni A, Alon R: Leukocyte adhesion deficiency III: A group of
integrin activation defects in hematopoietic lineage cells. Curr Opin
Allergy Clin Immunol 4:485-490, 2004.
73. Barbosa MD, Nguyen QA, Tchernev VT, et al: Identification of the
homologous beige and Chediak-Higashi syndrome genes. Nature
382:262-265, 1996.
74. Bohn G, Allroth A, Brandes G, et al: A novel human primary immu-
nodeficiency syndrome caused by deficiency of the endosomal ­adaptor
protein p14. Nat Med 13:38-45, 2007.
75. Lekstrom-Himes JA, Gallin JI: Immunodeficiency diseases caused by
defects in phagocytes. N Engl J Med 343:1703-1714, 2000.
76. Brinkmann V, Reichard U, Goosmann C, et al: Neutrophil extra-
cellular traps kill bacteria. Science 303:1532-1535, 2004.
77. Fuchs TA, Abed U, Goosmann C, et al: Novel cell death program
leads to neutrophil extracellular traps. J Cell Biol 176:231-241,
2007.
78. Duits AJ, Bootsma H, Derksen RH, et al: Skewed distribution of
IgG Fc receptor IIa (CD32) polymorphism is associated with renal
­disease in systemic lupus erythematosus patients. Arthritis Rheum 38:
1832-1836, 1995.
79. van de Velde NC, Mottram PL, Hogarth PM: FcgammaRII and ­
multi-system autoimmune disease. Springer Semin Immunopathol 28:
329-338, 2006.
80. Salmon JE, Millard S, Schachter LA, et al: Fc gamma RIIA alleles are
heritable risk factors for lupus nephritis in African Americans. J Clin
Invest 97:1348-1354, 1996.
81. Tse WY, Abadeh S, Jefferis R, et al: Neutrophil FcgammaRIIIb allelic
polymorphism in anti-neutrophil cytoplasmic antibody (ANCA)-
positive systemic vasculitis. Clin Exp Immunol 119:574-577,
2000.
82. Tse WY, Abadeh S, McTiernan A, et al: No association between
neutrophil FcgammaRIIa allelic polymorphism and anti-neutrophil
cytoplasmic antibody (ANCA)-positive systemic vasculitis. Clin Exp
Immunol 117:198-205, 1999.
83. Pawlik A, Ostanek L, Brzosko I, et al: FC gamma RIIa polymor-
phism in patients with rheumatoid arthritis. Clin Exp Rheumatol 20:
841-844, 2002.
84. Hepburn AL, Mason JC, Davies KA: Expression of Fcgamma and
complement receptors on peripheral blood monocytes in systemic
lupus erythematosus and rheumatoid arthritis. Rheumatology
(Oxford) 43:547-554, 2004.
85. Mandel NS: The structural basis of crystal-induced membranolysis.
Arthritis Rheum 19(Suppl 3):439-445, 1976.
86. Cross A, Bakstad D, Allen JC, et al: Neutrophil gene expression in
rheumatoid arthritis. Pathophysiology 12:191-202, 2005.
87. Lefkowitz DL, Gelderman MP, Fuhrmann SR, et al: Neutrophilic
myeloperoxidase-macrophage interactions perpetuate chronic
inflammation associated with experimental arthritis. Clin Immunol
91:145-155, 1999.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
88. Weissmann G, Spilberg I, Krakauer K: Arthritis induced in rabbits
by lysates of granulocyte lysosomes. Arthritis Rheum 12:103-116,
1969.
89. Befus AD, Mowat C, Gilchrist M, et al: Neutrophil defensins induce
histamine secretion from mast cells: mechanisms of action. J ­Immunol
163:947-953, 1999.
90. McCurdy L, Chatham WW, Blackburn WD Jr: Rheumatoid synovial
fibroblast adhesion to human articular cartilage: enhancement by
neutrophil proteases. Arthritis Rheum 38:1694-1700, 1995.
91. Kasama T, Kobayashi K, Yajima N, et al: Expression of vascular endo-
thelial growth factor by synovial fluid neutrophils in rheumatoid
arthritis (RA). Clin Exp Immunol 121:533-538, 2000.
92. Cross A, Bucknall RC, Cassatella MA, et al: Synovial fluid neutro-
phils transcribe and express class II major histocompatibility complex
molecules in rheumatoid arthritis. Arthritis Rheum 48:2796-2806,
2003.
93. Chen M, Lam BK, Kanaoka Y, et al: Neutrophil-derived leukotriene
B4 is required for inflammatory arthritis. J Exp Med 203:837-842,
2006.
94. Wipke BT, Allen PM: Essential role of neutrophils in the initiation
and progression of a murine model of rheumatoid arthritis. J Immunol
167:1601-1608, 2001.
95. Abramson SB, Dobro J, Eberle MA, et al: Acute reversible hypox-
emia in systemic lupus erythematosus. Ann Intern Med 114:941-947,
1991.
96. Goronzy JJ, Weyand CM: Cytokines in giant-cell arteritis. Cleve Clin
J Med 69(Suppl 2):SII-91-SII-94, 2002.
97. Belmont HM, Buyon J, Giorno R, et al: Up-regulation of endothe-
lial cell adhesion molecules characterizes disease activity in systemic
lupus erythematosus: The Shwartzman phenomenon revisited.
Arthritis Rheum 37:376-383, 1994.
98. Centola M, Wood G, Frucht DM, et al: The gene for familial Medi-
terranean fever, MEFV, is expressed in early leukocyte development
and is regulated in response to inflammatory mediators. Blood 95:
3223-3231, 2000.
99. Pillinger MH, Abramson SB: The neutrophil in rheumatoid arthritis.
Rheum Dis Clin N Am 21:691-714, 1995.
100. Cronstein BN, Van de Stouwe M, Druska L, et al: Nonsteroidal
antiinflammatory agents inhibit stimulated neutrophil adhesion to
endothelium: Adenosine dependent and independent mechanisms.
Inflammation 18:323-335, 1994.
101. Neal TM, Vissers MC, Winterbourn CC: Inhibition by nonsteroi-
dal anti-inflammatory drugs of superoxide production and granule
enzyme release by polymorphonuclear leukocytes stimulated with
immune complexes or formyl-methionyl-leucyl-phenylalanine.
Biochem Pharmacol 36:2511-2517, 1987.
102. Pillinger MH, Capodici C, Rosenthal P, et al: Modes of action of
aspirin-like drugs: Salicylates inhibit erk activation and integrin-
dependent neutrophil adhesion. Proc Natl Acad Sci U S A 95:
14540-14545, 1998.
103. Cronstein BN, Eberle MA, Gruber HE, et al: Methotrexate inhibits
neutrophil function by stimulating adenosine release from connec-
tive tissue cells. Proc Natl Acad Sci U S A 88:2441-2445, 1991.
104. Sperling RI, Benincaso AI, Anderson RJ, et al: Acute and chronic
suppression of leukotriene B4 synthesis ex vivo in neutrophils from
patients with rheumatoid arthritis beginning treatment with metho-
trexate. Arthritis Rheum 35:376-384, 1992.
105. Cronstein BN, Molad Y, Reibman J, et al: Colchicine alters the quan-
titative and qualitative display of selectins on endothelial cells and
neutrophils. J Clin Invest 96:994-1002, 1995.
106. Pillinger NH, Abramson SB: The neutrophil in rheumatoid arthritis.
Rheum Dis Clin North Am 21:691-714, 1995.
107. Moreland LW, Bucy RP, Weinblatt ME, et al: Immune function
in patients with rheumatoid arthritis treated with etanercept. Clin
Immunol 103:13-21, 2002.
108. Capsoni F, Sarzi-Puttini P, Atzeni F, et al: Effect of adalimumab on
neutrophil function in patients with rheumatoid arthritis. Arthritis
Res Ther 7:R250-R255, 2005.
109. Koizumi K, Poulaki V, Doehmen S, et al: Contribution of TNF-alpha
to leukocyte adhesion, vascular leakage, and apoptotic cell death
in endotoxin-induced uveitis in vivo. Invest Ophthalmol Vis Sci
44:2184-2191, 2003.
110. Rosenberg HF: Eosinophils. In Gallin JI, Snyderman R (eds): Inflam-
mation: Basic Principles and Clinical Correlates, 3rd ed. Philadel-
phia, Lippincott Williams & Wilkins, 1999, pp 61-76.
233
111. Mattes J, Foster PS: Regulation of eosinophil migration and Th2 cell
function by IL-5 and eotaxin. Curr Drug Targets Inflamm Allergy
2:169-174, 2003.
112. Ponath PD, Qin S, Post TW, et al: Molecular cloning and character-
ization of a human eotaxin receptor expressed selectively on eosino-
phils. J Exp Med 183:2437-2448, 1996.
113. Weller PF, Bach DS, Austen KF: Biochemical characterization of
human eosinophil Charcot-Leyden crystal protein (lysophospholi-
pase). J Biol Chem 259:15100-15105, 1984.
114. Abu-Ghazaleh RI, Dunnette SL, Loegering DA, et al: Eosinophil
granule proteins in peripheral blood granulocytes. J Leukoc Biol
52:611-618, 1992.
115. Rosenwasser LJ, Zimmermann N, Hershey GK, et al: Molecular
mechanisms in allergy and clinical immunology. J Allergy Clin
Immunol 111:227-242, 2003.
116. Fritz DK, Kerr C, Tong L, et al: Oncostatin-M up-regulates VCAM-1
and synergizes with IL-4 in eotaxin expression: involvement of STAT6.
J Immunol 176:4352-4360, 2006.
117. Nourshargh S: Mechanisms of neutrophil and eosinophil accumula-
tion in vivo. Am Rev Respir Dis 148(6 Pt 2):S60-S64, 1993.
118. Wines BD, Hogarth PM: IgA receptors in health and disease. Tissue
Antigens 68:103-114, 2006.
119. Gould HJ, Sutton BJ, Beavil AJ, et al: The biology of IGE and the
basis of allergic disease. Annu Rev Immunol 21:579-628, 2003.
120. Seminario MC, Saini SS, MacGlashan DW Jr, et al: Intracellular
expression and release of Fc epsilon RI alpha by human eosinophils.
J Immunol 162:6893-6900, 1999.
121. Weller PF, Rand TH, Barrett T, et al: Accessory cell function of
human eosinophils: HLA-DR-dependent, MHC-restricted antigen-
presentation and IL-1 alpha expression. J Immunol 150:2554-2562,
1993.
122. Klion AD, Nutman TB: The role of eosinophils in host defense
against helminth parasites. J Allergy Clin Immunol 113:30-37,
2004.
123. Grimaldi JC, Yu NX, Grunig G, et al: Depletion of eosinophils in
mice through the use of antibodies specific for C-C chemokine recep-
tor 3 (CCR3). J Leukoc Biol 65:846-853, 1999.
124. Bandeira-Melo C, Weller PF: Eosinophils and cysteinyl leukotrienes.
Prostaglandins Leukot Essent Fatty Acids 69:135-143, 2003.
125. Nagata M, Saito K, Tsuchiya K, et al: Leukotriene D4 upregulates
eosinophil adhesion via the cysteinyl leukotriene 1 receptor. J Allergy
Clin Immunol 109:676-680, 2002.
126. Ohshima N, Nagase H, Koshino T, et al: A functional study on
CysLT(1) receptors in human eosinophils. Int Arch Allergy Immunol
129:67-75, 2002.
127. Braccioni F, Dorman SC, O’Byrne PM, et al: The effect of cysteinyl
leukotrienes on growth of eosinophil progenitors from peripheral
blood and bone marrow of atopic subjects. J Allergy Clin Immunol
110:96-101, 2002.
128. Virchow JC Jr, Faehndrich S, Nassenstein C, et al: Effect of a specific
cysteinyl leukotriene-receptor 1-antagonist (montelukast) on the
transmigration of eosinophils across human umbilical vein endothe-
lial cells. Clin Exp Allergy 31:836-844, 2001.
129. Finsnes F, Lyberg T, Christensen G, et al: Leukotriene antagonism
reduces the generation of endothelin-1 and interferon-gamma and
inhibits eosinophilic airway inflammation. Respir Med 96:901-906,
2002.
130. Minoguchi K, Kohno Y, Minoguchi H, et al: Reduction of eosino-
philic inflammation in the airways of patients with asthma using
montelukast. Chest 121:732-738, 2002.
131. Daikh BE, Ryan CK, Schwartz RH: Montelukast reduces periph-
eral blood eosinophilia but not tissue eosinophilia or symptoms in
a patient with eosinophilic gastroenteritis and esophageal stricture.
Ann Allergy Asthma Immunol 90:23-27, 2003.
132. Capella GL, Grigerio E, Altomare G: A randomized trial of leuko­
triene receptor antagonist montelukast in moderate-to-severe atopic
dermatitis of adults. Eur J Dermatol 11:209-213, 2001.
133. Schmitt-Grohe S, Eickmeier O, Naujoks C, et al: Effects of ­ long-
term treatment with montelukast in mild cystic fibrosis (long term
treatment with montelukast in cystic fibrosis). Respir Med 101:684,
2007.
134. Jacoby DB, Gleich GJ, Fryer AD: Human eosinophil major basic
­protein is an endogenous allosteric antagonist at the inhibitory
­muscarinic M2 receptor. J Clin Invest 91:1314-1318, 1993.
 234 Pillinger  |  Neutrophils and Eosinophils
135. Kargili A, Bavbek N, Kaya A, et al: Eosinophilia in rheumatologic
diseases: A prospective study of 1000 cases. Rheumatol Int 24:
321-324, 2004.
136. Margolin L: Non-L-tryptophan related eosinophilia-myalgia syn-
drome with hypoproteinemia and hypoalbuminemia. J Rheumatol
30:628-629, 2003.
137. Clauw DJ, Crofford LJ: Eosinophilic rheumatic disorders. Rheum Dis
Clin N Am 21:231-246, 1995.
138. Fauci AS, Harley JB, Roberts WC, et al: NIH conference: The
­idiopathic hypereosinophilic syndrome: Clinical, pathophysiologic,
and therapeutic considerations. Ann Intern Med 97:78-92, 1982.
139. Cools J, DeAngelo DJ, Gotlib J, et al: A tyrosine kinase created by
fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of
imatinib in idiopathic hypereosinophilic syndrome. N Engl J Med
348:1201-1214, 2003.
140. Garrett JK, Jameson SC, Thomson B, et al: Anti-interleukin-5
(mepolizumab) therapy for hypereosinophilic syndromes. J Allergy
Clin Immunol 113:115-119, 2004.
141. Furuta GT: Emerging questions regarding eosinophil’s role in
the esophago-gastrointestinal tract. Curr Opin Gastroenterol 22:
658-663, 2006.
142. Blanchard C, Wang N, Rothenberg ME: Eosinophilic esophagi-
tis: Pathogenesis, genetics, and therapy. J Allergy Clin Immunol
118:1054-1059, 2006.
143. Konikoff MR, Noel RJ, Blanchard C, et al: A randomized, double-
blind, placebo-controlled trial of fluticasone propionate for pedi-
atric eosinophilic esophagitis. Gastroenterology 131:1381-1391,
2006.
144. Stein ML, Collins MH, Villanueva JM, et al: Anti-IL-5 (mepoli-
zumab) therapy for eosinophilic esophagitis. J Allergy Clin Immunol
118:1312-1319, 2006.
145. Butterfield JH, Leiferman KM, Gleich GJ: Nodules, eosinophilia,
rheumatism, dermatitis and swelling (NERDS): A novel eosinophilic
disorder. Clin Exp Allergy 23:571-580, 1993.
 13
KEY POINTS
Mast cells (MCs) arise in the bone marrow, circulate as im-
mature precursors, and develop into functional MCs after
entering peripheral tissues.
The phenotype of MCs is diverse, plastic, and governed by
signals from the local microenvironment.
In healthy tissues, MCs serve as immunologic sentinels and
participate in the defense against bacteria and parasites.
MCs accumulate in injured and inflamed tissue, where they
contribute to the inflammatory response and the remodeling
of surrounding tissues.
MCs have been implicated in autoimmune diseases, including
inflammatory arthritis.
Although the mast cell (MC) is best known for its role in
allergy and anaphylaxis, the immune function of this bone
marrow–derived lineage far outstrips its participation in
IgE-driven disease. MCs reside broadly in vascularized tis-
sues, but cluster near interfaces with the external world, in
the linings of vulnerable body cavities, and near blood ves-
sels and nerves. In these locations, MCs serve as immune
sentinels, equipped with an array of pathogen receptors and
an armamentarium of mediators capable of rapidly recruit-
ing immune effector cells. MCs also accumulate in sites
of tissue injury and chronic inflammation, although their
role in such locations is uncertain. Other functions for this
lineage, conserved by evolution for more than 500 million
years, continue to be defined.
Circumstantial and experimental evidence implicates
MCs in the pathogenesis of rheumatic diseases. MCs reside
constitutively in the normal synovium and are found in
large numbers in inflamed synovial tissue, whereas MC
mediators are identified in inflammatory joint fluid. Mod-
els have indicated that MCs may contribute importantly to
the pathogenesis of experimental arthritis. MCs also have
been implicated in other autoimmune conditions, including
multiple sclerosis, bullous pemphigoid, and systemic sclero-
sis. This chapter reviews the basic biology of MCs and their
potential role in human inflammatory diseases.
BASIC BIOLOGY OF MAST CELLS
DEVELOPMENT AND TISSUE DISTRIBUTION
MCs have a distinctive appearance. Ranging in size from 10
to 60 μM and with a centrally located round or oval nucleus,
their abundant cytoplasm is filled with multiple small gran-
ules. They were named Mastzellen in 1878 by the German
pathologist Ehrlich, who believed incorrectly that they were
Mast Cells
Peter A. Nigrovic  •  David M. Lee
overfed connective tissue cells (mästen, German, “to feed or
fatten an animal”).1 Electron microscopy reveals that the
plasma membrane of the MC exhibits multiple thin cyto-
plasmic extensions, providing a broad interface with the sur-
rounding tissue (Fig. 13-1). The tissue distribution of MCs is
extensive; within tissue, MCs tend to cluster around blood
vessels and nerves and near epithelial and mucosal surfaces.
They also are found in the lining of vulnerable body cavities,
such as the peritoneum and the diarthrodial joint. Given
this localization, MCs are among the first immune cells to
encounter pathogens invading into tissue from the external
world or via the bloodstream, consistent with their role as
immune sentinel cells.2
MCs are of hematopoietic origin, arising in the bone
marrow and depositing in tissues after migrating through
the bloodstream (Fig. 13-2).3,4 In contrast to most other
myeloid cells, such as monocytes and neutrophils, MCs do
not terminally differentiate in the bone marrow, but rather
circulate as committed progenitors, bearing the surface sig-
nature CD34+/c-kit+/CD13+.5 Further developmental details
have been worked out most extensively in mice. On enter-
ing the tissues, murine MCs may mature into classic granu-
lated cells or remain as ungranulated progenitors, awaiting
local signals to mature fully. Tissue homing and migration
is under the control of integrin adhesion molecules. Com-
parison of murine lung and intestine has shown that these
tissues use distinct pathways to regulate the constitutive and
inducible recruitment of MC progenitors, illustrating that
MC homing is a precisely controlled process.6
When MCs reside in tissues, they may live for many
months.7 In contrast to other myeloid lineage cells, such as
macrophages and neutrophils, mature MCs remain capable
of mitosis, although recruitment of circulating progenitors
seems to exceed greatly local replication as a pathway to
expand the number of MCs in a tissue.8 Mechanisms of
reducing MC numbers include apoptosis, shown in tissue
MCs deprived of the cytokine stem cell factor (SCF), a cru-
cial survival signal for MCs.9,10 Under certain conditions,
MCs also may emigrate via the lymphatics, appearing in
draining lymph nodes similar to dendritic cells.11
Mast Cell Heterogeneity: Common Progenitor,
Multiple Subsets, Phenotypic Plasticity
Although all types of MCs derive from a common progeni-
tor lineage, the phenotype of fully differentiated tissue MCs
is heterogeneous. Human MCs are conventionally divided
into two broad classes on the basis of the protease contents
of their granules (see Fig. 13-2).12 MCTC display rounded
granules containing the enzymes tryptase and chymase,
whereas the smaller and more irregularly shaped granules of
235
 236 NIGROVIC  |  MAST CELLS

A B
Figure 13-1  Mast cell morphology. A, Intact mast cell. B, Mast cell that has undergone anaphylactic degranulation; note how fusion of intracellular
granules has resulted in formation of a labyrinth of interconnected channels by which granule contents may be expelled from the cell. Arrows indicate
remaining granules. N, nucleus. (Courtesy of Dr. A. Dvorak, Beth Israel Deaconess Medical Center, Boston. From Dvorak AM, Schleimer RP, Lichtenstein LM:
Morphologic mast cell cycles. Cell Immunol 105:199, 1987; and Galli SJ, Dvorak AM, Dvorak HF: Basophils and mast cells: Morphologic insights into their biol-
ogy, secretory patterns, and function. In Ishizaka K [ed]: Progress in Allergy: Mast Cell Activation and Mediator Release, Vol 34. Basel, S. Karger, 1984, p 1.)

MCT contain tryptase, but not chymase.13 MCTC also express
other proteases, including carboxypeptidase and cathepsin
G. MCC, bearing only chymase, have been reported, but are
controversial. These subtypes differ in tissue distribution.
MCTC tend to be found in connective tissue, such as in
normal skin, muscle, intestinal submucosa, and synovium,
whereas MCT predominate in mucosal sites, including the
lining of the gut and respiratory tract; both are present in
many locations.14,15 Beyond protease signature, other differ-
ences between these subsets include their profile of cytokine
elaboration and cell surface receptor expression; however,
tissue-specific phenotypic differences are noted within each
type.
The relationship between MCTC and MCT is contro-
versial. Are they committed subsets, akin to CD4 and CD8
lymphocytes, or functional states that MCs assume under
the influence of the microenvironment? In mice, where
an analogous distinction exists between connective tissue
MCs and mucosal MCs, evidence for phenotypic plasticity
is strong. In culture and in vivo, single connective tissue
MCs may differentiate into (or give rise to) mucosal MCs,
and vice versa.16,17 MCs with intermediate protease expres-
sion are found, and serial observations suggest that expo-
sure to an inflammatory stimulus can induce progressive
change from one class to another, although it has not been
definitively established whether this occurs at a single-cell
level.18 Similarly, in murine and human mastocytosis, clon-
ally expanded MCs display divergent phenotypes depending
on tissue of residence.19,20 In aggregate, these data favor the
hypothesis that MCs assume a particular phenotype under
the control of the local environment, but can change radi-
cally if these conditions change.
Stem Cell Factor
One of the most important signals from tissue to local MCs
is SCF.9 The receptor for SCF, c-kit, is expressed widely on
hematopoietic lineages early in differentiation, but among
mature lineages only MCs express c-kit at a high level. Stim-
ulation of MCs by SCF promotes maturation and phenotypic
differentiation, blocks apoptosis, and induces chemotaxis. It
also may activate MCs directly to release mediators. In mice
and humans, SCF is an irreplaceable survival signal for tis-
sue MCs. Mice with defects in SCF or c-kit are strikingly
deficient in mature tissue MCs (e.g., W/Wv, Sl/Sld, and Wsash
strains). Similarly, clonal MCs obtained from patients with
systemic mastocytosis commonly exhibit activating muta-
tions in c-kit.21
SCF occurs in two alternate forms resulting from differ-
ential mRNA splicing: soluble and membrane-bound.9 The
importance of this latter form is clear from Sl/Sld mice, which
lack only the membrane-bound isoform, yet exhibit very few
tissue MCs.22 SCF is synthesized by multiple lineages, includ-
ing MCs themselves. Expression by fibroblasts is likely espe-
cially important, given the intimate physical contacts observed
between fibroblasts and MCs in situ. Rodent MCs cocultured
with fibroblasts show enhanced survival, connective tissue
phenotypic differentiation, and heightened capacity to elabo-
rate proinflammatory eicosanoids, effects mediated at least
partly by direct contact, including interactions between SCF
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
Bone
marrow
Circulation
Mast cell Tissue
progenitor
T cell
SCF
tissue
environment
MCTC
MCT
Phenotypic
plasticity
Figure 13-2  Mast cell origin and differentiation. Mast cells arise in the
bone marrow, circulate as committed progenitors, and differentiate into
mature mast cells on entering tissue. Human mast cells may be classified
on the basis of granule proteases into tryptase-positive mast cells (MCT)
and tryptase-positive/chymase-positive mast cells (MCTC), with charac-
teristic tissue localization and mediator production. SCF, stem cell factor.
(Adapted from Gurish MF, Austen KF: The diverse roles of mast cells. J Exp
Med 194:F1, 2001. Graphic design by Steven Moskowitz.)
and c-kit.23,24 The extent of similar regulation in human MCs
is uncertain.25 Expression of SCF also has been documented
on other lineages, including macrophages, vascular endothe-
lium, and airway epithelium, and is likely a crucial pathway by
which tissues modulate the local MC population.
T Lymphocytes
T lymphocytes exert a profound effect on the phenotype
of a major subset of tissue MCs. SCID mice lacking T cells
fail to develop mucosal MCs, a defect that may be corrected
by T cell engraftment.26 An analogous observation has
been made in human patients deficient in T cells second-
ary to congenital immunodeficiency or acquired immuno-
deficiency syndrome. Intestinal biopsy specimens in these
patients show that mucosal MCs (MCT) are strikingly
reduced, whereas connective tissue MCs (MCTC) are pres-
ent in normal numbers.27 The pathways by which T cells
exert this striking effect are undefined, although clearly T
cell cytokines, such as interleukin (IL)-3, IL-4, IL-6, and
IL-9, may have profound effects on MCs matured in cul-
ture, skewing toward either MCT or MCTC phenotype.28,29
By contrast, interferon-γ inhibits MC proliferation and
may induce apoptosis. These observations imply that cells
recruited to an inflamed tissue may have a profound impact
on the phenotype and survival of local MCs. The rheuma-
toid synovium may exemplify this phenomenon: normally
populated by MCTC, large numbers of MCT are identified
in the inflamed synovium, typically in the regions rich in
infiltrating leukocytes, whereas MCTC reside in the deeper,
more fibrotic areas of the joint.30
237
Different Functions for Mucosal Mast Cells
and Connective Tissue Mast Cells
The preservation of distinct types of MCs in multiple
species implies distinct and nonoverlapping roles for
these subtypes. The understanding of functional differ-
ences between MCT and MCTC is limited, however. One
hypothesis is that MCT play a proinflammatory role,
whereas MCTC specialize in matrix remodeling.31 This
hypothesis makes sense of the promotion of MCT devel-
opment by T cells patrolling the tissues, of the partition of
MCT and MCTC to inflamed (MCT) and fibrotic (MCTC)
areas, and of the preferential expression of the proinflam-
matory mediators IL-5 and IL-6 by MCT and the profi-
brotic mediator IL-4 by MCTC.32 Not all observations fit
comfortably into this dichotomy. The potently proinflam-
matory anaphylatoxin receptor C5aR (CD88) is expressed
on MCTC, but not MCT.33 Ultimately, too little is known
about the actual functional importance of these subsets to
permit firm conclusions.
MAST CELL ACTIVATION
IgE
The canonical pathway to MC activation is via IgE and its
receptor FcεRI. With an association constant Ka of 1010/M,
this receptor is essentially constantly saturated with IgE at
typical serum concentrations.34 Cross-linking FcεRI-bound
IgE by multivalent antigen induces a brisk and vigorous
response. Within minutes, granules within the MC fuse
together and with the surface membrane, creating a set
of labyrinthine channels that allow rapid release of gran-
ule contents (see Fig. 13-1B).35 This compound exocytosis
event, termed anaphylactic degranulation, is followed within
minutes by the elaboration of eicosanoids newly synthesized
from arachidonic acid cleaved from internal membrane lip-
ids. Finally, signals transduced via FcεRI induce the tran-
scription of new genes and elaboration of a wide range of
chemokines and cytokines (Fig. 13-3). On termination of
the stimulation event, the surface membrane closes over
the granule-formed channels, and these subsequently bud
off within the cytoplasm, recreating discrete granules using
the original membranes.35 These granules become recharged
with mediators in a process that occurs gradually over days
to weeks.36
IgG and Immune Complexes
IgE is only one among many pathways of MC activation.
One key trigger for MC activation in humans and mice is
IgG, acting via receptors for the Fc portion of IgG (FcγR).
The importance of this pathway was shown first in mice ren-
dered genetically deficient in IgE. Contrary to expectations,
these animals remained susceptible to anaphylaxis mediated
through IgG and the low-affinity IgG receptor FcγRIII.37,38
The human counterpart of this receptor, FcγRIIa, is equally
capable of inducing activation of human MCs.39 Human
MCs exposed to interferon-γ also may be induced to express
the high-affinity IgG receptor FcγRI, rendering them sus-
ceptible to IgG-mediated activation, although expression of
this receptor in vivo has not been shown.40
 238 NIGROVIC  |  MAST CELLS

Granule contents are implicated in the response of MCs to pathogens.49 MCs

Proteases also may be activated through complement, including the
Tryptase, chymase, anaphylatoxins C3a and C5a.46 MCs can respond directly to
carboxypeptidase-A physical stimuli, including trauma, temperature, and osmotic
Proteoglycans
Heparin, chondroitin sulfate
stress.50 Together, these receptors enable MC involvement in
Mast Vasoactive amines a broad range of immune and nonimmune processes.
cell Histamine, serotonin
IgE Cytokines
IgG TNF, IL-4, bFGF, VEGF, IL-16
Inhibitory Signals for Mast Cells
Complement As with other immune lineages, MCs are subject to neg-
TLR agonists Lipid metabolites
SCF, cytokines
ative and positive regulation. Examples of inhibitory
PGD2, LTC4, LTB4, PAF
Cell-cell contact receptors on the surface of MCs include the IgG receptor
Trauma Newly synthesized mediators FcγRIIb and the integrin-binding immunoglobulin super-
Cytokines family member gp49b1. The importance of these receptors
IL-1, IL-3, IL-6, IL-8, IL-16, IL-18 is shown in genetically deficient animals. Mice lacking
TNF, SCF, TGF-β
FcγRIIb show a striking propensity to activation via IgG
Chemokines and IgE (which binds with low affinity to FcγRIIb as well
MCP-1, MCP-1α, MCP-1β, as strongly to FcεRI, respectively),51,52 whereas gp49b1-null
RANTES mice are unusually susceptible to IgE-mediated anaphy-
Eotaxin, TARC, Lymphotactin laxis.53 Modulating the surface expression of inhibitory
receptors serves as an important mechanism for regulation
Growth factors
of the activation threshold of MCs in tissues.54
GM-CSF, M-CSF,
bFGF, PDGF, VEGF
Figure 13-3  Mediator production by human mast cells (partial list). The MAST CELL MEDIATORS
set of mediators liberated on activation varies depending on the state of dif-
ferentiation of the mast cell and the nature of the stimulus. See Galli and col- Granule Contents: Proteases, Amines,
leagues80 for a complete mediator list and references. bFGF, basic fibroblast Proteoglycans, and Cytokines
growth factor; GM-CSF, granulocyte-macrophage colony-stimulating fac-
tor; IL, interleukin; LTB4, leukotriene B4; LTC4, leukotriene C4; MCP, monocyte Mature MCs package a range of mediators in their gran-
chemoattractant protein; M-CSF, macrophage colony-stimulating factor;
PAF, platelet-activating factor; PDGF, platelet-derived growth factor; PGD2,
ules, ready for immediate release through fusion with the
prostaglandin D2, RANTES, released on activation, normal T cell expressed surface membrane. The most abundant of these are the neu-
and secreted; SCF, stem cell factor; TARC, thymus and activation-related tral proteases, named for their enzymatic activity at neutral
chemokine TGF-β; transforming growth factor-β; TLR, Toll-like receptor; (physiologic extracellular) pH, but vasoactive amines, pro-
TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor. teoglycans such as heparin, and preformed cytokines play
distinct roles in the biologic consequences of MC degranu-
These IgG receptors contribute to involvement of MCs in lation. The release of these mediators is not all or none. In
IgG-driven diseases. In mice, MCs participate in IgG-medi- addition to anaphylactic degranulation, MCs may release
ated immune complex peritonitis, the cutaneous Arthus only a few granules at a time in a process termed piece-
reaction, and experimental murine bullous pemphigoid.41-43 meal degranulation.55 Alternatively, under other conditions,
Activation via Fc receptors also mediates MC participation MCs may elaborate cytokines and chemokines without any
in antibody-mediated murine arthritis.44 release of granule contents, as illustrated by activation via
CD30L.47 Although the MC is well equipped to release of
large volumes of preformed mediators, it is equally capable
Soluble Mediators and Cell-Cell Contact
of responses tailored to the activating stimulus.
MCs may coordinate with immune and nonimmune lin-
eages via mechanisms beyond antibody response, including Tryptase. Named for its enzymatic similarity to pancreatic
soluble mediators and surface receptors. Examples of such trypsin, tryptase is the most abundant granule protein in
signals include the cytokine tumor necrosis factor (TNF)-α human MCs.56 It also is an essentially specific marker for
and the neurogenic peptide substance P, which can induce MCs, synthesized in scant amounts by basophils but by
MC degranulation.45,46 Physical contact with other cells no other lineage.57 The enzyme found in granules is the
also can induce MC activation. CD30 on lymphocytes can β isomer, which is enzymatically active on formation of a
interact with CD30L on MCs to induce the production of a homotetramer that relies on the scaffolding function of the
range of chemokines.47 Ligation of CD30L does not induce proteoglycan heparin.58 MCs also synthesize α-tryptase, a
release of granule contents or lipid mediators, illustrating protein incapable of forming homotetramers and so enzy-
the selectivity of response of which MCs are capable. matically inactive. In contrast to β-tryptase, the α isomer is
not stored in granules, but constitutively released into circu-
lation, where its function is unknown. The distinction be-
Danger and Injury
tween tryptase isomers is important for diagnostic reasons:
MCs are equipped to recognize danger in the absence of guid- As a marker of degranulation, systemic levels of β-tryptase
ance from other lineages via a range of pathogen receptors, constitute a marker of recent anaphylaxis.59 By contrast,
including multiple Toll-like receptors and CD48, a surface pro- α-trypsin levels reflect total body MC load and serve as a
tein recognizing the fimbrial antigen FimH.48 These receptors useful biomarker in systemic mastocytosis.60
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
Tryptase directly cleaves structural proteins, such as fibro-
nectin and type IV collagen, and enzymatically ­ activates
stromelysin, an enzyme responsible for activating collage-
nase.61 Tryptase also promotes hyperplasia and activation
of fibroblasts, airway smooth muscle cells, and epithelium.
Cleavage of protease-activated receptors, such as PAR2,
likely contributes to these activities.62-64 In aggregate, these
effects suggest an important role for tryptase in matrix
remodeling. A further contribution to the inflammatory
milieu is suggested by the capacity of tryptase to promote
neutrophil and eosinophil recruitment and to cleave C3 to
generate the anaphylatoxin C3a.65,66
Chymase. Chymase is a chymotrypsin-like neutral protease
found in the MCTC subset of human MCs, packaged within
the same granules as tryptase.13 Similar to tryptase, chymase
can cleave matrix components and activate stromeolysin;
it also can activate collagenase directly, suggesting a role in
matrix remodeling.67 Chymase also can affect cytokine func-
tion, with the capacity to cleave pro-IL-1β to generate ac-
tive cytokines and to inactivate proinflammatory cytokines,
such as IL-6 and TNF-α.68,69
Vasoactive Amines. Human MCs are capable of syn-
thesizing and storing the biogenic amines histamine and
serotonin, implicated in vascular leaks.70 Histamine, by
far the more abundant, is a vasoactive amine found in
MCT and MCTC, although it is not unique to this lineage.
Histamine is involved in the wheal-and-flare response
to cutaneous allergen challenge, via augmented vascular
permeability, transendothelial vesicular transport, and
neurogenic vasodilation. These effects are mediated prin-
cipally via the H1 receptor. Three other histamine surface
receptors, H2 through H4, are distributed widely on im-
mune and nonimmune lineages, with effects as diverse as
gastric acid secretion, Langerhans cell migration, and B
cell proliferation.71
Heparin and Chondroitin Sulfate E. Heparin and chon-
droitin sulfate E are large proteoglycans that enable the or-
dered packing of mediators within human MC granules.72,73
Negatively charged carbohydrate side chains complex
tightly with positively charged proteins, allowing very high
concentrations of β-tryptase and other proteases. Heparin,
produced exclusively by MCs, facilitates the activity of
tryptase by making possible proteolytic self-activation with-
in the granule and stabilizing the active tetrameric form of
this enzyme.74 Heparin also has a wide range of effects be-
yond the MC, and is potently angiogenic.75 Heparin bind-
ing activates antithrombin III, providing the basis for use
as an anticoagulant, and inhibits chemokines and classic
and alternative pathways of complement activation.76 The
physiologic role of these extracellular activities of MC-
derived heparin is uncertain.
Preformed Cytokines. MCs are able to store certain cyto-
kines in their granules for rapid release. The first of these to
be documented was TNF-α.77 In mice, this pool of TNF-α
is implicated in the rapid recruitment of neutrophils to the
peritoneum during peritonitis.41,78 Other cytokines that may
be stored in granules include IL-4, IL-16, basic fibroblast
growth factor, and vascular endothelial growth factor.
239
Newly Synthesized Mediators: Lipid Mediators,
Cytokines, Chemokines, and Growth Factors
Beyond preformed mediators stored within granules, acti-
vated MCs elaborate a range of mediators that are generated
de novo. These mediators are released minutes to hours after
stimulation, broadening and extending the impact of acti-
vated MCs on surrounding tissues.
Lipid Mediators. Within minutes of activation, MCs begin
to release metabolites of membrane phospholipids. This pro-
cess is rapid because the relevant enzymes, beginning with
phospholipase A2 responsible for harvesting phospholipids
from the outer leaflet of the nuclear membrane, already
are present in the cytoplasm and need only to be activated
through signals mediated by calcium flux and the phosphory-
lation of intracellular messengers. The hallmark prostaglandin
of human MCs is prostaglandin D2, capable of inducing bron-
choconstriction, vascular leak, and neutrophil recruitment.
Smaller amounts of other prostaglandins and thromboxane
also are made. MC-derived leukotrienes have similar but
generally more potent activity. Leukotriene C4 is the major
leukotriene species generated by human MCs; leukotriene C4
and its metabolites leukotriene D4 and leukotriene E4 serve
as potent inducers of vascular leak. Smaller amounts of the
chemotaxins leukotriene B4 and platelet-activating factor
also are generated. The particular profile of lipid mediators
produced by MCs can change with local environmental sig-
nals and the resulting state of differentiation. MCs from skin
generate more prostaglandin D2 than leukotriene C4, whereas
both species are elaborated in roughly equal proportion by
MCs isolated from lung and osteoarthritic synovium.79
Cytokines, Chemokines, and Growth Factors. Within 2
hours of activation, MCs begin to elaborate newly synthe-
sized mediators as the end result of induced gene transcrip-
tion and translation. The range of such mediators is broad
(see Fig. 13-3). They include the canonical proinflammatory
mediators TNF-α, IL-1, and IL-6; the T helper cytokines IL-4,
IL-5, IL-10, and IL-13; chemotactic factors including IL-8,
macrophage inflammatory protein (MIP)-1α, and RANTES
(released on activation, normal T cell expressed and se-
creted); and growth factors for fibroblasts, blood vessels, and
other cells such as basic fibroblast growth factor, vascular en-
dothelial growth factor, and platelet-derived growth factor.80
As noted earlier, some of these also may be stored preformed
in granules for rapid release. The panel of mediators gener-
ated depends on the state of differentiation and the activat-
ing signal and may occur in the absence of degranulation.
ROLE OF MAST CELLS IN HEALTH
AND DISEASE
Understanding of the role of MCs in health and disease has
been aided greatly by the availability of mice lacking MCs
through defects in the SCF/c-kit axis. Although these mice
exhibit multiple phenotypic abnormalities, they are viable,
excluding an obligate basal role for MCs in the structure and
function of most tissues. Yet under physiologic stress, such as
imposed by experimental models of disease, multiple differences
from wild-type mice become evident. In many cases, these
abnormalities may be corrected by engraftment with cultured
 240 NIGROVIC  |  MAST CELLS

Table 13-1  Participation of Mast Cells in Murine Models of Disease

Beneficial to Host References Harmful to Host References
Angiogenesis 122 Anaphylaxis* 82
Bacterial cystitis 49 Arthritis* 138, 139
Bacterial peritonitis* 78, 87 Asthma* 84
Bone remodeling 117 Bullous pemphigoid* 43
Envenomation* 145 Cardiomyopathy 146
Glomerulonephritis* 147 Dermatitis, irritant* 148
Graft tolerance* 108 Dermatitis, sunburn 149
Parasites, intestine 89, 91 Gastritis 150
Parasites, skin 151 Glomerulonephritis* 152
Thromboembolism 153 Immune complex peritonitis* 41
Wound healing* 111 Ischemia-reperfusion injury 154, 155
Multiple sclerosis* 102
Neurogenic inflammation* 100, 101
Peritonitis, irritant* 156
Peritoneal adhesions 157
Pneumonitis 158
Scleroderma 114, 115
Tumor angiogenesis 159
Note: Mast cells are implicated in these processes by virtue of phenotypic abnormalities in mast cell–deficient mice.
*The phenotype has been shown to be reversible by engraftment with cultured mast cells, providing more direct evidence for a role for this lineage.

MCs,81 directly implicating MCs in a remarkably broad range
of disease processes (Table 13-1). Interpretation of such exper-
iments is limited by incomplete physiologic restoration of the
MC compartment and by residual effects of deficient c-kit sig-
naling in other lineages. Together with in vitro experiments
and careful observation of normal ­subjects, animal experiments
in MC-deficient mice have contributed greatly to progress in
understanding MC physiology and pathophysiology.
MAST CELLS IN ALLERGIC DISEASE:
ANAPHYLAXIS, ALLERGIC DISEASE, AND ASTHMA
MCs are the primary mediators of systemic anaphylaxis. This is
shown in MC-deficient mice, in which resistance to IgE-medi-
ated anaphylaxis may be restored by engraftment with MCs.82
In humans, participation of MCs in anaphylaxis has been
documented through the detection of elevated serum levels of
β-tryptase, a specific marker of MC degranulation.59 MCs accu-
mulate in atopic mucosal tissues, where they degranulate on
exposure to antigen and contribute prominently to tissue edema
and the overproduction of mucus.34 MCs also accumulate in
the asthmatic airway, including within the smooth muscle lin-
ing the airways, and have been implicated by human and ani-
mal data in airway hyperreactivity and mucosal changes.83,84
MAST CELLS IN NONALLERGIC INFLAMMATION
Pathogen Defense: Mast Cells as Sentinels of Innate
Immunity
The involvement of MCs in atopic disease is well docu-
mented, but it does not explain their remarkable evolution-
ary conservation. MCs must somehow contribute to the
survival of the organism. The most probable mechanism by
which MCs convey a survival advantage is in the defense
against infection. This role is reflected in the localization of
MCs near epithelial surfaces, around blood vessels, and in
other locations of potential invasion by pathogens.
MCs are competent defensive cells against bacteria.
They express Toll-like receptors and other receptors
against bacterial antigens, and on activation they are
able to phagocytose bacteria and generate antimicrobial
molecules, such as cathelicidin.85,86 Given their relatively
small numbers, however, the most important function of
MCs in immune defense is to serve as sentinels, moni-
toring for early traces of infection and rapidly mobilizing
neutrophils and other inflammatory cells when needed.
Such a role has been shown in mouse models of bacte-
rial peritonitis, where MC-deficient animals exhibit a
high mortality. This susceptibility correlates with delayed
recruitment of neutrophils via TNF-α and leukotrienes;
neutrophil influx and survival may be restored by correc-
tion of the MC deficit.78,87,88 Clearance of bacteria from
lung also is delayed in MC-deficient mice and can be
similarly restored.78 Analogous observations have been
made in other models of bacterial infection.48 MCs play
an important role in the defense of the host against bacte-
rial infection.
MCs also are implicated in the defense against parasites.
MC-deficient animals exhibit abnormal clearance of mul-
tiple parasites from gut and skin, in a manner promoted by
IgE.89,90 The mechanism of this defense is uncertain, but may
include direct attack on pathogens, recruitment of inflam-
matory lineages such as neutrophils and eosinophils, and
lysis of tight junctions in the mucosal lining to facilitate the
expulsion of helminths.89,91
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
Mast Cells and the Adaptive Immune Response
In addition to recruiting innate effector cells, MCs mobi-
lize T lymphocytes and B lymphocytes, the adaptive arm
of the immune system.80 MCs may express major histocom-
patibility complex II proteins and costimulatory molecules,
such as CD80 and CD86, rendering them effective antigen
presenting cells for CD4 T cells. They may migrate from
peripheral tissues to lymph nodes carrying antigen and
contribute to the recruitment of T cells to lymph nodes
via mediators, including MIP-1β and TNF-α.11,92 Infec-
tion-induced lymph node hyperplasia is abrogated in the
absence of MCs. MCs can recruit CD4 and CD8 effec-
tor T cells to peripheral tissues via leukotriene B4, among
other mediators.93-95 MCs can contribute to the migration
of cutaneous Langerhans cells (dendritic cells) to lymph
nodes via mediators, including histamine.96,97 By means of
the inducible expression of CD40L and cytokines, MCs
may stimulate B cells and induce class-switching to IgE
entirely in the absence of T cell help.98 The physiologic
importance of these effects varies with circumstances.
Under some conditions, delayed-type hypersensitivity
responses in skin are MC-dependent, whereas under oth-
ers MCs seem to play no role.80
Neurogenic Inflammation
In addition to their perivascular localization, MCs cluster
near and even within peripheral nerves. A discrete func-
tion for MCs in these locations has not yet been identi-
fied, although the potential for bidirectional neuroimmune
interaction is clear. MC mediators such as histamine may
activate neurons directly, whereas MCs residing near stim-
ulated neurons may be induced to degranulate.99 Vascular
leak and neutrophil infiltration arising from infiltration of
skin with the neurogenic mediator substance P are mediated
by MCs.100,101 Neurons may recruit MCs as local effectors to
initiate neurogenic inflammation.
Autoimmune Disease
Reconstitution experiments in MC-deficient mice have
implicated MCs in a variety of pathologic inflammatory
states (see Table 13-1). These include murine models of
multiple autoimmune diseases, such as bullous pemphi-
goid, multiple sclerosis, scleroderma, and inflammatory
arthritis. In pemphigoid, MCs triggered via IgG antibod-
ies against a hemidesmosomal antigen recruit neutrophils
that are responsible for blister formation.43 The role of
MCs in murine experimental autoimmune encephalomy-
elitis is more complex. Although the resistance of W/Wv
mice to experimental autoimmune encephalomyelitis cor-
rects with MC engraftment, these cells fail to repopulate
the brain and spinal cord, indicating that MCs are not
obligate local effector cells in this model.102,103 One mech-
anism for this activity seems to be the promotion of the
adaptive immune response because MC engraftment into
W/Wv animals improves T cell responses to immunization
with the inciting myelin antigen.104,105 The contribution
of MCs to human multiple sclerosis is unknown. The par-
ticipation of MCs in scleroderma and arthritis is discussed
subsequently.
241
Mast Cells as Anti-inflammatory Cells
In recent years, it has become evident that MCs also may
help to moderate the immune response. One mechanism for
this effect is by degradation of proinflammatory mediators.
MC proteases may cleave and inactivate the cytokines IL-5,
IL-6, IL-13, TNF-α and endothelin-1 and the anaphylatoxin
C3a.66,69,106,107 The importance of this activity has been shown
in a murine sepsis model, where MCs reduced mortality by
restraining excess inflammation in a protease-dependent
manner.107 Data also suggest that MCs may interact with
regulatory T cells to promote immunologic tolerance to skin
grafts, although the pathway by which MCs prevent rejection
is undefined.108
MAST CELLS AND CONNECTIVE TISSUE
Wound Healing and Tissue Fibrosis
MCs have long been noted to accumulate at the borders
of healing wounds.109 In normal human subjects undergoing
experimental wounding and recurrent biopsies, MC num-
bers increase sixfold by day 10 after initial incision. These
MCs localize preferentially to fibrotic areas of the wound
and strongly express IL-4, a cytokine capable of inducing
fibroblast proliferation and collagen synthesis.8 In vitro
studies confirm the stimulatory effects of MCs on fibroblast
growth110; candidate fibroblast mitogens in addition to IL-4
include tryptase, histamine, leukotriene C4, and basic fibro-
blast growth factor. MC-deficient W/Wv animals exhibit
delayed contracture and healing of skin wounds in a manner
reparable by local engraftment with cultured MCs.111
MCs also accumulate in sites of pathologic fibro-
sis, including the skin and lungs of patients with sclero-
derma.112,113 Because experimental skin fibrosis proceeds in
MC-deficient mice with only relatively subtle differences in
intensity or kinetics, it is unlikely that MCs are an obligate
effector lineage in human scleroderma, although they may
contribute to disease progression.114,115
Bone
MCs also are implicated in the remodeling of bone. MCs
accumulate at sites of healing fracture, and under normal
circumstances they may contribute productively to normal
bone turnover.116,117 MCs accumulate in osteoporotic bone,
however, and systemic osteoporosis is a known complication
of systemic mastocytosis.118,119 Heparin is one potentially
important mediator of bone loss because it directly promotes
differentiation and activation of osteoclasts.120 MC products
such as IL-1, TNF-α, and MIP-1α have similar activity.
Angiogenesis
A final and potentially quite important activity of MCs on
the stroma is the promotion of angiogenesis. MCs are not
required for the development of the normal vasculature, as
evident in the viability of MC-deficient mice. MCs cluster
at sites of early blood vessel growth in tumors, however, and
contribute appreciably to physiologic angiogenesis under
certain experimental conditions.121,122 Heparin was the first
proangiogenic MC mediator identified75; basic fibroblast
 242 NIGROVIC  |  MAST CELLS
growth factor and vascular endothelial growth factor are
other potent stimulators of endothelial migration and pro-
liferation.
MAST CELLS IN ARTHRITIS
The normal synovium features a small population of resi-
dent MCs. These cells are not found in the immediate lin-
ing layer, but rather are found in the synovial sublining,
near blood vessels and nerves, constituting almost 3% of
cells within 70 μM of the intra-articular space.123 In mice
and humans, their phenotype is principally MCTC, similar
to MCs found in most other connective tissue sites.30,124
The severalfold increased density of MCs in the immediate
vicinity of the synovial lining, compared with more distant
connective tissue, supports the hypothesis that MCs con-
tribute to surveillance of the articular cavity.30 Extrapolating
from the activity of MCs near other vulnerable body cavi-
ties, such as the peritoneum, it is likely that one function of
synovial MCs is to monitor the joint for early evidence of
infection. No direct support for a protective effect of MCs
against joint sepsis is available, however.
In arthritis, the population of synovial MCs may expand
remarkably (Fig. 13-4). More than two thirds of synovial
specimens from patients with rheumatoid arthritis (RA)
exhibit abnormal numbers of MCs, averaging more than
10-fold above normal.125 Consistent with these histologic
findings, synovial fluid from rheumatoid joints contains
appreciable quantities of histamine and tryptase.126,127 In
contrast to normal joints, in joints with RA, both subtypes
of MCs are present in roughly equal numbers; MCT are
located nearer to the pannus and infiltrating leukocytes,
whereas MCTC cluster in deeper, more fibrotic areas of the
synovium.30 MCs have been noted near the junction of pan-
nus and cartilage.128 Rarely, MCs also are identified in syno-
vial fluid.129 The absence of mitotic figures and of staining
for the proliferation antigen Ki-67 in this population sug-
gests that they arise not from local replication, but rather
by recruitment of circulating progenitors.130 Although the
Figure 13-4  Mast cells in the rheumatoid synovium. Stained red by an
antibody against tryptase, mast cells are abundant in this synovial biopsy
specimen from a patient with chronic rheumatoid arthritis. Note the pro-
liferation of mast cells in the synovial sublining. (From Nigrovic PA, Lee
DM: Synovial mast cells: Role in acute and chronic arthritis. Immunol Rev,
217: 19-37, 2007.)
signals driving this recruitment are unknown, inflammatory
cytokines, such as TNF-α, enhance expression of the MC
chemotactic and survival factor SCF on synovial fibroblasts,
suggesting one mechanism for this dramatic expansion.131
Degree of inflammation is the best predictor of the number
of MCs within the joint.125,132,133 Incompletely identified fac-
tors in RA synovial fluid can potently promote MC differ-
entiation and growth.134
Hyperplasia of the MC population is not specific for RA,
but is observed in a wide range of inflammatory joint disorders
(Table 13-2). Expansion also is noted in osteoarthritis, often
to numbers seen in RA.30,130,135,136 The levels of histamine and
tryptase in osteoarthritis synovial fluid also are comparable.
In contrast to in RA, the expansion in osteoarthritis results
from an increase in numbers of MCT, the subtype generally
associated with T cells and inflammation.30,137
Mast Cells in Acute Arthritis: Insights from Animal
Models
Experimental work in mice has begun to shed light on the
role of MCs in inflammatory arthritis. Several MC-deficient
strains show striking resistance to arthritis induced by IgG
autoantibodies, a defect that may be repaired by engraft-
ment with cultured MCs expressing receptors for IgG.44,138,139
Numerous mechanisms contribute to this arthritogenic
activity. First, MCs induce vascular permeability, facilitat-
ing entry of autoantibody into the joint.140,141 Second, MCs
release proinflammatory mediators, including IL-1, that
help to establish inflammation, presumably via effects on
endothelium and other local populations, such as macro-
phages and fibroblasts.44 These actions seem to be most cru-
cial at the initiation of disease, constituting a “jump start”
for acute inflammation within the joint. This function is
in line with the activity of MCs in other models of IgG-
mediated disease, such as IgG-mediated immune complex
peritonitis, murine bullous pemphigoid, and anaphylaxis. In
each of these models, MCs resident in tissue for the purpose
of immune defense become co-opted by autoantibodies to
initiate inflammatory pathology (Fig. 13-5).
Mast Cells in Chronic Arthritis
In contrast to the acute phase of joint inflammation, the
contribution of MCs to established arthritis has not been
explored in animal models. The sheer numbers of these cells
Table 13-2  Joint Diseases with Documented  
Synovial Mastocytosis
Chronic infection
Gout
Juvenile rheumatoid arthritis
Osteoarthritis
Psoriatic arthritis
Rheumatoid arthritis
Rheumatic fever
Traumatic arthritis
See Nigrovic and Lee142 for tuberculous arthritis relevant references.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION 243

Histamine,
Endothelium
Leukotrienes, Synovial
Permeability TNF, IL-1 mast cell
Adhesion
molecules ( )
Tryptase, Normal bone
IL-4, TNF,
Leukocyte

Acute arthritis
bFGF
recruitment TNF, IL-1,
and activation Cartilage
Chemokines, Synoviocyte
Eicosanoids activation
PMNs
(fibroblast,
Elastase
PMN Monocyte macrophage)
Lymphocyte
Chondrocyte
activation
bFGF, PDGF, Histamine, IL-1
LTC4, histamine MMPs
tryptase
Heparin,

Chronic arthritis
Angiogenesis Fibroblast,
bFGF,
VEGF proliferation
Tryptase, Pannus Osteoclast
Chymase, differentiation
IL-4 and bone
Heparin, MIP-1α,
remodeling
TNF, IL-1

Matrix remodeling
and fibrosis

Figure 13-5  Potential roles of mast cells in acute and chronic arthritis. In the acute phase of joint inflammation, mast cells may contribute to initia-
tion of arthritis by inducing vascular permeability, recruiting and activating circulating leukocytes, and stimulating local fibroblasts and macrophages.
In established arthritis, these activities may be joined by effects on the stroma, including the promotion of pannus formation, angiogenesis, fibro-
sis, and injury to cartilage and bone. Potential anti-inflammatory effects of mast cells are not depicted. The mediators listed are representative and
do not constitute a complete list. bFGF, basic fibroblast growth factor; IL, interleukin; LTC4, leukotriene C4; MIP, macrophage inflammatory protein;
MMP, matrix metalloproteinase; PDGF, platelet-derived growth factor; PMN, polymorphonuclear neutrophil; TNF, tumor necrosis factor; VEGF, vascular 
endothelial growth factor. (From Nigrovic PA, Lee DM: Synovial mast cells: Role in acute and chronic arthritis. Immunol Rev, 217: 19-37, 2007. Illustration by
Steven Moskowitz.)

in arthritic synovium implies a substantial role. Taking into
account the spectrum of MC activity elsewhere, it is likely
that MCs participate both in the inflammatory process and
in the mesenchymal response (see Fig. 13-5).142
An ongoing contribution of MCs to inflammatory arthri-
tis is suggested by several observations. First, as noted,
prominent among infiltrating synovial MCs are MCT, typi-
cally associated elsewhere with the elaboration of cytokines,
such as IL-6, with documented pathogenic activity in RA.
Immunofluorescence staining also has identified TNF-α in
RA synovial MCs,10 and the elaboration of other proinflam-
matory mediators is probable. Second, MCs from RA, but
not osteoarthritis, synovium express the receptor for the
anaphylatoxin C5a, a mediator readily documented in syno-
vial fluid.143 Immune complexes within RA joints provide
another likely pathway to activation of synovial MCs.144
Ultrastructural data support ongoing degranulation of MCs
in the RA synovium.137
The effect of MCs on the established inflammatory syno-
vial infiltrate is difficult to predict. As in acute arthritis,
activated MCs may promote the recruitment and activation
of leukocytes, although the proinflammatory contribution of
this lineage relative to macrophages and other synoviocytes
remains to be determined. Alternatively, protease cleavage
of inflammatory mediators and the elaboration of mediators
such as TGF-β could down-modulate inflammation, poten-
tially under the direction of regulatory T cells, as has been
observed in tolerance of skin grafts in mice.108
MCs likely modulate the stromal response to inflamma-
tion as well. Expansion and activation of synovial fibroblasts
is a key pathogenic process within RA, and the capacity of
MCs to promote such changes is well established. By their
interaction with osteoclasts, MCs also may promote focal
erosions and periarticular osteopenia. Together, these effects
may contribute to joint injury. Finally, by producing pro-
angiogenic mediators, MCs may enable the growth of the
vascular supply required for the profound expansion of the
thin synovial layer into thick pannus. Confirmation of these
roles awaits further experimental data.
SUMMARY
MCs are potent immune cells characterized by phenotypic
diversity and an extremely broad range of functions in health
and disease. In addition to mediating atopic disease, MCs
represent important sentinels against pathogen invasion.
Under certain conditions, it is likely that they also partici-
pate in the remodeling of tissue matrix. Aberrant activation
of MCs by antoantibodies and potentially other signals has
been identified in a variety of inflammatory diseases, including
 244 NIGROVIC  |  MAST CELLS

arthritis. Such activation may represent a key pathologic   22. Flanagan JG, Chan DC, Leder P: Transmembrane form of the kit
step in the development of tissue inflammation and injury ligand growth factor is determined by alternative splicing and is miss-
ing in the Sld mutant. Cell 64:1025, 1991.
and could present an interesting target for the development   23. Levi-Schaffer F, Austen KF, Caulfield JP, et al: Fibroblasts maintain
of anti-inflammatory therapies. the phenotype and viability of the rat heparin-containing mast cell
in vitro. J Immunol 135:3454, 1985.
  24. Fujita J, Nakayama H, Onoue H, et al: Fibroblast-dependent growth
REFERENCES of mouse mast cells in vitro: Duplication of mast cell depletion in
mutant mice of W/Wv genotype. J Cell Physiol 134:78, 1988.
   1. Ehrlich P: Beiträge zur Theorie und Praxis der Histologischen Fär-
  25. Sellge G, Lorentz A, Gebhardt T, et al: Human intestinal fibroblasts
bung [Thesis]. Leipzig University, 1878.
prevent apoptosis in human intestinal mast cells by a mechanism
   2. Galli SJ, Maurer M, Lantz CS: Mast cells as sentinels of innate immu-
independent of stem cell factor, IL-3, IL-4, and nerve growth factor.
nity. Curr Opin Immunol 11:53, 1999.
J Immunol 172:260, 2004.
   3. Kitamura Y., Go S., Hatanaka K.: Decrease of mast cells in W/Wv mice
  26. Ruitenberg EJ, Elgersma A: Absence of intestinal mast cell response
and their increase by bone marrow transplantation. Blood 52:447, 1978.
in congenitally athymic mice during Trichinella spiralis infection.
   4. Rodewald HR, Dessing M, Dvorak AM, et al: Identification of a com-
Nature 264:258, 1976.
mitted precursor for the mast cell lineage. Science 271:818, 1996.
  27. Irani AM, Craig SS, DeBlois G, et al: Deficiency of the tryptase-posi-
   5. Rottem M, Okada T, Goff JP, et al: Mast cells cultured from the
tive, chymase-negative mast cell type in gastrointestinal mucosa of
peripheral blood of normal donors and patients with mastocyto-
patients with defective T lymphocyte function. J Immunol 138:4381,
sis originate from a CD34+/Fc epsilon RI− cell population. Blood
1987.
84:2489, 1994.
  28. Toru H, Eguchi M, Matsumoto R, et al: Interleukin-4 promotes the
   6. Gurish MF, Tao H, Abonia JP, et al: Intestinal mast cell progenitors
development of tryptase and chymase double-positive human mast
require CD49dbeta7 (alpha4beta7 integrin) for tissue-specific hom-
cells accompanied by cell maturation. Blood 91:187, 1998.
ing. J Exp Med 194:1243, 2001.
  29. Ochi H, Hirani WM, Yuan Q, et al: T helper cell type 2 cytokine-
   7. Padawer J: Mast cells: Extended lifespan and lack of granule turnover
mediated comitogenic responses and CCR3 expression during
under normal in vivo conditions. Exp Mol Pathol 20:269, 1974.
differentiation of human mast cells in vitro. J Exp Med 190:267,
   8. Trautmann A, Toksoy A, Engelhardt E, et al: Mast cell involvement
1999.
in normal human skin wound healing: Expression of monocyte che-
  30. Gotis-Graham I, McNeil HP: Mast cell responses in rheumatoid
moattractant protein-1 is correlated with recruitment of mast cells
synovium: Association of the MCTC subset with matrix turnover
which synthesize interleukin-4 in vivo. J Pathol 190:100, 2000.
and clinical progression. Arthritis Rheum 40:479, 1997.
   9. Galli SJ, Zsebo KM, Geissler EN: The kit ligand, stem cell factor.
  31. McNeil HP, Gotis-Graham I: Human mast cell subsets—distinct
Adv Immunol 55:1, 1994.
functions in inflammation? Inflamm Res 49:3, 2000.
  10. Juurikivi A, Sandler C, Lindstedt KA, et al: Inhibition of c-kit tyro-
  32. Bradding P, Okayama Y, Howarth PH, et al: Heterogeneity of human
sine kinase by imatinib mesylate induces apoptosis in mast cells in
mast cells based on cytokine content. J Immunol 155:297, 1995.
rheumatoid synovia: A potential approach to the treatment of arthri-
  33. Oskeritzian CA, Zhao W, Min HK, et al: Surface CD88 functionally
tis. Ann Rheum Dis 64:1126, 2005.
distinguishes the MCTC from the MCT type of human lung mast
  11. Wang HW, Tedla N, Lloyd AR, et al: Mast cell activation and migra-
cell. J Allergy Clin Immunol 115:1162, 2005.
tion to lymph nodes during induction of an immune response in
  34. Gould HJ, Sutton BJ, Beavil AJ, et al: The biology of IgE and the
mice. J Clin Invest 102:1617, 1998.
basis of allergic disease. Annu Rev Immunol 21:579, 2003.
  12. Irani AA, Schechter NM, Craig SS, et al: Two types of human mast
  35. Dvorak AM, Schleimer RP, Schulman ES, et al: Human mast cells
cells that have distinct neutral protease compositions. Proc Natl
use conservation and condensation mechanisms during recovery from
Acad Sci U S A 83:4464, 1986.
degranulation: In vitro studies with mast cells purified from human
  13. Craig SS, Schechter NM, Schwartz LB: Ultrastructural analysis of
lungs. Lab Invest 54:663, 1986.
human T and TC mast cells identified by immunoelectron micros-
  36. Hammel I, Lagunoff D, Kruger PG: Recovery of rat mast cells after
copy. Lab Invest 58:682, 1988.
secretion: A morphometric study. Exp Cell Res 184:518, 1989.
  14. Weidner N, Austen KF: Heterogeneity of mast cells at multiple body
  37. Oettgen HC, Martin TR, Wynshaw-Boris A, et al: Active anaphy-
sites: Fluorescent determination of avidin binding and immunofluo-
laxis in IgE-deficient mice. Nature 370:367, 1994.
rescent determination of chymase, tryptase, and carboxypeptidase
  38. Miyajima I, Dombrowicz D, Martin TR, et al: Systemic anaphylaxis in
content. Pathol Res Pract 189:156, 1993.
the mouse can be mediated largely through IgG1 and Fc gammaRIII:
  15. Irani AM, Bradford TR, Kepley CL, et al: Detection of MCT and
Assessment of the cardiopulmonary changes, mast cell degranulation,
MCTC types of human mast cells by immunohistochemistry using
and death associated with active or IgE- or IgG1-dependent passive
new monoclonal anti-tryptase and anti-chymase antibodies. J Histo-
anaphylaxis. J Clin Invest 99:901, 1997.
chem Cytochem 37:1509, 1989.
  39. Zhao W, Kepley CL, Morel PA, et al: Fc gamma RIIa, not Fc gamma
  16. Sonoda S, Sonoda T, Nakano T, et al: Development of mucosal mast
RIIb, is constitutively and functionally expressed on skin-derived
cells after injection of a single connective tissue-type mast cell in
human mast cells. J Immunol 177:694, 2006.
the stomach mucosa of genetically mast cell-deficient W/Wv mice.
  40. Tkaczyk C, Okayama Y, Woolhiser MR, et al: Activation of human
J Immunol 137:1319, 1986.
mast cells through the high affinity IgG receptor. Mol Immunol
  17. Kanakura Y, Thompson H, Nakano T, et al: Multiple bidirectional
38:1289, 2002.
alterations of phenotype and changes in proliferative potential dur-
41. Zhang Y, Ramos BF, Jakschik BA: Neutrophil recruitment by
ing the in vitro and in vivo passage of clonal mast cell populations
tumor necrosis factor from mast cells in immune complex peritoni-
derived from mouse peritoneal mast cells. Blood 72:877, 1988.
tis. Science 258:1952, 1997.
  18. Friend DS, Ghildyal N, Austen KF, et al: Mast cells that reside at
  42. Sylvestre DL, Ravetch JV: A dominant role for mast cell Fc receptors
different locations in the jejunum of mice infected with Trichinella
in the Arthus reaction. Immunity 5:387, 1996.
spiralis exhibit sequential changes in their granule ultrastructure and
  43. Chen R, Ning G, Zhao ML, et al: Mast cells play a key role in neutro-
chymase phenotype. J Cell Biol 135:279, 1996.
phil recruitment in experimental bullous pemphigoid. J Clin Invest
19. Gurish MF, Pear WS, Stevens RL, et al: Tissue-regulated differen-
108:1151, 2001.
tiation and maturation of a v-abl-immortalized mast cell-committed
  44. Nigrovic PA, Binstadt BA, Monach PA, et al: Mast cells contribute
progenitor. Immunity 3:175, 1995.
to initiation of autoantibody-mediated arthritis via IL-1. Proc Natl
  20. Longley BJ, Tyrrell L, Lu S, et al: Chronically KIT-stimulated clon-
Acad Sci U S A, 104:2325-2330, 2007.
ally-derived human mast cells show heterogeneity in different tissue
  45. van Overveld FJ, Jorens PG, Rampart M, et al: Tumour necrosis fac-
microenvironments. J Invest Dermatol 108:792, 1997.
tor stimulates human skin mast cells to release histamine and trypt-
  21. Nagata H, Worobec AS, Oh CK, et al: Identification of a point muta-
ase. Clin Exp Allergy 21:711, 1991.
tion in the catalytic domain of the protooncogene c-kit in peripheral
  46. Lawrence ID, Warner JA, Cohan VL, et al: Purification and charac-
blood mononuclear cells of patients who have mastocytosis with an
terization of human skin mast cells: Evidence for human mast cell
associated hematologic disorder. Proc Natl Acad Sci U S A 92:10560,
heterogeneity. J Immunol 139:3062, 1987.
1995.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
  47. Fischer M, Harvima IT, Carvalho RF, et al: Mast cell CD30 ligand is  
upregulated in cutaneous inflammation and mediates degranulation-
independent chemokine secretion. J Clin Invest 116:2748, 2006.
48. Marshall JS: Mast-cell responses to pathogens. Nat Rev Immunol  
4:787, 2004.
  49. Malaviya R, Ikeda T, Abraham SN, et al: Contribution of mast cells  
to bacterial clearance and their proliferation during experimental
cystitis induced by type 1 fimbriated E. coli. Immunol Lett 91:103,
2004.  
  50. Stokes AJ, Shimoda LM, Koblan-Huberson M, et al: A TRPV2-PKA
signaling module for transduction of physical stimuli in mast cells.
J Exp Med 200:137, 2004.  
  51. Takai T, Ono M, Hikida M, et al: Augmented humoral and anaphy-
lactic responses in Fc gamma RII-deficient mice. Nature 379:346,
1996.  
  52. Ujike A, Ishikawa Y, Ono M, et al: Modulation of immunoglobulin
(Ig)E-mediated systemic anaphylaxis by low-affinity Fc receptors for
IgG. J Exp Med 189:1573, 1999.
  53. Daheshia M, Friend DS, Grusby MJ, et al: Increased severity of local
and systemic anaphylactic reactions in gp49B1-deficient mice. J Exp  
Med 194:227, 2001.
  54. Shushakova N, Skokowa J, Schulman J, et al: C5a anaphylatoxin is a
major regulator of activating versus inhibitory FcgammaRs in immune
complex-induced lung disease. J Clin Invest 110:1823, 2000.
  55. Dvorak AM, Kissell S: Granule changes of human skin mast cells  
characteristic of piecemeal degranulation and associated with recov-
ery during wound healing in situ. J Leukoc Biol 49:197, 1991.
  56. Schwartz LB, Irani AM, Roller K, et al: Quantitation of histamine,
tryptase, and chymase in dispersed human T and TC mast cells.
J Immunol 138:2611, 1987.  
  57. Castells MC, Irani AM, Schwartz LB: Evaluation of human peripheral
blood leukocytes for mast cell tryptase. J Immunol 138:2184, 1987.
  58. Schwartz LB, Bradford TR: Regulation of tryptase from human lung
mast cells by heparin: Stabilization of the active tetramer. J Biol  
Chem 261:7372, 1986.
  59. Schwartz LB, Metcalfe DD, Miller JS, et al: Tryptase levels as an indi-  
cator of mast-cell activation in systemic anaphylaxis and mastocyto-
sis. N Engl J Med 316:1622, 1987.
  60. Schwartz LB, Sakai K, Bradford TR, et al: The alpha form of human  
tryptase is the predominant type present in blood at baseline in nor-
mal subjects and is elevated in those with systemic mastocytosis.  
J Clin Invest 96:2702, 1995.
  61. Gruber BL, Marchese MJ, Suzuki K, et al: Synovial procollagenase acti-
vation by human mast cell tryptase dependence upon matrix metal-
loproteinase 3 activation. J Clin Invest 84:1657, 1989.
  62. Cairns JA, Walls AF: Mast cell tryptase stimulates the synthesis of type
I collagen in human lung fibroblasts. J Clin Invest 99:1313, 1997.  
  63. Berger P, Perng DW, Thabrew H, et al: Tryptase and agonists of
PAR-2 induce the proliferation of human airway smooth muscle cells.
J Appl Physiol 91:1372, 2001.  
  64. Cairns JA, Walls AF: Mast cell tryptase is a mitogen for epithelial
cells: Stimulation of IL-8 production and intercellular adhesion mol-  
ecule-1 expression. J Immunol 156:275, 1996.
  65. He S, Peng Q, Walls AF: Potent induction of a neutrophil and eosin-
ophil-rich infiltrate in vivo by human mast cell tryptase: Selective  
enhancement of eosinophil recruitment by histamine. J Immunol
159:6216, 1997.
  66. Schwartz LB, Kawahara MS, Hugli TE, et al: Generation of C3a ana-  
phylatoxin from human C3 by human mast cell tryptase. J Immunol
130:1891, 1983.
  67. Saarinen J, Kalkkinen N, Welgus HG, et al: Activation of human  
interstitial procollagenase through direct cleavage of the Leu83-
Thr84 bond by mast cell chymase. J Biol Chem 269:18134, 1994.
  68. Mizutani H, Schechter N, Lazarus G, et al: Rapid and specific con-  
version of precursor interleukin 1 beta (IL-1 beta) to an active IL-1
species by human mast cell chymase. J Exp Med 174:821, 1991.
  69. Zhao W, Oskeritzian CA, Pozez AL, et al: Cytokine production by  
skin-derived mast cells: Endogenous proteases are responsible for deg-
radation of cytokines. J Immunol 175:2635, 2005.
  70. Kushnir-Sukhov NM, Brown JK, Wu Y, et al: Human mast cells are  
capable of serotonin synthesis and release. J Allergy Clin Immunol
119:498-499, 2007.
  71. Tanaka S, Ichikawa A: Recent advances in molecular pharmacology  
of the histamine systems: Immune regulatory roles of histamine pro-
duced by leukocytes. J Pharmacol Sci 101:19, 2006.
245
72. Humphries DE, Wong GW, Friend DS, et al: Heparin is essential for
the storage of specific granule proteases in mast cells. Nature 400:769,
1999.
73. Forsberg E, Pejler G, Ringvall M, et al: Abnormal mast cells in mice
deficient in a heparin-synthesizing enzyme. Nature 400:773, 1999.
74. Sakai K, Ren S, Schwartz LB: A novel heparin-dependent process-
ing pathway for human tryptase: Autocatalysis followed by activation
with dipeptidyl peptidase I. J Clin Invest 97:988, 1996.
75. Azizkhan RG, Azizkhan JC, Zetter BR, et al: Mast cell heparin
stimulates migration of capillary endothelial cells in vitro. J Exp Med
152:931, 1980.
76. Church MK, Holgate ST, Schute JK, et al: Mast cell-derived media-
tors. In Middleton E, Reed CE, Ellis EF, et al: Allergy: Principle and
Practice, Vol 1, St Louis, Mosby, 1998, p 146.
77. Gordon JR, Galli SJ: Mast cells as a source of both preformed and immu-
nologically inducible TNF-a-alpha/cachectin. Nature 346:274, 1990.
78. Malaviya R, Ikeda T, Ross E, et al: Mast cell modulation of neu-
trophil influx and bacterial clearance at sites of infection through
TNF-a-alpha. Nature 381:77, 1996.
79. de Paulis A, Marino I, Ciccarelli A, et al: Human synovial mast cells,
I: Ultrastructural in situ and in vitro immunologic characterization.
Arthritis Rheum 39:1222, 1996.
80. Galli SJ, Nakae S, Tsai M: Mast cells in the development of adap-
tive immune responses. Nat Immunol 6:135, 2005.
81. Nakano T, Sonoda T, Hayashi C, et al: Fate of bone marrow-derived
cultured mast cells after intracutaneous, intraperitoneal, and intrave-
nous transfer into genetically mast cell-deficient W/Wv mice: Evi-
dence that cultured mast cells can give rise to both connective tissue
type and mucosal mast cells. J Exp Med 162:1025, 1985.
82. Martin TR, Galli SJ, Katona IM, et al: Role of mast cells in anaphy-
laxis: Evidence for the importance of mast cells in the cardiopulmo-
nary alterations and death induced by anti-IgE in mice. J Clin Invest
83:1375, 1989.
83. Brightling CE, Bradding P, Symon FA, et al: Mast-cell infiltration of
airway smooth muscle in asthma. N Engl J Med 346:1699, 2002.
84. Yu M, Tsai M, Tam SY, et al: Mast cells can promote the develop-
ment of multiple features of chronic asthma in mice. J Clin Invest
116:1633, 2006.
85. Malaviya R, Abraham SN: Mast cell modulation of immune responses
to bacteria. Immunol Rev 179:16, 2001.
86. Di Nardo A, Vitiello A, Gallo RL: Cutting edge: Mast cell antimicro-
bial activity is mediated by expression of cathelicidin antimicrobial
peptide. J Immunol 170:2274, 2003.
87. Echtenacher B, Mannel DN, Hultner L: Critical protective role of
mast cells in a model of acute septic peritonitis. Nature 381:75,
1996.
88. Malaviya R, Abraham SN: Role of mast cell leukotrienes in neu-
trophil recruitment and bacterial clearance in infectious peritonitis.
J Leukoc Biol 67:841, 2000.
89. Ha TY, Reed ND, Crowle PK: Delayed expulsion of adult Trichinella spi-
ralis by mast cell-deficient W/Wv mice. Infect Immun 41:445, 1983.
90. Gurish MF, Bryce PJ, Tao H, et al: IgE enhances parasite clearance
and regulates mast cell responses in mice infected with Trichinella spi-
ralis. J Immunol 172:1139, 2004.
91. McDermott JR, Bartram RE, Knight PA, et al: Mast cells disrupt epi-
thelial barrier function during enteric nematode infection. Proc Natl
Acad Sci U S A 100:7761, 2003.
92. McLachlan JB, Hart JP, Pizzo SV, et al: Mast cell-derived tumor
necrosis factor induces hypertrophy of draining lymph nodes during
infection. Nat Immunol 4:1199, 2003.
93. Ott VL, Cambier JC, Kappler J, et al: Mast cell-dependent migration
of effector CD8+ T cells through production of leukotriene B4. Nat
Immunol 4:974, 2003.
94. Tager AM, Bromley SK, Medoff BD, et al: Leukotriene B4 receptor
BLT1 mediates early effector T cell recruitment. Nat Immunol 4:982,
2003.
95. Rumsaeng V, Cruikshank WW, Foster B, et al: Human mast cells
produce the CD4+ T lymphocyte chemoattractant factor, IL-16.
J Immunol 159:2904, 1997.
96. Bryce PJ, Miller ML, Miyajima I, et al: Immune sensitization in the
skin is enhanced by antigen-independent effects of IgE. Immunity
20:1, 2004.
97. Jawdat DM, Albert EJ, Rowden G, et al: IgE-mediated mast cell
activation induces Langerhans cell migration in vivo. J Immunol
173:5275, 2004.
 246 NIGROVIC  |  MAST CELLS
  98. Gauchat JF, Henchoz S, Mazzei G, et al: Induction of human IgE syn-
thesis in B cells by mast cells and basophils. Nature 365:340, 1993.
  99. Kowalski ML, Kaliner MA: Neurogenic inflammation, vascular per-
meability, and mast cells. J Immunol 140:3905, 1988.
100. Yano H, Wershil BK, Arizono N, et al: Substance P-induced augmen-
tation of cutaneous vascular permeability and granulocyte infiltration
in mice is mast cell dependent. J Clin Invest 84:1276, 1989.
101. Matsuda H, Kawakita K, Kiso Y, et al: Substance P induces granu-
locyte infiltration through degranulation of mast cells. J Immunol
142:927, 1989.
102. Secor VH, Secor WE, Gutekunst CA, et al: Mast cells are essential
for early onset and severe disease in a murine model of multiple scle-
rosis. J Exp Med 191:813, 2000.
103. Tanzola MB, Robbie-Ryan M, Gutekunst CA, et al: Mast cells exert
effects outside the central nervous system to influence experimental
allergic encephalomyelitis disease course. J Immunol 171:4385, 2003.
104. Gregory GD, Robbie-Ryan M, Secor VH, et al: Mast cells are required
for optimal autoreactive T cell responses in a murine model of mul-
tiple sclerosis. Eur J Immunol 35:3478, 2005.
105. Gregory GD, Raju SS, Winandy S, et al: Mast cell IL-4 expression is
regulated by Ikaros and influences encephalitogenic Th1 responses in
EAE. J Clin Invest 116:1327, 2006.
106. Mallen-St Clair J, Pham CT, Villalta SA, et al: Mast cell dipepti-
dyl peptidase I mediates survival from sepsis. J Clin Invest 113:628,
2004.
107. Maurer M, Wedemeyer J, Metz M, et al: Mast cells promote homeo-
stasis by limiting endothelin-1-induced toxicity. Nature 432:512,
2004.
108. Lu LF, Lind EF, Gondek DC, et al: Mast cells are essential intermedi-
aries in regulatory T-cell tolerance. Nature 442:997, 2006.
109. Wichmann B-E: The mast cell count during the process of wound
healing. Acta Pathol Microb Scand 108(Suppl):7, 1955.
110. Dayton ET, Caulfield JP, Hein A, et al: Regulation of the growth rate
of mouse fibroblasts by IL-3-activated mouse bone marrow-derived
mast cells. J Immunol 142:4307, 1989.
111. Weller K, Foitzik K, Paus R, et al: Mast cells are required for normal
healing of skin wounds in mice. FASEB J 20:2366, 2006.
112. Hawkins RA, Claman HN, Clark RA, et al: Increased dermal mast
cell populations in progressive systemic sclerosis: A link in chronic
fibrosis? Ann Intern Med 102:182, 1985.
113. Chanez P, Lacoste JY, Guillot B, et al: Mast cells’ contribution to
the fibrosing alveolitis of the scleroderma lung. Am Rev Respir Dis
147:1497, 1993.
114. Everett ET, Pablos JL, Harley RA, et al: The role of mast cells in
the development of skin fibrosis in tight-skin mutant mice. Comp
Biochem Physiol A Physiol 110:159, 1995.
115. Yamamoto T, Takahashi Y, Takagawa S, et al: Animal model of scle-
rotic skin, II: Bleomycin induced scleroderma in genetically mast cell
deficient WBB6F1-W/W(V) mice. J Rheumatol 26:2628, 1999.
116. Severson AR: Mast cells in areas of experimental bone resorption and
remodelling. Br J Exp Pathol 50:17, 1969.
117. Silberstein R, Melnick M, Greenberg G, et al: Bone remodeling in
W/Wv mast cell deficient mice. Bone 12:227, 1991.
118. Frame B, Nixon RK: Bone-marrow mast cells in osteoporosis of aging.
N Engl J Med 279:626, 1968.
119. Fallon MD, Whyte MP, Teitelbaum SL: Systemic mastocytosis asso-
ciated with generalized osteopenia: Histopathological characteriza-
tion of the skeletal lesion using undecalcified bone from two patients.
Hum Pathol 12:813, 1981.
120. Chowdhury MH, Hamada C, Dempster DW: Effects of heparin on
osteoclast activity. J Bone Miner Res 7:771, 1992.
121. Kessler DA, Langer RS, Pless NA, et al: Mast cells and tumor angio-
genesis. Int J Cancer 18:703, 1976.
122. Heissig B, Rafii S, Akiyama H, et al: Low-dose irradiation promotes tis-
sue revascularization through VEGF release from mast cells and MMP-
9-mediated progenitor cell mobilization. J Exp Med 202:739, 2005.
123. Castor W: The microscopic structure of normal human synovial tis-
sue. Arthritis Rheum 3:140, 1960.
124. Shin K, Gurish MF, Friend DS, et al: Lymphocyte-independent con-
nective tissue mast cells populate murine synovium. Arthritis Rheum
54:2863, 2006.
125. Crisp A.J., Chapman C.M., Kirkham S.E., et al: Articular masto-
cytosis in rheumatoid arthritis. Arthritis Rheum 27:845, 1984.
126. Frewin DB, Cleland LG, Jonsson JR, et al: Histamine levels in human
synovial fluid. J Rheumatol 13:13, 1986.
127. Buckley MG, Walters C, Wong WM, et al: Mast cell activation in
arthritis: Detection of alpha- and beta-tryptase, histamine and eosino-
phil cationic protein in synovial fluid. Clin Sci (Lond) 93:363, 1997.
128. Bromley M, Fisher WD, Woolley DE: Mast cells at sites of cartilage
erosion in the rheumatoid joint. Ann Rheum Dis 43:76, 1984.
129. Malone DG, Irani AM, Schwartz LB, et al: Mast cell numbers and
histamine levels in synovial fluids from patients with diverse arthriti-
des. Arthritis Rheum 29:956, 1986.
130. Ceponis A, Konttinen YT, Takagi M, et al: Expression of stem cell
factor (SCF) and SCF receptor (c-kit) in synovial membrane in
arthritis: Correlation with synovial mast cell hyperplasia and inflam-
mation. J Rheumatol 25:2304, 1998.
131. Kiener HP, Hofbauer R, Tohidast-Akrad M, et al: Tumor necrosis
factor alpha promotes the expression of stem cell factor in synovial
fibroblasts and their capacity to induce mast cell chemotaxis. Arthri-
tis Rheum 43:164, 2000.
132. Malone DG, Wilder RL, Saavedra-Delgado AM, et al: Mast cell
numbers in rheumatoid synovial tissues: Correlations with quantita-
tive measures of lymphocytic infiltration and modulation by antiin-
flammatory therapy. Arthritis Rheum 30:130, 1987.
133. Gotis-Graham I, Smith MD, Parker A, et al: Synovial mast cell
responses during clinical improvement in early rheumatoid arthritis.
Ann Rheum Dis 57:664, 1998.
134. Firestein GS, Xu WD, Townsend K, et al: Cytokines in chronic
inflammatory arthritis, I: Failure to detect T cell lymphokines (inter-
leukin 2 and interleukin 3) and presence of macrophage colony-
stimulating factor (CSF-1) and a novel mast cell growth factor in
rheumatoid synovitis. J Exp Med 168:1573, 1988.
135. Fritz P, Muller J, Reiser H, et al: Distribution of mast cells in human
synovial tissue of patients with osteoarthritis and rheumatoid arthri-
tis. Z Rheumatol 43:294, 1984.
136. Kopicky-Burd JA, Kagey-Sobotka A, Peters SP, et al: Characteriza-
tion of human synovial mast cells. J Rheumatol 15:1326, 1988.
137. Buckley MG, Gallagher PJ, Walls AF: Mast cell subpopulations in
the synovial tissue of patients with osteoarthritis: Selective increase
in numbers of tryptase-positive, chymase-negative mast cells. J Pathol
186:67, 1998.
138. Lee DM, Friend DS, Gurish MF, et al: Mast cells: A cellular
link between autoantibodies and inflammatory arthritis. Science
297:1689, 2002.
139. Corr M, Crain B: The role of FcgammaR signaling in the K/B × N
serum transfer model of arthritis. J Immunol 169:6604, 2002.
140. Wipke BT, Wang Z, Nagengast W, et al: Staging the initiation of
autoantibody-induced arthritis: A critical role for immune com-
plexes. J Immunol 172:7694, 2004.
141. Binstadt BA, Patel PR, Alencar H, et al: Particularities of the vascu-
lature can promote the organ specificity of autoimmune attack. Nat
Immunol 7:284, 2006.
142. Nigrovic PA, Lee DM: Synovial mast cells: Role in acute and chronic
arthritis. Immunol Rev, 217:19-37, 2007.
143. Kiener HP, Baghestanian M, Dominkus M, et al: Expression of the
C5a receptor (CD88) on synovial mast cells in patients with rheuma-
toid arthritis. Arthritis Rheum 41:233, 1998.
144. Nigrovic PA, Lee DM: Immune complexes and innate immunity
in rheumatoid arthritis. In Firestein GS, Panayi GS, Wollheim FA
(eds): Rheumatoid Arthritis: New Frontiers in Pathogenesis and
Treatment. Oxford, Oxford University Press, 2006, p 135.
145. Metz M, Piliponsky AM, Chen CC, et al: Mast cells can enhance
resistance to snake and honeybee venoms. Science 313:526, 2006.
146. Hara M, Ono K, Hwang MW, et al: Evidence for a role of mast cells in
the evolution to congestive heart failure. J Exp Med 195:375, 2002.
147. Kanamaru Y, Scandiuzzi L, Essig M, et al: Mast cell-mediated remod-
eling and fibrinolytic activity protect against fatal glomerulonephri-
tis. J Immunol 176:5607, 2006.
148. Wershil BK, Murakami T, Galli SJ: Mast cell-dependent amplifica-
tion of an immunologically nonspecific inflammatory response: Mast
cells are required for the full expression of cutaneous acute inflam-
mation induced by phorbol 12-myristate 13-acetate. J Immunol
140:2356, 1988.
149. Ikai K, Danno K, Horio T, et al: Effect of ultraviolet irradiation on mast
cell-deficient W/Wv mice. J Invest Dermatol 85:82, 1985.
150. Higa A, Yoshida T, Tanaka K, et al: Natural resistance of W/Wv mice
to ethanol-induced gastric lesions and its abrogation by bone mar-
row grafting: Possible role of mast cells and LTC4. Gastroenterol Jpn
26:277, 1991.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
151. Matsuda H, Fukui K, Kiso Y, et al: Inability of genetically mast cell-
deficient W/Wv mice to acquire resistance against larval Haemaphy-
salis longicornis ticks. J Parasitol 71:443, 1985.
152. Timoshanko JR, Kitching R, Semple TJ, et al: A pathogenetic role for
mast cells in experimental crescentic glomerulonephritis. J Am Soc
Nephrol 17:150, 2006.
153. Kitamura Y, Taguchi T, Yokoyama M, et al: Higher susceptibility of
mast-cell-deficient W/WV mutant mice to brain thromboembolism
and mortality caused by intravenous injection of India ink. Am J
Pathol 122:469, 1986.
154. Lazarus B, Messina A, Barker JE, et al: The role of mast cells in isch-
aemia-reperfusion injury in murine skeletal muscle. J Pathol 191:443,
2000.
247
155. Abonia JP, Friend DS, Austen WG Jr, et al: Mast cell protease 5
mediates ischemia-reperfusion injury of mouse skeletal muscle.
J Immunol 174:7285, 2005.
156. Qureshi R, Jakschik BA: The role of mast cells in thioglycollate-
induced inflammation. J Immunol 141:2090, 1988.
157. Cahill RA, Wang JH, Soohkai S, et al: Mast cells facilitate local
VEGF release as an early event in the pathogenesis of postoperative
peritoneal adhesions. Surgery 140:108, 2006.
158. Takizawa H, Ohta K, Hirai K, et al: Mast cells are important in the
development of hypersensitivity pneumonitis: a study with mast-cell-
deficient mice. J Immunol 143:1982, 1989.
159. Starkey JR, Crowle PK, Taubenberger S: Mast-cell-deficient W/Wv mice
exhibit a decreased rate of tumor angiogenesis. Int J Cancer 42:48, 1988.
 14
KEY POINTS
Platelets are small circulating cytoplasmic fragments that play
a crucial role in hemostasis.
Platelets release a variety of factors that contribute to inflam-
mation, including chemotactic factors for leukocytes; factors
that alter vascular tone and permeability; and transforming
growth factor-β, a potent stimulus of fibrosis.
Platelet surface proteins also participate in inflammation by
serving as sites for leukocyte adhesion (e.g., P-selectin, Glyco-
protein Ibα [GPIbα]) or as agonists for counter-receptors on
leukocytes (e.g., CD40 ligand, platelet-activating factor).
Platelets have been implicated in the pathogenesis of several
rheumatic diseases, including rheumatoid arthritis and sys-
temic lupus erythematosus; in the latter case, they have been
particularly implicated in atherothrombotic complications.
The ongoing development of antiplatelet agents offers the
promise of new therapeutic modalities.
Platelets are small circulating cytoplasmic fragments that
play a crucial role in hemostasis. They are produced in the
bone marrow by megakaryocytes. Single platelets circulate
freely in the bloodstream; after vascular injury, platelets
adhere to the subendothelium resulting in responses that
contribute to the formation of the hemostatic plug. These
responses include aggregation, secretion of bioactive com-
pounds, and production of procoagulant activity. Platelets
also secrete soluble factors that contribute to wound repair
by altering vascular tone and permeability, promoting cell
growth, and stimulating scavenger cells such as monocytes.
During the inflammatory response, many of the activities
that lead to the hemostasis contribute to inflammation.1
Inflammation initiates clotting, decreasing the activity
of natural anticoagulant mechanisms and impairing the
fibrinolytic system. Conversely, activated platelets release
­chemotactic factors that promote leukocyte adhesion,
which facilitates their extravasation into inflammatory foci.
Platelets secrete a variety of factors that can alter vascular
tone and permeability. Lastly, platelets are a major source
of transforming growth factor (TGF)-β, a potent stimulus
of fibrosis. Taken together, these activities make platelets
­contributors to the inflammatory response and to the patho-
genesis of ­systemic rheumatic diseases.2 This chapter focuses
on platelet functions, particularly as they pertain to the
inflammatory response and rheumatic diseases.
Platelets and Rheumatic
Diseases
Federico Díaz-González  • 
Mark H. Ginsberg
GENERAL CHARACTERISTICS
OF PLATELETS
Platelets are the smallest blood cells; they are cytoplasmic
fragments derived from their bone marrow precursor, the
megakaryocyte. Resting platelets have a smooth disk shape
and are 3.6 ± 0.7 μm in diameter. On activation, platelets
undergo a shape change becoming a compact sphere with
numerous long dendritic extensions markedly increas-
ing their surface area. In humans, normal platelet counts
range from 150,000/μL to 450,000/μL. The main function
of platelets is to maintain the vascular integrity, playing a
crucial role in hemostasis.
The plasma membrane of platelets is a typical lipid
bilayer, having an extensive series of complex invagi-
nations termed the canalicular system. The role of this
surface-connected tubular system seems to be to facilitate
the quick release of secreted substances to the extracellu-
lar environment. The platelet membrane bears numerous
­glycoprotein (GP) receptors.3 Platelet surface phospholipids
play an important role in coagulation4 and are a source of
arachidonic acid, a precursor of important vasoactive sub-
stances such as thromboxane A2, a potent vasoconstrictor
and platelet-aggregating agent, and of leukotrienes, which
can amplify the inflammatory response. Platelet surface
GPs are receptors that mediate the adhesion to subendo-
thelial tissue and the subsequent aggregation to form the
hemostatic plug.5-7 The largest GP is termed I and the
smallest Ix. The a and b distinguish between two separate
electrophoretic bands that initially were considered one
(e.g., GPI became GPIa and GPIb). The platelet GPIb-IX-V
is an important receptor that binds to von Willebrand’s fac-
tor (vWF) exposed in the subendothelial matrix, causing the
attachment of platelets.8 Deficiency of any component of
the GPIb-IX-V complex or of vWF leads to the congenital
bleeding disorders Bernard-Soulier disease (GPIb-IX-V com-
plex)7 and von Willebrand’s disease (vWF).9 ADAMTS-13
(a disintegrin-like and metalloprotease with thrombospon-
din type I repeats 13) is a plasma protease that cleaves vWF
into smaller multimers, reducing its hemostatic potency.10
Mutations in the ADAMTS-13 gene11 and autoantibodies
against ADAMTS-1312,13 have been shown to cause famil-
ial and acquired thrombotic thrombocytopenic purpura. In
mice, ADAMTS-13 has a powerful natural antithrombotic
activity, and recombinant ADAMTS-13 has the potential
to be used as an antithrombotic agent.14
Other interactions that contribute to the initial plate-
let adhesion are mediated by collagen receptors GPIa-IIa
(integrin α2β1) and GPVI, which bind to collagen in the
subendothelial matrix.15 The most abundant platelet sur-
face receptor, GPIIb-IIIa (integrin αIIbβ3), is activated by
adhesion to collagen or vWF or by soluble agonists, such
249
 250 Díaz-González  |  Platelets and Rheumatic Diseases
as thrombin. After activation, GPIIb-IIIa binds fibrinogen,
leading to platelet aggregation.5 Deficiency of this GP results
in Glanzmann thrombasthenia, a disorder characterized by
petechial bleeding and the absence of platelet aggregation
and clot retraction.16
The cytoplasm of platelets is rich in actin and myosin,
which provide platelets the ability to change shape and to
retract clots. Platelet cytoplasm has mitochondria, lysosomes,
glycogen stores, and three types of granules that ­ contain
numerous biologically active molecules (Table 14-1). These
granules are classified according to their ultrastructure, den-
sity, and contents as alpha granules, lysosomes, and dense
granules. Although most of the contents of these granules
are made in megakaryocytes, some are taken up from the
plasma by megakaryocytes and platelets.
Alpha granules contain numerous proteins and growth
factors, such as platelet-derived growth factor (PDGF),
TGF-β, platelet factor-4 (also referred as CXCL4), and
vWF, which are synthesized in the megakaryocyte.17 Other
proteins, such as fibrinogen, enter the alpha granules from
the plasma via GPIIb-IIIa receptor-mediated endocytosis.18,19
P-selectin (CD62P), an adhesion molecule, also is ­localized
in the membrane of alpha granules20,21 and redistributes to
the cell surface during platelet activation. Platelet P-­selectin
has been implicated in stabilizing platelet ­ aggregates.22
The best documented high-affinity counter-receptor for
P-­selectin is P-selectin glycoprotein ligand-1 (PSGL-1),
a transmembrane sialomucin found on leukocytes and
­lymphoid cells,23 through whose interaction ­ platelets par-
ticipate in the inflammatory response.24
Dense granules contain serotonin, adenosine diphosphate
(ADP), adenosine triphosphate, and calcium. The dense
granule membrane bodies are made in megakaryocytes, but
they do not acquire their content of serotonin and calcium
until platelets are released into the circulation.25 Another
series of intracellular membrane vesicles serves as a reserve
to increase membrane surface area on platelet activation.
As stated previously, platelets are small cytoplasmic frag-
ments derived from megakaryocytes. Although megakaryo-
cytes are rare in the bone marrow (approximately 0.1% of
Table 14-1  Platelet Granule Compounds and   consequently, thrombopoietin concentrations increase
Granule Membrane Components with Role   and megakaryocyte growth is stimulated. In conditions of
in the Hemostatic/Inflammatory Response
Platelet Granules Actions Contents
Dense granule Proaggregating Serotonin, histamine, ADP,
factors ATP, Ca2+, Mg2+
Alpha granule Adhesive   P-selectin, CD31, GPIIb-IIIa,
glycoproteins fibronectin, vitronectin,
thrombospondin
Growth factors TGF-β, PDGF, EGF, VEGF
Platelet   β-Thromboglobulin, PF4
aggregation   (CXCL4), CC and CXC
and chemotaxis chemokines
Hemostasis factors Fibrinogen, vWF
Lysosome Tissue destruction Hydrolases, collagenase,
cathepsins D and E
ADP, adenosine diphosphate; ATP, adenosine triphosphate; EGF, epidermal
growth factor; GPIIb-IIIa, glycoprotein IIb-IIIa; PDGF, platelet-derived growth
factor; PF4 platelet factor-4; TGF, transforming growth factor; VEGF, vascular
endothelial growth factor; vWF, von Willebrand factor.
Modified from Rendu F, Brohard-Bohn B: The platelet release reaction:
Granules’ constituents, secretion and functions. Platelets 12:261, 2001.
all nucleated cells), megakaryocytes are easily recognized by
their giant size (50 to 100 μm diameter) and large, multilobed
nucleus. Megakaryocytes have two unique characteristics:
(1) They undergo a process known as endomitosis, in which
the nucleus accumulates many times the normal number of
chromosomes, and (2) they have specialized structures in
the cytoplasm that permit fragments to be shed, as platelets,
into the bloodsteam.26
Every day, about 2 × 1011 platelets are released into the
bloodsteam of healthy adults by mature megakaryocytes.
This quantity can be increased 10-fold under specific con-
ditions.27 In humans, as in other species, there is an inverse
relationship between the platelet count and the mean
platelet volume.28 This suggests that platelet production
by bone marrow megakaryocytes is regulated to maintain a
constant total platelet mass. The tendency toward a stable
platelet mass explains the wide variation in the platelet
count in healthy donors (150,000/μL to 450,000/μL).29
Megakaryocytes normally replace about 10% of the platelet
mass daily.30 In response to the increased need for platelets,
megakaryocytes modify their number, size, and ploidy.31,32
Changes in free thrombopoietin levels, the main physi-
ologic regulator of platelet production, are responsible for
these morphologic and functional adaptations in mega-
karyocytes.
Thrombopoietin is an 80- to 90-kD GP produced mainly
by the liver33 and released at a constant rate into the circula-
tion. Thrombopoietin acts through its receptor, the throm-
bopoietin receptor, also known as c-Mpl, which is present
in platelets, megakaryocytes, and, to a lesser extent, most
other hematopoietic precursor cells. Thrombopoietin pre-
vents apoptosis of megakaryocytes, while increasing their
number, size, and maturation,31,32 but it does not seem to
increase the rate of shedding of platelets into circulation.34
On circulating platelets, thrombopoietin is not a sufficiently
strong stimulus to trigger platelet function, but reduces the
threshold for activation by other agonists, such as ADP.35
Binding to the platelet thrombopoietin receptor is the major
route of catabolism, however, of circulating thrombopoietin.
When the platelet production rate decreases, the platelet
mass and the quantity of thrombopoietin receptor decrease;
high platelet mass (e.g., hypertransfusion of platelets), the
amount of thrombopoietin receptors increases, thrombo-
poietin concentrations decrease, and megakaryocyte growth
decreases. In addition to thrombopoietin, other soluble fac-
tors, such as interleukin (IL)-3 and IL-11, seem to promote
megakaryocyte maturation, and these cytokines may play a
relevant role in thrombocytosis conditions.36,37
The life span of platelets in circulation is 7 to 10 days.
Under normal conditions, the spleen stores about one third
of circulating platelets. Circumstances that increase splenic
volume, such as hepatic cirrhosis or portal hypertension,
cause a reduction in the circulating platelet count by a
sequestration within the splenic sinusoids.38 Hypersplenism
does not reduce platelet life span, however; rather, it reduces
the circulating platelets available for effective hemostasis.
After senescence, platelets are removed from circulation
by the reticuloendothelial system. Only a small fraction
of ­circulating platelets is consumed in forming hemostatic
plugs to maintain vascular integrity.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
FUNCTION OF PLATELETS
In response to vascular injury, platelets adhere to subendo-
thelium, secreting a variety of potent agonists and aggre-
gating to form a hemostatic plug. During the inflammatory
response, these physiologic responses of platelets can pro-
mote and exacerbate inflammation. In this sense, platelets
are authentic inflammatory cells.
HEMOSTASIS
When a blood vessel is injured, a complex process, which
involves biochemical reactions and cell-cell and cell-matrix
interactions, termed hemostasis, occurs. The initial hemo-
static response is mediated by platelets that form the plate-
let plug (Fig. 14-1).
Under physiologic conditions, the undamaged endo-
thelium prevents the adherence of platelets by several
mechanisms. These mechanisms include a cell-associated
ecto-ADPase (CD39) and the production of nitric oxide
and prostacyclin.39 When blood vessel integrity is disrupted,
the first reaction is vasoconstriction, which reduces blood
loss. Simultaneously, subendothelial matrix elements are
exposed, and platelets are rapidly transformed into sticky
cellular elements capable of adhering to the underlying
surface. Platelet adhesion is initially mediated by the inter-
action of GPIb-IX-V receptor complex with vWF in the
subendothelial matrix.8 This interaction transduces signals
through the GPIb-IX-V complex that activate platelet inte-
grins.40,41 The activation of GPIa-IIa and GPIIb-IIIa inte-
grins allows the binding to collagen (GPIa-IIa) and vWF
(GPIIb-IIIa), mediating the stable adhesion of platelets to
subendothelial surface. In addition to vWF, the active form of
GPIIb-IIIa binds fibrinogen.42 The association of soluble fibrin-
ogen with GPIIb-IIIa creates bridges between platelets that
result in platelet aggregation and thrombus growth. In concert
Amplification
Adhesion Secretion
Aggregation
Exposure of
subendothelial
matrix
Figure 14-1  Platelet plug formation. Platelet activation can be initiated
by several mechanical (vessel wall injury, disruption of atherosclerotic
plaques) or chemical (adenosine diphosphate, epinephrine, thrombox-
ane A2, thrombin) stimuli. In response to vessel wall injury, platelets at-
tach to subendothelial matrix (adhesion), which is followed by fibrinogen-
­mediated, platelet-platelet interaction (aggregation). Simultaneously,
platelets release their intracellular granule contents (secretion), which
leads to the recruitment of additional circulating platelets (amplification).
251
with aggregation, platelets release their intracellular gran-
ules, amplifying the hemostatic response (see Table 14-1).43,44
The outcome is the formation of a platelet plug and the trig-
gering of the coagulation cascade, which leads to thrombin
generation and resulting fibrin clot formation (Fig. 14-2).
One response of platelets to activation by stimuli such
as shear stress or collagen is the release of vesicles called
platelet microparticles, fragments 0.1 to 0.2 μm in diameter
that carry antigens present in intact platelets. These plate-
let-derived microparticles may play a role in normal hemo-
stasis.45,46 The number of clinical disorders associated with
elevated platelet microparticles is increasing,47,48 including
several rheumatic diseases.49-52 The relevance of platelet-
derived microparticles in the physiopathology of those dis-
orders needs to be fully clarified, however.
GLYCOPROTEIN IIb-IIIa
GPIIb-IIIa is a member of a family of cell-adhesion receptors
termed integrins. It also is referred to as integrin αIIbβ3 or
CD41/CD61. Although integrins are expressed on virtually
all nucleated cells, GPIIb-IIIa is restricted to megakaryo-
cytes and platelets. It is the most abundant receptor on the
platelet surface, averaging 80,000 copies per platelet. GPIIb-
IIIa recognizes at least five different adhesive ligands53: fibro-
nectin, fibrinogen, vWF, thrombospondin, and vitronectin.
Cells can modify integrin functions through dynamic modu-
lation of receptor affinity.53 On resting platelets, GPIIb-IIIa
does not bind soluble fibrinogen. After platelet stimulation
(e.g., by thrombin, collagen, or ADP), GPIIb-IIIa undergoes
a conformational change, however, and is converted from a
low-affinity to a high-affinity fibrinogen receptor, a process
known as “inside-out” signaling. In this situation, fibrino-
gen bridges the activated platelets, and platelet aggregation
occurs. Simultaneously, the cytosolic portion of the activated
GPIIb-IIIa binds to platelet cytoskeleton proteins and medi-
ates platelet spreading and clot retraction in what is referred
to as “outside-in” integrin signaling. GPIIb-IIIa integrates
receptor-ligand interactions on the external face of the
membrane with cytosolic events in a bidirectional fashion.5
This is the final common pathway for platelet aggregation,
P P
P d
P
EC
P
Figure 14-2  Anatomy of a platelet plug. Electron micrograph of a
group of platelets (P) attached to an endothelial cell (EC) in the initial
platelet plug formation. Several dense granules (d) and alpha granules
(α) are visible. The central platelet shows long dendritic extensions or 
filopodia (F). (Courtesy of Dr. Lucio Díaz-Flores.)
 252 Díaz-González  |  Platelets and Rheumatic Diseases
regardless of the mode of platelet stimulation. The impor-
tance of GPIIb-IIIa integrin is illustrated by Glanzmann
thrombasthenia, a bleeding disorder caused by mutations in
the gene for either the αIIb or the β3 subunit,16 and by the
clinical utility of GPIIb-IIIa antagonists as antithrombotic
agents in the treatment of thrombotic diseases.
ROLE OF PLATELETS IN THE INFLAMMATORY
RESPONSE
The accumulation of leukocytes in tissue is an essential
event for the inflammatory response. The current paradigm
of leukocyte extravasation requires a multistep cascade of
sequential leukocyte–endothelial cell interactions, in which
members of three different families of adhesion receptors
participate: selectins, integrins, and the immunoglobulin
superfamily.54 Platelets contribute in many ways to leuko-
cyte accumulation in the inflammatory foci (Table 14-2).
In flowing blood, leukocytes roll on adherent activated
platelets, mainly through the interaction of platelet P-
selectin with its major leukocyte ligand, PSGL-1.55-57
This initial rolling of leukocytes on platelet P-selectin
is followed by their firm adhesion and subsequent migra-
tion, processes that depend on the leukocyte integrin
Mac-1 (αMβ2, CD11b/CD18).57,58 Mac-1 adheres firmly
to platelets through direct binding to Glycoprotein Ibα
(GPIbα, CD42b).59 These interactions provide molecu-
lar mechanisms for leukocyte recruitment to hemostatic
plugs where platelets have been previously deposited in
response to vascular injury.60,61 Parallel lines of investi-
gation have shown that resting platelets are able to roll
on activated endothelial cells,62 apparently through an
interaction between PSGL-1 expressed in platelets and
the endothelial P-selectin.63 The physiologic function of
platelet rolling on stimulated endothelial cells needs to
be clarified. If this contact results in activation of plate-
lets, however, those platelets may release proinflamma-
tory mediators, such as cytokines, chemokines,64,65 and
eicosanoid precursors,66-68 or growth factors that stimulate
tissue healing. Activated platelets in circulation stimu-
late secretion of Weibel-Palade bodies from endothelial
cells in vivo, which leads to P-selectin-mediated leuko-
cyte rolling.69 Considering the important role of platelet
P-selectin in the chronic inflammatory processes,70,71 this
effect of activated platelets might represent an important
pathway of platelet-induced inflammation.
In addition to the adhesion molecules, activated platelets
express on their surface two major proinflammatory media-
tors: platelet-activating factor (PAF) and CD40 ligand
(CD154). PAF is a potent platelet aggregating phospholipid
produced by macrophages, mast cells, platelets, endothelial
cells, neutrophils, and monocytes. On cell activation, PAF
is rapidly synthesized and translocated to the plasma mem-
brane of endothelial cells, where it recognizes its receptor
in neutrophils, resulting in β2 integrin–mediated adhesion
of leukocytes to the endothelial surface.72 In the same way,
PAF can signal neutrophils when it is displayed on the sur-
face of adherent activated platelets acting in cooperation
with P-selectin to tether neutrophils.72 The biologic action
of PAF is physiologically inactivated by a plasma and cel-
lular acetylhydrolase.73 A role of PAF in the pathogenesis
of chronic inflammatory arthritis has been proposed74,75;
Table 14-2  Platelet Components Implicated  
in the Inflammatory Response
Platelet Component Actions
Surface molecules P-selectin (CD62-P), Adhesive targets for
PECAM (CD31), GPIbα leukocytes
PAF, ROS Neutrophil activation
CD154 (CD40 ligand) Agonist for  
endothelial cells
Soluble factors Serotonin, histamine Regulators of vascular
permeability
β-Thromboglobulin, PF4 Chemotaxis
Acid hydrolases, ROS Tissue destruction
PDGF, TGF-β Cellular mitogens,
chemoattractant
End products of Thrombin, fibrin Promote leukocyte
platelet proco- accumulation
agulant activity
GPI, glycosylphosphatidylinositol; PAF, platelet-activating factor; PDGF,
platelet-derived growth factor; PECAM, platelet-endothelial cell adhe-
sion molecule; PF4, platelet factor-4; ROS, reactive oxygen species; TGF,
transforming growth factor.
however, a well-controlled clinical trial failed to show any
beneficial effect of a PAF antagonist in patients with active
rheumatoid arthritis (RA).76
CD40 is a transmembrane protein member of the tumor
necrosis factor receptor family. CD40 is present on many
cells, including B cells, monocytes, macrophages, den-
dritic cells, and vascular endothelial cells.77 Platelets are
the major peripheral blood source of CD154, the ligand of
CD40, and they express it on their surface within seconds
of exposure to an agonist.78 The interaction of CD154 on
activated platelets with CD40 on endothelial cells causes a
proinflammatory reaction of the endothelium characterized
by the expression of inflammatory adhesion molecules, such
as E-selectin, vascular cell adhesion molecule-1 (CD106),
and intercellular adhesion molecule-1 (CD54), and the
secretion of the chemokines IL-8 (CXCL8) and monocyte
chemotactic protein-1 (CCL2).78 CD154 expressed on acti-
vated platelets can provide a potent stimulus to the inflam-
matory response. Clinical data suggest that the blockade of
CD154 may induce a prothrombotic state in patients with
lupus79 through a mechanism that needs to be clarified.
When platelets adhere, they release numerous growth
factors, such as PDGF, TGF-β, and other factors that are
chemotactic for monocytes, macrophages, and fibroblasts.
These growth factors may play an important role in the
chronic inflammatory response by mediating a fibroprolifer-
ative response. PDGF is a homodimer or heterodimer mol-
ecule of A and B chains80 produced by platelets, monocytes
or macrophages, endothelial cells, and vascular smooth
muscle cells (under some conditions). This molecule plays
an essential role in tissue repair and wound healing.81 PDGF
is a potent mitogen and chemoattractant for smooth muscle
cells, connective tissue cells, and macrophages,82-85 which
­contributes to the formation of lesions of atherosclerosis,85,86
a disorder strongly related to the inflammatory response.87
It has been shown that PDGF is a potent mitogen for syno-
vial fibroblasts isolated from patients with RA.88
TGF-β has three isoforms (TGF-β1, TGF-β2, and TGF-
β3) secreted by virtually all cell types as latent complexes
that need to be processed to exhibit biologic activity.89
Several effects have been associated with TGF-β: (1) It is
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
chemotactic for various cell types, including leukocytes;
(2) it inhibits proliferation of most cells; (3) it induces the
synthesis and deposition of extracellular matrix, and (4) it
stimulates the formation of granulation tissue.90 The net
result is that TGF-β is mainly an inhibitor of the inflamma-
tory response.91,92 The systemic administration of TGF-β1
has antagonized the development of polyarthritis in suscep-
tible rats.93 Carefully regulated expression of active TGF-β
is essential for resolution of inflammation and repair. Over-
production of this cytokine has been associated with several
fibrosis processes,94-96 including scleroderma.
Several reactive oxygen species are released from unstim-
ulated platelets and after platelet stimulation with agonists
such as collagen or thrombin.97,98 Because reactive oxygen
species have been implicated in direct tissue injury and in
inflammatory reactions through the promotion of adhesive
interactions between inflammatory and endothelial cells,99
reactive oxygen species originating from platelets may act as
an autocrine or paracrine mediator that participates in the
amplification of the inflammatory response in disorders such
as rheumatic diseases.
PLATELETS AND RHEUMATIC DISEASES
ALTERATIONS IN PLATELET NUMBERS
IN RHEUMATIC DISEASES
Increases in platelet counts have three major causes: (1)
reactive or secondary thrombocytosis; (2) familial thrombo-
cytosis; and (3) clonal thrombocytosis, including essential
thrombocythemia and related myeloproliferative disorders.
The platelet count is frequently elevated in patients with
active RA and juvenile chronic arthritis, owing to reactive
thrombocytosis. The level of thrombocytosis correlates
with clinical and laboratory parameters of disease activ-
ity. Relapses of RA are often accompanied by increases in
platelet count, whereas remissions are associated with their
reduction, to normal limits.100 This activity indicates that
the thrombocytosis observed in patients with rheumatic
disease is reactive or secondary to the chronic inflammatory
process. Although the mechanism involved in this throm-
bocytosis is uncertain, increased intravascular coagulation
with a compensatory increase in platelet production has
been suggested as a possible cause.101 More recently, several
studies have suggested that inflammatory cytokines with a
minor role in the physiologic production of platelets, such
as IL-6, IL-1, or tumor necrosis factor-α, among others,102-105
may be active mediators in the regulation of thrombopoi-
esis during the reactive thrombocytosis that occurs in the
inflammatory process.
Reduced platelet count, or thrombocytopenia, is ­common
in rheumatic diseases. The mechanisms involved in throm-
bocytopenic states are reduction in platelet ­ production,
sequestration, and rapid platelet destruction. Several drugs
used in rheumatic diseases are able to suppress the bone
marrow. Among drugs that can produce thrombocytopenia
because of megakaryocytic hypoplasia are gold, cyclophos-
phamide, methotrexate, penicillamine, and azathioprine.
The effect these compounds have on suppressing mega-
karyocyte replication depends on the time and dose of expo-
sure; reduced elimination of these drugs places patients at
increased risk for this complication.106
253
The normal spleen contains about 30% of the platelet
mass, and splenomegaly can result in a low circulating count
without reduction in the platelet life span.107 Several rheu-
matic diseases may lead to this type of thrombocytopenia.
The most well known is Felty’s syndrome, an uncommon
but severe subset of seropositive RA complicated by granu-
locytopenia and splenomegaly. In this disorder, thrombocy-
topenia usually is not life-threatening.
Another related disease is immune-mediated platelet
destruction,108 a disorder termed idiopathic thrombocytopenic
purpura. Autoantibodies cause idiopathic thrombocytope-
nic purpura, and platelet surface proteins, including GPIIb-
IIIa, GPIb-IX, GPIa-IIa, GPIV, and GPV can be antigenic
targets of such autoantibodies.109,110 Circulating platelets
coated with IgG autoantibodies undergo an accelerated
clearance through Fcγ receptors expressed by macrophages
in the spleen and liver. In some cases of idiopathic thrombo-
cytopenic purpura, platelet production seems to be reduced,
either by intramedullary destruction of antibody-coated
platelets or by the inhibition of megakaryocytopoiesis.111
The level of thrombopoietin is not increased,112 suggesting a
normal megakaryocyte mass. Idiopathic thrombocytopenic
purpura is present in 15% to 25% of patients with systemic
lupus erythematosus113 and in about 25% of patients with
antiphospholipid syndrome.114 The outcome in these cases
is rarely severe.
In contrast, the thrombocytopenia that occurs during
episodes of systemic vasculitis has a more complex patho-
genesis, a worse clinical course, and a poorer outcome.115,116
Immune thrombocytopenia is rare in RA except when related
to therapy. Among the drugs that can produce thrombocy-
topenia in RA, intramuscular gold salts are the most clearly
associated with drug-induced immune thrombocytopenia.
About 1% to 3% of patients receiving intramuscular gold
salts for the treatment of RA develop a thrombocytopenia,
which may be life-threatening. Although, as stated previ-
ously, bone marrow suppression can occur in patients under-
going gold treatment, thrombocytopenia is usually due to
immune destruction of platelets associated with an active
marrow.117,118
ROLE OF PLATELETS IN THE PATHOGENESIS
OF RHEUMATIC DISEASES
The role that platelets play in the amplification of the
inflammatory response provides a basis for their involve-
ment in rheumatic diseases. Most of the available evidence
implicating platelets in the pathogenesis of rheumatic disor-
ders is indirect and circumstantial, however.
Platelets have been implicated in the pathogenesis of
RA2 based on several studies that have documented the
presence of platelets in the synovial fluid of RA patients119,120
and mainly on the observation that labeled platelets localize
only to joints with clinically active inflammation.121 Levels
of plasma-soluble P-selectin are increased in RA patients
compared with controls,122 indicating platelet activation in
this disease. A direct correlation has been described between
platelet-derived microparticle levels and disease activity in
RA patients, which suggests that generation of platelet mi­
cro­particles49 contributes to the pathogenesis of RA. Several
studies have focused on the presence of activated platelets
in patients with systemic lupus erythematosus.123-125 The risk
 254 Díaz-González  |  Platelets and Rheumatic Diseases
for thrombosis is increased significantly in these patients,  
and platelets have been implicated in the prothrombotic
 
state of systemic lupus erythematosus through the release of
microparticles52 and by the increased deposition of comple-  
ment-activation product C4d on platelet surface.126 Patients
with essential thrombocythemia have an increased preva-  
lence of antiphospholipid antibodies, which may be asso-  
ciated with a higher risk of thrombosis.127 The presence of
activated platelets and enhanced aggregation of platelets
have been described in patients with antiphospholipid
syndrome,124 systemic sclerosis,128,129 and primary Raynaud’s
phenomenon.128
INHIBITION OF PLATELET FUNCTION
BY PHARMACOLOGIC AGENTS
Nonsteroidal anti-inflammatory drugs (NSAIDs) are a
foundation of therapy in many rheumatic diseases. NSAIDs
inhibit prostaglandin synthesis130 through the blockade of
cyclooxygenase. These agents can interfere with platelet
aggregation and secretion131,132 through the inactivation
of platelet cyclooxygenase-1. In platelets, this enzyme is a
rate-limiting step in the transformation of arachidonic acid
into thromboxane A2, a potent platelet-aggregating agent.
In addition, some NSAIDs reduce platelet aggregation by
interfering with the activation of GPIIb-IIIa, through a
cyclooxygenase-independent mechanism.133 NSAIDs inhibit
platelet function and can lead to bleeding complications in
patients with rheumatic diseases.
The development of potent new antithrombotic agents
also may provide new weapons in the treatment of rheumatic
diseases. Among these are ticlopidine and its analogue, clop-
idogrel, two inhibitors of the of the P2Y12 ADP receptor.
These agents have greater efficacy than aspirin for prevention
of recurrent stroke134 and may find a place in the antirheu-
matic armamentarium as a result of their anti-inflamma-
tory properties.135,136 Agents that interfere directly with the
adhesive function of integrin GPIIb-IIIa137 have come into
therapeutic use. This new group of agents includes mono-
clonal antibodies, peptides, and other small molecules that
have been approved for intravenous coronary angioplasty
and stent procedures. Orally active GPIIb-IIIa blockers have
been developed for long-term therapy, including secondary
and even primary prevention of thrombotic diseases. Avail-
able data from the clinical trials of these oral agents have
failed to show clinical benefits, however, whereas they have
shown unexplained increased mortality.138
Acknowledgments
This work was supported by grants from the NIH and FIS FIS04/1275 from
Instituto de Salud Carlos III of Spain.
REFERENCES
  1. Esmon CT: The interactions between inflammation and coagulation.
Br J Haematol 131:417, 2005.
  2. Ginsberg MH: Role of platelets in inflammation and rheumatic dis-
ease. Adv Inflamm Res 2:53, 1986.
  3. Isenberg WH, Bainton DF: Megakaryocyte and platelet structure. In
Hoffman R, Benz EJ, Shattil SJ, et al (eds): Hematology: Basic Prin-
ciples and Practice. New York, Churchill Livingstone, 1994, p 1516.
  4. Shattil SJ, Bennett JS: Platelets and their membranes in hemostasis:
Physiology and pathophysiology. Ann Intern Med 94:108, 1981.
5. Shattil SJ, Kashiwagi H, Pampori N: Integrin signaling: The platelet
paradigm. Blood 91:2645, 1998.
6. Santoro SA, Zutter MM: The alpha 2 beta 1 integrin: A collagen
receptor on platelets and other cells. Thromb Haemost 74:813, 1995.
7. Lopez JA, Andrews RK, Afshar-Kharghan V, et al: Bernard-Soulier
syndrome. Blood 91:4397, 1998.
8. Clemetson KJ: Platelet GPIb-V-IX complex. Thromb Haemost
78:266, 1997.
9. Schneppenheim R, Budde U, Ruggeri ZM: A molecular approach to
the classification of von Willebrand disease. Best Pract Res Clin Hae-
matol 14:281, 2001.
10. Chung DW, Fujikawa K: Processing of von Willebrand factor by
ADAMTS-13. Biochemistry 41:11065, 2002.
11. Levy GG, Nichols WC, Lian EC, et al: Mutations in a member of the
ADAMTS gene family cause thrombotic thrombocytopenic purpura.
Nature 413:488, 2001.
12. Tsai HM, Lian EC: Antibodies to von Willebrand factor-cleaving
protease in acute thrombotic thrombocytopenic purpura. N Engl J
Med 339:1585, 1998.
13. Scheiflinger F, Knobl P, Trattner B, et al: Nonneutralizing IgM and IgG
antibodies to von Willebrand factor-cleaving protease (ADAMTS-13)
in a patient with thrombotic thrombocytopenic purpura. Blood
102:3241, 2003.
14. Chauhan AK, Motto DG, Lamb CB, et al: Systemic antithrombotic
effects of ADAMTS13. J Exp Med 203:767, 2006.
15. Sixma JJ, van Zanten GH, Huizinga EG, et al: Platelet adhesion to
collagen: An update. Thromb Haemost 78:434, 1997.
16. Tomiyama Y: Glanzmann thrombasthenia: Integrin alpha IIb beta 3
deficiency. Int J Hematol 72:448, 2000.
17. Greenberg SM, Kuter DJ, Rosenberg RD: In vitro stimulation of
megakaryocyte maturation by megakaryocyte stimulatory factor.
J Biol Chem 262:3269, 1987.
18. Handagama PJ, Amrani DL, Shuman MA: Endocytosis of fibrino-
gen into hamster megakaryocyte alpha granules is dependent on a
dimeric gamma A configuration. Blood 85:1790, 1995.
19. Harrison P, Wilbourn B, Debili N, et al: Uptake of plasma fibrino-
gen into the alpha granules of human megakaryocytes and platelets.
J Clin Invest 84:1320, 1989.
20. Stenberg PE, McEver RP, Shuman MA, et al: A platelet alpha-gran-
ule membrane protein (GMP-140) is expressed on the plasma mem-
brane after activation. J Cell Biol 101:880, 1985.
21. Berman CL, Yeo EL, Wencel-Drake JD, et al: A platelet alpha
granule membrane protein that is associated with the plasma
membrane after activation: Characterization and subcellular
localization of platelet activation-dependent granule-external
membrane protein. J Clin Invest 78:130, 1986.
22. Smyth SS, Reis ED, Zhang W, et al: Beta(3)-integrin-deficient mice
but not P-selectin-deficient mice develop intimal hyperplasia after
vascular injury: Correlation with leukocyte recruitment to adherent
platelets 1 hour after injury. Circulation 103:2501, 2001.
23. Norman KE, Moore KL, McEver RP, et al: Leukocyte rolling
in vivo is mediated by P-selectin glycoprotein ligand-1. Blood
86:4417, 1995.
24. McEver RP: Adhesive interactions of leukocytes, platelets, and the
vessel wall during hemostasis and inflammation. Thromb Haemost
86:746, 2001.
25. Schick PK, Weinstein M: A marker for megakaryocytes: Serotonin accu-
mulation in guinea pig megakaryocytes. J Lab Clin Med 98:607, 1981.
26. Cramer EM: Megakaryocyte structure and function. Curr Opin
Hematol 6:354, 1999.
27. Kaushansky K: Regulation of megakaryopoiesis. In Loscalzo J,
Schafer A (eds): Thrombosis and Hemorrhage. Baltimore, Williams &
Wilkins, 1998, p 173.
28. Bessman JD, Williams LJ, Gilmer PR Jr: Mean platelet volume: The
inverse relation of platelet size and count in normal subjects, and an
artifact of other particles. Am J Clin Pathol 76:289, 1981.
29. Giles C: The platelet count and mean platelet volume. Br J Haema-
tol 48:31, 1981.
30. Gurney AL, Carver-Moore K, de Sauvage FJ, et al: Thrombocy­
topenia in c-mpl-deficient mice. Science 265:1445, 1994.
31. Penington DG, Olsen TE: Megakaryocytes in states of altered plate-
let production: Cell numbers, size and DNA content. Br J Haematol
18:447, 1970.
32. Kuter DJ, Rosenberg RD: Regulation of megakaryocyte ploidy in vivo
in the rat. Blood 75:74, 1990.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
33. Nomura S, Ogami K, Kawamura K, et al: Cellular localization of
thrombopoietin mRNA in the liver by in situ hybridization. Exp
Hematol 25:565, 1997.
34. Choi ES, Hokom MM, Chen JL, et al: The role of megakaryocyte
growth and development factor in terminal stages of thrombopoiesis.
Br J Haematol 95:227, 1996.
35. Akkerman JW: Thrombopoietin and platelet function. Semin
Thromb Hemost 32:295, 2006.
36. Kobayashi S, Teramura M, Hoshino S, et al: Circulating megakaryo-
cyte progenitors in myeloproliferative disorders are hypersensitive to
interleukin-3. Br J Haematol 83:539, 1993.
37. Cairo MS, Plunkett JM, Nguyen A, et al: Effect of interleukin-11
with and without granulocyte colony-stimulating factor on in vivo
neonatal rat hematopoiesis: Induction of neonatal thrombocytosis by
interleukin-11 and synergistic enhancement of neutrophilia by inter-
leukin-11 + granulocyte colony-stimulating factor. Pediatr Res 34:56,
1993.
38. Aster RH: Pooling of platelets in the spleen: Role in the pathogenesis
of “hypersplenic” thrombocytopenia. J Clin Invest 45:645, 1966.
39. Marcus AJ, Safier LB, Broekman MJ, et al: Thrombosis and inflam-
mation as multicellular processes: Significance of cell-cell interac-
tions. Thromb Haemost 74:213, 1995.
40. Zaffran Y, Meyer SC, Negrescu E, et al: Signaling across the platelet
adhesion receptor glycoprotein Ib-IX induces alpha IIbbeta 3 activa-
tion both in platelets and a transfected Chinese hamster ovary cell
system. J Biol Chem 275:16779, 2000.
41. Yap CL, Hughan SC, Cranmer SL, et al: Synergistic adhesive interac-
tions and signaling mechanisms operating between platelet glycopro-
tein Ib/IX and integrin alpha IIbbeta 3: Studies in human platelets
and transfected Chinese hamster ovary cells. J Biol Chem 275:41377,
2000.
42. Savage B, Saldivar E, Ruggeri ZM: Initiation of platelet adhesion
by arrest onto fibrinogen or translocation on von Willebrand fac-
tor. Cell 84:289, 1996.
43. Kroll MH, Schafer AI: Biochemical mechanisms of platelet activa-
tion. Blood 74:1181, 1989.
44. Harrison P, Savidge GF, Cramer EM: The origin and physiological
relevance of alpha-granule adhesive proteins. Br J Haematol 74:125,
1990.
45. Sims PJ, Wiedmer T, Esmon CT, et al: Assembly of the platelet pro-
thrombinase complex is linked to vesiculation of the platelet plasma
membrane: Studies in Scott syndrome: an isolated defect in platelet
procoagulant activity. J Biol Chem 264:17049, 1989.
46. Castaman G, Yu-Feng L, Battistin E, et al: Characterization of a
novel bleeding disorder with isolated prolonged bleeding time and
deficiency of platelet microvesicle generation. Br J Haematol 96:458,
1997.
47. Jy W, Horstman LL, Arce M, et al: Clinical significance of platelet
microparticles in autoimmune thrombocytopenias. J Lab Clin Med
119:334, 1992.
48. Nomura S, Suzuki M, Katsura K, et al: Platelet-derived micropar-
ticles may influence the development of atherosclerosis in diabetes
mellitus. Atherosclerosis 116:235, 1995.
49. Knijff-Dutmer EA, Koerts J, Nieuwland R, et al: Elevated levels of
platelet microparticles are associated with disease activity in rheuma-
toid arthritis. Arthritis Rheum 46:1498, 2002.
50. Nomura S, Yanabu M, Fukuroi T, et al: Anti-phospholipid antibodies
bind to platelet microparticles in idiopathic (autoimmune) thrombo-
cytopenic purpura. Ann Hematol 65:46, 1992.
51. Brogan PA, Shah V, Brachet C, et al: Endothelial and platelet mic-
roparticles in vasculitis of the young. Arthritis Rheum 50:927, 2004.
52. Pereira J, Alfaro G, Goycoolea M, et al: Circulating platelet-derived
microparticles in systemic lupus erythematosus: Association with
increased thrombin generation and procoagulant state. Thromb Hae-
most 95:94, 2006.
53. Ginsberg MH, Partridge A, Shattil SJ: Integrin regulation. Curr Opin
Cell Biol 17(5): 509-516, 2005.
54. Springer TA: Traffic signals for lymphocyte recirculation and leuko-
cyte emigration: The multistep paradigm. Cell 76:301, 1994.
55. Buttrum SM, Hatton R, Nash GB: Selectin-mediated rolling of neu-
trophils on immobilized platelets. Blood 82:1165, 1993.
56. Yeo EL, Sheppard JA, Feuerstein IA: Role of P-selectin and leukocyte
activation in polymorphonuclear cell adhesion to surface adherent
activated platelets under physiologic shear conditions (an injury ves-
sel wall model). Blood 83:2498, 1994.
255
57. Diacovo TG, Roth SJ, Buccola JM, et al: Neutrophil rolling, arrest,
and transmigration across activated, surface-adherent platelets via
sequential action of P-selectin and the beta 2-integrin CD11b/
CD18. Blood 88:146, 1996.
58. Evangelista V, Manarini S, Rotondo S, et al: Platelet/polymorpho-
nuclear leukocyte interaction in dynamic conditions: Evidence of
adhesion cascade and cross talk between P-selectin and the beta 2
integrin CD11b/CD18. Blood 88:4183, 1996.
59. Simon DI, Chen Z, Xu H, et al: Platelet glycoprotein Ibalpha is a
counterreceptor for the leukocyte integrin Mac-1 (CD11b/CD18).
J Exp Med 192:193, 2000.
60. Rinder CS, Bonan JL, Rinder HM, et al: Cardiopulmonary bypass
induces leukocyte-platelet adhesion. Blood 79:1201, 1992.
61. Rogers C, Edelman ER, Simon DI: A mAb to the beta2-leukocyte
integrin Mac-1 (CD11b/CD18) reduces intimal thickening after
angioplasty or stent implantation in rabbits. Proc Natl Acad Sci U S A
95:10134, 1998.
62. Frenette PS, Johnson RC, Hynes RO, et al: Platelets roll on stimu-
lated endothelium in vivo: An interaction mediated by endothelial
P-selectin. Proc Natl Acad Sci U S A 92:7450, 1995.
63. Frenette PS, Denis CV, Weiss L, et al: P-Selectin glycoprotein ligand
1 (PSGL-1) is expressed on platelets and can mediate platelet-endo-
thelial interactions in vivo. J Exp Med 191:1413, 2000.
64. Schenk BI, Petersen F, Flad HD, et al: Platelet-derived chemokines
CXC chemokine ligand (CXCL)7, connective tissue-activating pep-
tide III, and CXCL4 differentially affect and cross-regulate neutrophil
adhesion and transendothelial migration. J Immunol 169:2602, 2002.
65. Schober A, Manka D, von Hundelshausen P, et al: Deposition of
platelet RANTES triggering monocyte recruitment requires P-selectin
and is involved in neointima formation after arterial injury. Circula-
tion 106:1523, 2002.
66. Marcus AJ, Hajjar DP: Vascular transcellular signaling. J Lipid Res
34:2017, 1993.
67. Serhan CN, Haeggstrom JZ, Leslie CC: Lipid mediator networks in cell
signaling: Update and impact of cytokines. FASEB J 10:1147, 1996.
68. Marcus AJ, Broekman MJ, Safier LB, et al: Formation of leukotrienes
and other hydroxy acids during platelet-neutrophil interactions in
vitro. Biochem Biophys Res Commun 109:130, 1982.
69. Dole VS, Bergmeier W, Mitchell HA, et al: Activated platelets
induce Weibel-Palade-body secretion and leukocyte rolling in vivo:
Role of P-selectin. Blood 106:2334, 2005.
70. Huo Y, Schober A, Forlow SB, et al: Circulating activated platelets
exacerbate atherosclerosis in mice deficient in apolipoprotein E. Nat
Med 9:61, 2003.
71. Burger PC, Wagner DD: Platelet P-selectin facilitates atherosclerotic
lesion development. Blood 101:2661, 2003.
72. Zimmerman GA, McIntyre TM, Prescott SM: Adhesion and signal-
ing in vascular cell-cell interactions. J Clin Invest 98:1699, 1996.
73. Prescott SM: Inflammatory actions of platelet-activating factor: Con-
trol by PAF acetylhydrolase. J Invest Allergol Clin Immunol 7:416,
1997.
74. Gutierrez S, Palacios I, Egido J, et al: IL-1 beta and IL-6 stimulate
the production of platelet-activating factor (PAF) by cultured rabbit
synovial cells. Clin Exp Immunol 99:364, 1995.
75. Palacios I, Miguelez R, Sanchez-Pernaute O, et al: A platelet activat-
ing factor receptor antagonist prevents the development of chronic
arthritis in mice. J Rheumatol 26:1080, 1999.
76. Hilliquin P, Chermat-Izard V, Menkes CJ: A double blind, placebo
controlled study of a platelet activating factor antagonist in patients
with rheumatoid arthritis. J Rheumatol 25:1502, 1998.
77. Karmann K, Hughes CC, Schechner J, et al: CD40 on human endothe-
lial cells: Inducibility by cytokines and functional regulation of adhe-
sion molecule expression. Proc Natl Acad Sci U S A 92:4342, 1995.
78. Henn V, Slupsky JR, Grafe M, et al: CD40 ligand on activated
platelets triggers an inflammatory reaction of endothelial cells.
Nature 391:591, 1998.
79. Boumpas DT, Furie R, Manzi S, et al: A short course of BG9588 (anti-
CD40 ligand antibody) improves serologic activity and decreases
hematuria in patients with proliferative lupus glomerulonephritis.
Arthritis Rheum 48:719, 2003.
80. Hart CE, Bailey M, Curtis DA, et al: Purification of PDGF-AB and
PDGF-BB from human platelet extracts and identification of all
three PDGF dimers in human platelets. Biochemistry 29:166, 1990.
81. Pierce GF, Mustoe TA, Altrock BW, et al: Role of platelet-derived
growth factor in wound healing. J Cell Biochem 45:319, 1991.
 256 Díaz-González  |  Platelets and Rheumatic Diseases
82. Ross R, Raines EW, Bowen-Pope DF: The biology of platelet-
derived growth factor. Cell 46:155, 1986.
83. Grotendorst GR, Chang T, Seppa HE, et al: Platelet-derived growth
factor is a chemoattractant for vascular smooth muscle cells. J Cell
Physiol 113:261, 1982.
84. Deuel TF, Senior RM, Huang JS, et al: Chemotaxis of monocytes and
neutrophils to platelet-derived growth factor. J Clin Invest 69:1046,
1982.
85. Ross R, Masuda J, Raines EW, et al: Localization of PDGF-B protein
in macrophages in all phases of atherogenesis. Science 248:1009,
1990.
86. Wilcox JN, Smith KM, Williams LT, et al: Platelet-derived growth
factor mRNA detection in human atherosclerotic plaques by in situ
hybridization. J Clin Invest 82:1134, 1988.
87. Ridker PM, Rifai N, Rose L, et al: Comparison of C-reactive protein
and low-density lipoprotein cholesterol levels in the prediction of
first cardiovascular events. N Engl J Med 347:1557, 2002.
88. Pohlers D, Huber R, Ukena B, et al: Expression of platelet-derived
growth factors C and D in the synovial membrane of patients with
rheumatoid arthritis and osteoarthritis. Arthritis Rheum 54:788,
2006.
89. Lawrence DA: Transforming growth factor-beta: A general review.
Eur Cytokine Netw 7:363, 1996.
90. Wahl SM: Transforming growth factor-β (TGF-β) in the resolution
and repair of inflammation. In Gallin JI, Snyderman R (eds): Inflam-
mation: Basic Principles and Clinical Correlates, 3rd ed. Philadel-
phia, Lippincott Williams & Wilkins, 1999, p 883.
91. McCartney-Francis NL, Mizel DE, Redman RS, et al: Autoimmune
Sjogren’s-like lesions in salivary glands of TGF-beta1-deficient mice
are inhibited by adhesion-blocking peptides. J Immunol 157:1306,
1996.
92. Christ M, McCartney-Francis NL, Kulkarni AB, et al: Immune
dysregulation in TGF-beta 1-deficient mice. J Immunol 153:1936,
1994.
93. Brandes ME, Allen JB, Ogawa Y, et al: Transforming growth fac-
tor beta 1 suppresses acute and chronic arthritis in experimental
animals. J Clin Invest 87:1108, 1991.
94. Bernasconi P, Torchiana E, Confalonieri P, et al: Expression of trans-
forming growth factor-beta 1 in dystrophic patient muscles correlates
with fibrosis: Pathogenetic role of a fibrogenic cytokine. J Clin Invest
96:1137, 1995.
95. Border WA, Noble NA, Yamamoto T, et al: Natural inhibitor of
transforming growth factor-beta protects against scarring in experi-
mental kidney disease. Nature 360:361, 1992.
96. Varga J: Scleroderma and Smads: Dysfunctional Smad family dynam-
ics culminating in fibrosis. Arthritis Rheum 46:1703, 2002.
97. Caccese D, Pratico D, Ghiselli A, et al: Superoxide anion and
hydroxyl radical release by collagen-induced platelet aggregation—
role of arachidonic acid metabolism. Thromb Haemost 83:485,
2000.
98. Wachowicz B, Olas B, Zbikowska HM, et al: Generation of reactive
oxygen species in blood platelets. Platelets 13:175, 2002.
99. Nagata M: Inflammatory cells and oxygen radicals. Curr Drug Targets
Inflamm Allergy 4:503, 2005.
100. Hutchinson RM, Davis P, Jayson MI: Thrombocytosis in rheumatoid
arthritis. Ann Rheum Dis 35:138, 1976.
101. Ehrenfeld M, Penchas S, Eliakim M: Thrombocytosis in rheuma-
toid arthritis: Recurrent arterial thromboembolism and death. Ann
Rheum Dis 36:579, 1977.
102. Wang JC, Chen C, Novetsky AD, et al: Blood thrombopoietin lev-
els in clonal thrombocytosis and reactive thrombocytosis. Am J Med
104:451, 1998.
103. Dan K, Gomi S, Inokuchi K, et al: Effects of interleukin-1 and tumor
necrosis factor on megakaryocytopoiesis: Mechanism of reactive
thrombocytosis. Acta Haematol 93:67, 1995.
104. Hsu HC, Tsai WH, Jiang ML, et al: Circulating levels of thrombopoi-
etic and inflammatory cytokines in patients with clonal and reactive
thrombocytosis. J Lab Clin Med 134:392, 1999.
105. Ertenli I, Kiraz S, Ozturk MA, et al: Pathologic thrombopoiesis of
rheumatoid arthritis. Rheumatol Int 23:49, 2003.
106. Calvo-Romero JM: Severe pancytopenia associated with low-dose
methotrexate therapy for rheumatoid arthritis. Ann Pharmacother
35:1575, 2001.
107. Warkentin TE, Trimble MS, Kelton JG: Thrombocytopenia due
to platelet destruction and hypersplenism. In Hoffman R, Benz EJ,
Shattil SF, et al (eds): Hematology: Basic Principles and Practice.
New York, Churchill Livingstone, 1995, p 1889.
108. Cines DB, Blanchette VS: Immune thrombocytopenic purpura.
N Engl J Med 346:995, 2002.
109. McMillan R: Autoantibodies and autoantigens in chronic immune
thrombocytopenic purpura. Semin Hematol 37:239, 2000.
110. He R, Reid DM, Jones CE, et al: Spectrum of Ig classes, specificities,
and titers of serum antiglycoproteins in chronic idiopathic thrombo-
cytopenic purpura. Blood 83:1024, 1994.
111. Gernsheimer T, Stratton J, Ballem PJ, et al: Mechanisms of response
to treatment in autoimmune thrombocytopenic purpura. N Engl J
Med 320:974, 1989.
112. Emmons RV, Reid DM, Cohen RL, et al: Human thrombopoietin
levels are high when thrombocytopenia is due to megakaryocyte defi-
ciency and low when due to increased platelet destruction. Blood
87:4068, 1996.
113. Gladman DD, Urowitz MB, Tozman EC, et al: Haemostatic abnor-
malities in systemic lupus erythematosus. QJM 52:424, 1983.
114. Cuadrado MJ, Hughes GR: Hughes (antiphospholipid) syndrome:
Clinical features. Rheum Dis Clin North Am 27:507, 2001.
115. Pistiner M, Wallace DJ, Nessim S, et al: Lupus erythematosus in the
1980s: A survey of 570 patients. Semin Arthritis Rheum 21:55, 1991.
116. Reveille JD, Bartolucci A, Alarcon GS: Prognosis in systemic lupus
erythematosus: Negative impact of increasing age at onset, black
race, and thrombocytopenia, as well as causes of death. Arthritis
Rheum 33:37, 1990.
117. Adachi JD, Bensen WG, Kassam Y, et al: Gold induced thrombo-
cytopenia: 12 cases and a review of the literature. Semin Arthritis
Rheum 16:287, 1987.
118. von dem Borne AE, Pegels JG, van der Stadt RJ, et al: Thrombocy-
topenia associated with gold therapy: A drug-induced autoimmune
­disease? Br J Haematol 63:509, 1986.
119. Farr M, Wainwright A, Salmon M, et al: Platelets in the synovial fluid
of patients with rheumatoid arthritis. Rheumatol Int 4:13, 1984.
120. Endresen GK: Evidence for activation of platelets in the synovial
fluid from patients with rheumatoid arthritis. Rheumatol Int 9:19,
1989.
121. Farr M, Scott DL, Constable TJ, et al: Thrombocytosis of active
rheumatoid disease. Ann Rheum Dis 42:545, 1983.
122. Ertenli I, Kiraz S, Arici M, et al: P-selectin as a circulating molecu-
lar marker in rheumatoid arthritis with thrombocytosis. J Rheumatol
25:1054, 1998.
123. Nagahama M, Nomura S, Ozaki Y, et al: Platelet activation mark-
ers and soluble adhesion molecules in patients with systemic lupus
erythematosus. Autoimmunity 33:85, 2001.
124. Joseph JE, Harrison P, Mackie IJ, et al: Increased circulating plate-
let-leucocyte complexes and platelet activation in patients with
antiphospholipid syndrome, systemic lupus erythematosus and rheu-
matoid arthritis. Br J Haematol 115:451, 2001.
125. Ekdahl KN, Bengtsson AA, Andersson J, et al: Thrombotic disease
in systemic lupus erythematosus is associated with a maintained sys-
temic platelet activation. Br J Haematol 125:74, 2004.
126. Navratil JS, Manzi S, Kao AH, et al: Platelet C4d is highly specific
for systemic lupus erythematosus. Arthritis Rheum 54:670, 2006.
127. Harrison CN, Donohoe S, Carr P, et al: Patients with essential
thrombocythaemia have an increased prevalence of antiphospho-
lipid antibodies which may be associated with thrombosis. Thromb
Haemost 87:802, 2002.
128. Silveri F, De Angelis R, Poggi A, et al: Relative roles of endothelial
cell damage and platelet activation in primary Raynaud’s phenome-
non (RP) and RP secondary to systemic sclerosis. Scand J Rheumatol
30:290, 2001.
129. Chiang TM, Takayama H, Postlethwaite AE: Increase in platelet
non-integrin type I collagen receptor in patients with systemic scle-
rosis. Thromb Res 117:299, 2006.
130. Vane JR: Inhibition of prostaglandin synthesis as a mechanism of
nonsteroidal antiinflammatory drugs. Nature 231:232, 1971.
131. O’Brien JR: Effect of anti-inflammatory agents on platelets. ­Lancet
1:894, 1968.
132. McQueen EG, Facoory B, Faed JM: Non-steroidal anti-inflammatory
drugs and platelet function. N Z Med J 99:358, 1986.
 PART 2  |  CELLS INVOLVED IN AUTOIMMUNE DISEASES AND INFLAMMATION
133. Dominguez-Jimenez C, Diaz-Gonzalez F, Gonzalez-Alvaro I, et al:
Prevention of alphaII(b)beta3 activation by non-steroidal antiin-
flammatory drugs. FEBS Lett 446:318, 1999.
134. Sharis PJ, Cannon CP, Loscalzo J: The antiplatelet effects of ticlopi-
dine and clopidogrel. Ann Intern Med 129:394, 1998.
135. Graff J, Harder S, Wahl O, et al: Anti-inflammatory effects of clopido-
grel intake in renal transplant patients: Effects on platelet-leukocyte
interactions, platelet CD40 ligand expression, and proinflammatory
biomarkers. Clin Pharmacol Ther 78:468, 2005.
257
136. Heitzer T, Rudolph V, Schwedhelm E, et al: Clopidogrel improves
systemic endothelial nitric oxide bioavailability in patients with cor-
onary artery disease: Evidence for antioxidant and antiinflammatory
effects. Arterioscler Thromb Vasc Biol 26:1648, 2006.
137. Topol EJ, Byzova TV, Plow EF: Platelet GPIIb-IIIa blockers. Lancet
353:227, 1999.
138. Newby LK, Califf RM, White HD, et al: The failure of orally
administered glycoprotein IIb/IIIa inhibitors to prevent recurrent
cardiac events. Am J Med 112:647, 2002.
 Part effector mechanisms
3 in autoimmunity
and inflammation
15
KEY POINTS
Autoimmune diseases can be classified along two
axes—organ-specific versus systemic and adaptive versus innate.
Autoreactivity is much more common than autoimmune
disease and does not always result in illness.
Animal models of autoimmune disease allow detailed study of
specific features of autoimmunity, but rarely model an entire
disease.
Autoimmune diseases can best be understood as
resulting from multistep processes beginning with loss of
self-tolerance and requiring the development of auto­
reactivity and the initiation of effector mechanisms.
Genetic and environmental factors contribute to autoimmune
diseases.
Common genetic polymorphisms modulate susceptibility to
multiple autoimmune diseases.
Single-gene autoimmune diseases are rare, but give insights
into disease pathogenesis.
Successful biologic therapies often target key pathways in
autoimmune disease pathogenesis.
OVERVIEW OF AUTOIMMUNE DISEASE
PATHOGENESIS
The human immune system has evolved to recognize and con-
tain a wide variety of potentially pathogenic micro­organisms.
Operationally, the immune system can be divided into innate
and adaptive arms. The innate immune system, consisting of
phagocytes, natural killer (NK) cells, dendritic cells (DCs),
epithelial surfaces, and a variety of antigen-nonspecific soluble
molecules, functions as a first line of host defense, and greatly
influences the type of immune response that is ultimately gen-
erated.1 The adaptive immune system, consisting of B cells
and T cells and their secreted products, amplifies, focuses, and
allows recall of antigen-specific immune responses. Innate
immune recognition is “hard-wired” by a fixed number of sur-
face and intracellular receptors, whereas the cells of the adap-
tive immune system recognize antigen with surface receptors
encoded by DNA segments that can rearrange and, in the
Autoimmunity
Richard M. Siegel  •  Peter E. Lipsky
case of B cells, mutate to enable specific recognition of an
almost limitless variety of antigens (see Chapters 16 and 17).
T cells are distinguished by the presence of the T cell receptor
(TCR) for antigen and recognize antigens presented by major
histocompatibility complex (MHC) molecules on antigen-
presenting cells. B cells (see Chapter 10) are identified by the
expression of surface immunoglobulin, which functions as the
specific B cell receptor (BCR) for antigen.
Adaptive immunity also has the special property of
immunologic memory, allowing a more prompt and specific
immune response to occur with repeated exposure to a patho-
gen. The type of priming provided by innate immune cells
significantly influences the specificity, type, and strength of
an adaptive immune response.2 Conversely, cytokines and
antibodies produced by adaptive immune cells can activate
innate immune cells, creating a potential positive feed-
back loop that can maintain autoimmunity. Normally, the
immune system does not make pathogenic responses to the
individual’s own tissues. This selective unresponsiveness to
self-antigens, termed immunologic tolerance, has long been
recognized as a fundamental feature of the normal immune
system. The balance between the need to respond to the
universe of potential pathogens and the need to prevent
autoimmunity is imperfect, however. Consequently, about
5% of humans have autoimmune and chronic inflamma-
tory diseases that stem from unregulated activation of the
immune system and failure of self-tolerance.
REQUIREMENTS FOR SELF-RECOGNITION
AND TISSUE DAMAGE
Autoimmune diseases are a heterogeneous group of disor-
ders in which recognition of self-antigens by lymphocytes
is centrally involved in pathologic organ damage. Not all
immune-mediated destruction of self-organs is autoim-
mune in nature. The liver damage that occurs in hepatitis
B infection is primarily due to immune responses directed
at the virus and not at liver self-antigens. In contrast, some
autoimmune diseases, such as rheumatic heart disease, may
be triggered initially by the immune response to an infec-
tious organism, but the pathologic response is autoimmune
in nature because of cross-reactivity between bacterial and
host antigens. In chronic Lyme arthritis, disease progression
259
 260 SIEGEL  |  Autoimmunity
may be antibiotic resistant, and Borrelia burgdorferi anti-
gens may be undetectable, but the T cells isolated from
joints continue to show reactivity to this bacterium’s outer
surface protein A antigens.3 A subset of these T cells can
cross-react with a normal lymphocyte surface protein, lym-
phocyte function–associated protein 1.4 Whether chronic
Lyme arthritis depends on T cells directed to undetectable
but present bacterial antigens or cross-reactive autoimmune
responses has yet to be determined.
Autoantibodies associated with systemic autoimmune
diseases, such as anti–double-stranded DNA (dsDNA) in
systemic lupus erythematosus (SLE), are present in geneti-
cally diverse individuals with the disease and in many
different animal models. After the discovery that innate
and adaptive immune cells express invariant receptors for
pathogen-­encoded molecular patterns, it was found that
many autoantigens have the ability to stimulate these recep-
tors, activating immune cells in parallel with their recogni-
tion by the TCR or BCR. This type of molecule has been
termed an autoadjuvant because of similarity to immune adju-
vants that are derived from or mimic infectious agents.5 This
principle may apply to innate immune cells as well; certain
chemical structures, such as crystalline uric acid, can initiate
inflammation in neutrophils through cross-reactive activa-
tion with the intracellular receptor CIAS1 (also known as
NALP3 or NLR family, pyrin domain containing 3), which
normally recognizes pathogen-derived muramyl dipeptides
and nucleic acids.6,7 As is well known clinically, however,
autoimmune responses do not follow uric acid–induced
inflammation in gout, suggesting that other steps are neces-
sary to progress from inflammation to autoimmunity.
CLASSIFYING AUTOIMMUNE
AND AUTOINFLAMMATORY DISEASES BY PATTERN
OF ORGAN DAMAGE AND EFFECTOR CELL TYPE
Autoimmune diseases traditionally have been categorized
as organ specific or systemic or both. Examples of common
organ-specific autoimmune diseases include thyroiditis, in
which damage to thyroid cells may lead to hypothyroid-
ism; type 1 diabetes, in which the insulin-secreting beta
cells in pancreatic islets are destroyed; and multiple scle-
rosis, in which central nervous system myelin is damaged.
Many organ systems are targeted by organ-specific autoim-
mune diseases (Table 15-1). In these diseases, it is rare to
find evidence of systemic inflammation or production of
autoantibodies to antigens other than those found in the
target organ, although expanded clones of T cells and B
cells directed against the target organ have been detected in
peripheral blood by sensitive means.8,9 The organ-­specific
autoimmune diseases may represent examples of normal
immune responses that produce disease because they are
“misdirected” against a self-antigen or organ.
Organ-specific autoimmune diseases can be classified into
groups depending on whether pathology is mediated primarily
through autoantibodies or autoreactive T cells (see Table 15-1).
In Graves’ disease, antibodies bind to the thyroid-stimulating
hormone receptor and cause hyperthyroidism. In contrast,
T cells seem to be primarily responsible for the myelin damage
and neurologic deficits in multiple sclerosis, although antibod-
ies to myelin-associated antigens also may play a role. The pres-
ence of autoantibodies does not imply a role in pathogenesis
because autoantibodies can just be a marker of the underlying
T helper cell response or reflect benign responses to damaged
tissue. Such a scenario may occur in type 1 diabetes, in which
IgG autoantibodies to insulin and other pancreatic islet cell
antigens are generated, but islet cell destruction seems to be
mediated primarily by CD4+ and CD8+ T cells.
By contrast, in systemic autoimmune diseases, multiple
organs are targets for immune attack, and chronic activa-
tion of innate and adaptive immune cells is usually present.
SLE is considered to be the prototypic systemic autoimmune
disease, and IgG autoantibodies directed to different autoan-
tigens are primarily responsible for the multitude of possible
clinical manifestations (see Chapter 74). Although CD4+ T
cells may not directly cause tissue damage in SLE, they can
provide immunologic “help” for the generation of patho-
genic autoantibodies. Rheumatoid arthritis (RA) is com-
monly characterized as an organ-selective or joint-selective
disease process, but it is systemic in that autoantibodies to
organ-nonselective autoantigens, such as rheumatoid factor
and citrulline-containing proteins, are usually present. Severe
arthritis in RA is usually associated with these autoantibod-
ies and often accompanied by extra-articular manifestations
(see Chapter 66).
Just as the immune system can be thought of as divided
into innate and adaptive arms, rheumatic and other
immune-mediated diseases can be thought of as lying on
a spectrum in which the primary pathology is mediated by
cells of the innate or adaptive immune system, or a com-
bination of both.10 Rheumatic diseases can be placed on
these two classification axes (Fig. 15-1). In organ-specific
autoimmune diseases and SLE, tissue damage is primarily
mediated by T cells and B cells, putting this disease in the
autoimmune/adaptive category. There are many rheumatic
diseases, however, in which there is inflammation and tissue
damage without evidence of abnormal adaptive immune cell
activation or involvement of autoantigen-specific T cells
or B cells in the pathophysiology of the disease. Numerous
single-gene diseases that share the symptoms of periodic
fevers, episodic arthritis, and chronic elevation of acute-phase
proteins, such as familial Mediterranean fever, tumor necro-
sis factor (TNF) receptor–associated periodic syndrome, and
neonatal-onset multisystem inflammatory disease, involve
activation of innate immune cells and tissue damage related
to amyloid deposition without any evidence of lympho-
cyte activation or autoantibody production. Because of the
prominent inflammatory component and lack of B cell or
T cell involvement, these diseases have been termed auto-
inflammatory to distinguish them from other autoimmune
conditions. Systemic juvenile RA, adult Still’s disease, and
gout are polygenic diseases in which innate immune system
activation predominates and are being recognized as part of
the spectrum of autoinflammatory disease.
Often, immune damage seems to result from collabora-
tion between adaptive and innate immune mechanisms.
In RA, activation of the innate immune system can lead
to tissue damage through the action of inflammatory cyto-
kines on tissue cells, but there also is evidence to suggest
the involvement of T cells and B cells in the pathogenesis
of RA. Similar data exist for inflammatory bowel disease.
As discussed subsequently, autoimmune disease can be sus-
tained by a positive feedback loop reinforcing the activation
of adaptive and innate immune cells.
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION 261

Table 15-1  Examples of Organ-Specific Autoimmune Diseases

Organ Disease(s) Self-Antigen (Example) Major Autoimmune Mechanism
Adrenal cells Addison’s disease Cytochrome P-450 antigens Autoantibodies
Brain/spinal cord Multiple sclerosis Myelin proteins (myelin basic   T cells
protein)
Eye Uveitis Uveal antigens T cells
Gastrointestinal tract
  Stomach Pernicious anemia Gastric parietal cell antigens   Autoantibodies/T cells
(H+/ATPase, intrinsic factor)
  Small bowel Celiac sprue (gluten enteropathy) Transglutaminase Autoantibodies/T cells
  Large bowel Ulcerative colitis or Crohn’s disease Unknown T cells
  Heart Myocarditis Myocardial cell proteins (myosin) Autoantibodies/T cells
Rheumatic heart disease Myocardial antigens Autoantibodies
Hematopoiteic system
  Platelets Idiopathic thrombocytopenic   Platelet antigens (GP llb/llla) Autoantibodies
purpura
  Red blood cells Autoimmune hemolytic anemia Red blood cell membrane proteins Autoantibodies
  Neutrophils Autoimmune neutropenia Neutrophil membrane proteins Autoantibodies
  Kidney/lung Goodpasture’s syndrome Basement membrane antigens   Autoantibodies
(type IV collagen α3 chain)
Liver
  Bile duct cells Primary biliary cirrhosis Intrahepatic bile duct/­mitochondrial Autoantibodies/ T cells
antigens/2-oxoacid  
dehydrogenase complexes
(­pyruvate dehydrogenase  
complex protein)
  Hepatocytes Autoimmune hepatitis Hepatocyte antigens   T cells/antibodies
(cytochrome P450IID6)
Muscle Myasthenia gravis Acetylcholine receptors Autoantibodies
Pancreatic islets Type 1 diabetes Beta cell antigens (glutamic acid T cells (autoantibodies present)
decarboxylase, insulin)
Skin Pemphigus/other bullous diseases Desmogleins Autoantibodies
Testes/ovaries Orchitis/oophoritis Unknown/multiple Autoantibodies
Thyroid Hashimoto’s thyroiditis Thyroid cell antigens   T cells/autoantibodies
(e.g., thyroglobulin)
Graves’ disease Thyroid-simulating hormone   Autoantibodies
­receptor
ATPase, adenosine triphosphatase; GP, glycoprotein.

AUTOIMMUNE DISEASE VERSUS
AUTOREACTIVITY
Although many self-reactive T cells and B cells are elimi-
nated during immune cell development during the estab-
lishment of central immune tolerance, it is clear from many
lines of evidence that many self-reactive cells do survive to
populate the immune system. These self-reactive lympho-
cytes can become pathogenic if not held in check by numer-
ous mechanisms collectively referred to as peripheral immune
tolerance. In experimental animal models of organ-specific
autoimmune disease, peripheral tolerance can be “broken”
through immunization with self-antigens in the presence of
strong innate immune stimulants, termed adjuvants, which
provoke sustained activation of self-reactive lymphocytes
and autoimmune disease. Regulatory T cells are enriched
in autoreactive specificities and play a role in maintaining
peripheral tolerance, as discussed subsequently.
Nonpathogenic, autoreactive B cells exist in the normal
peripheral immune system, and they can be identified more
easily by the antibodies that their progeny secrete. A large
fraction of serum immunoglobulins in healthy animals and
humans binds to self-antigens, and these have been labeled
“natural” autoantibodies (see Chapter 10). Most of these
antibodies are IgM, bind to self-antigens with low affinity,
frequently cross-react with multiple antigens, and are inde-
pendent of T cell help for production. Natural autoantibodies
seem to be an important component of the normal immune
system, and B cells with these self-specificities are posi-
tively selected during development. Their cross-reactivity
to bacterial glycoproteins or glycolipids may be part of the
early immune response to pathogens. Natural autoanti-
bodies also function to help in the clearance of senescent
cells or cell constituents after cell death or in the removal
of immune complexes. Studies in humans and mice also
suggest that a major portion of these autoantibodies are
secreted by the B-1 B cell lineage, including CD5+ B cells
(see Chapter 10).
In mice, the genetic origin and characteristics of patho-
genic autoantibodies are thought to be different from those
of natural autoantibodies. The origin of natural autoanti-
bodies in humans is less clear, however. B cells whose prog-
eny produce natural autoantibodies should be recognized
as another component of the normal immune system and
distinguished from potentially pathogenic B cells.
Even autoantibodies thought of as being associated with
autoimmune disease, such as rheumatoid factor or antinu-
clear antibodies, are found in a small subset of the ­normal
 262 SIEGEL  |  Autoimmunity

Polygenic Monogenic Autoimmune/ Environment Individual Genetic factors

diseases diseases adaptive
E.g., smoking, Innate immune
responsiveness Gender, ethnic
UV light,
Organ-specific IPEX ALPS Systemic origin
infectious agents
autoimmune lupus
diseases erythematosus Self-limiting
APCED
(see Table 15-1) nonspecific
inflammation Host defense
Organ-specific

genes
Multiple (TLR7, FcR, TNF)

Systemic
sclerosis
Adaptive immune
Rheumatoid system activation
arthritis Loss of self- Immune-response
tolerance genes
Inflammatory Systemic (HLA-DR,
bowel disease JRA PTPN22,
Immunoregulation FcRIIB, IL-10,
Familial fever Proinflammatory
Gout and other (Treg, clonal CTLA4,
syndromes cytokines
crystal arthropathies deletion, anergy) PD-1, Fas)

Autoinflammatory/innate
Figure 15-1  Classification of autoimmune and rheumatic diseases. Autoaggressive Autoantibody
Specific diseases are grouped according to the two axes described. T cells production
­Organ-specific autoimmune diseases are described in more detail in
Table 15-1. ALPS, autoimmune lymphoproliferative syndrome; APECED, Autoimmunity
autoimmune polyendocrinopathy syndrome, with candidiasis and ecto-
dermal dysplasia; IPEX, immunodysregulation, polyendocrinopathy, and
enteropathy, X-linked (syndrome); JRA, juvenile rheumatoid arthritis. Tissue-response
Tissue damage
genes

Figure 15-2  Steps and pathways influencing the development and

population without apparent consequence, particularly older
individuals and first-degree relatives of patients with autoim-
mune disease. Other autoantibodies, such as anticitrullinated
peptide antibodies in rheumatoid arthritis, and anti-DNA
and anti-Sm in SLE, seem to be more specifically associated
with a particular autoimmune disease. This finding indicates
that the mere presence of these antibodies does not produce
autoimmune disease. In individuals who do develop autoim-
mune disease, retrospective studies have shown that char-
acteristic autoantibodies can precede the development of
autoimmune disease by years.11 Potentially pathogenic auto-
antibodies can be induced by certain infections, drugs, or tis-
sue injury. Examples include the production of antinuclear
antibodies after treatment with drugs such as procainamide
and hydralazine and the development of anticitrullinated
peptide antibodies in smokers with certain HLA haplotypes
linked to RA susceptibility.12 Some of these antibodies, as
in the case of drug-induced SLE, can result in pathologic
consequences, but in these cases, autoantibodies usually are
transient and disappear in weeks to months.
MODEL FOR THE PATHOGENESIS
OF AUTOIMMUNE DISEASE
Because autoreactive lymphocytes are present in the normal
mature immune system, the pathogenesis of autoimmune
disease must involve escape by these cells from normal
immune regulation, the perpetuation of these responses,
and induction of immunopathology. Figure 15-2 presents a
conceptual framework for the pathogenesis of autoimmune
disease. Considerable evidence from genetic epidemiology,
association, and linkage studies indicates that the develop-
ment of most autoimmune diseases has a strong genetic basis
(see Chapter 18). Except in certain rare diseases, autoimmune
perpetuation of autoimmune diseases. Environmental (external, e.g., UV
light, and internal, e.g., cytokine milieu) factors are listed in the left col-
umn, and genetic factors are listed in the right column.
disease is inherited as a complex trait, with multiple loci
controlling various aspects of disease susceptibility. More
recently, some of these susceptibility genes have been
identified, and these are listed in the right-hand column
of Figure 15-2. The contribution of susceptibility genes
may occur at several levels, from the breakdown of toler-
ance to immune-mediated end-organ damage (Table 15-2).
Certain environmental influences, such as cigarette smoke,
ultraviolet light, or infectious agents, may interact with this
genetic predisposition to result in disease, although for most
autoimmune diseases, the exact environmental triggers are
undefined. As in conventional immune responses, evidence
is emerging that activation of autoimmune B cells and
T cells can be influenced by innate immune receptors, such
as Toll-like receptors (TLRs), which primarily recognize
pathogen-derived molecular structures but may cross-react
with host molecules, particularly nucleic acids.
A second phase of autoimmune disease pathogenesis
involves conversion from transient to sustained T cell or
B cell activation, which may perpetuate autoimmune dis-
ease in a “vicious circle” of positive feedback. As depicted
in the lower part of Figure 15-2, autoreactive T cell and B
cell activation can lead to tissue damage through autoanti-
bodies, direct attack of tissues by autoreactive T cells, and
cytokine production. Signals released from damaged tissues,
such as cytokines, recruit and activate macrophages and
neutrophils. Unscheduled necrotic cell death secondary to
tissue damage or production of apoptotic blebs containing
nuclear antigens may lead to more release of self-antigens.
In the presence of autoantibodies, these antigens can form
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION 263

Table 15-2  Selected Susceptibility Genes for Autoimmune Diseases

Gene Product Animal Model Human Disease Function Principle Illustrated References
ZAP-70 SKG mouse — T cell receptor signal Weak T cell receptor signaling can   28
­transduction block deletion of autoreactive
­thymocytes
Fas MRL lpr/lpr   ALPS Induction of programmed cell Apoptosis eliminates   159, 160
mouse death autoreactive immune cells
TLR7 BSXB yaa   — Sensing single-stranded RNA Increased levels of TLR7 can lead to 22, 23
mouse in endosome B cell hyperactivation
AIRE AIRE knockout APECED Regulates transcription of   Tissue-restricted antigens need 27
mouse self-antigen genes in the to be expressed in the thymus
thymus for deletion of autoreactive
­thymocytes
Roquin San Roque   — Ubiquitin ligase, suppresses Follicular helper T cells and   48
mouse follicular helper T cells and interleukin-21 can promote
interleukin-21 ­autoimmunity
FoxP3 Scurfy mouse; IPEX Transcription factor ­crucial for Regulatory T cells essential in 119, 120
FoxP3   induction and maintenance ­maintaining self-tolerance of
knockout of regulatory T cells ­peripheral T cells
Complement   C4 knockout C4 deficiency Classic complement compo- Clearance of immune complexes   161
C4 nent, aids in rapid clearance suppresses autoimmunity
of ­immune complexes
CTLA4 CTLA4- Linked to multiple Negative regulator of T cell Regulation of T cell activation 77
­knockout autoimmune activation ­suppresses autoimmunity
diseases
ALPS, autoimmune lymphoproliferative syndrome; APECED, autoimmune polyendocrinopathy syndrome, with candidiasis and ectodermal dysplasia;
IPEX, immunodysregulation, polyendocrinopathy, and enteropathy, X-linked (syndrome).

immune complexes and become immunostimulatory, pro-
viding “fuel for the fire” of autoimmunity.13
Numerous immunoregulatory mechanisms can dampen
or potentially terminate autoimmune tissue damage (see
Fig. 15-2, lower left). Regulatory T cells (see Chapter 9) can
inhibit T cell activation and cytokine production or limit
the function of effector cells. Reduction of regulatory T cell
function or numbers, in genetic diseases or experimentally,
results in the unmasking of multiple T cell–mediated auto-
immune pathologies. Elimination of autoreactive T cells
through programmed cell death or rendering them “anergic”
or unresponsive to TCR stimulation also has been shown
to ameliorate autoimmune disease. Inflammatory cytokines,
such as TNF-α and interleukin (IL)-6, have been shown to
inhibit the functionality of regulatory T cells.14,15 This inhi-
bition may sustain or allow autoimmunity to develop in the
setting of chronic infection or inflammation.
INSIGHTS FROM ANIMAL MODELS
AND HUMAN GENETIC DISEASES
Studies using animal models have contributed greatly to
understanding of the immunopathogenesis of autoimmune
disease, although each model generally captures only one
or a few aspects of a particular human disease. Models of
disease can be divided into three broad categories based on
how disease occurs, as follows: (1) spontaneous diseases,
occurring because of a single gene mutation or interaction
of many genes; (2) diseases induced by immunization, cell
transfer, or other exogenous agent; or (3) diseases devel-
oping after genetic manipulation, usually by transgenic
expression or knockout of single genes. In addition to these
models of disease, genetically manipulated TCR and BCR
transgenic mice have been invaluable in tracing the fate of
self-­reactive T cells and B cells. In recent years, mutations
underlying an increasing number of human single-gene dis-
eases that involve autoimmunity and inflammation have
been identified, providing parallel insights into the mecha-
nisms of autoimmunity.
ANIMAL MODELS WITH SPONTANEOUS DISEASE
AND HUMAN DISEASE COUNTERPARTS
The animal models most similar to human autoimmune dis-
ease include strains of mice that develop a high incidence of
spontaneous autoimmune disease. Polygenic models include
the nonobese diabetic (NOD) mouse, which develops a dis-
ease similar to type 1 diabetes,16 and several models of lupus-
like disease.17 New Zealand hybrid mice, in which crosses or
inbred recombinant strains of New Zealand black (NZB) and
New Zealand white (NZW) mice develop lupus-like disease,
have been intensively studied as a polygenic spontaneous
autoimmune disease model.17,18 As mapping of complex loci
responsible for autoimmune disease models has progressed, it
has become clear that many loci that strongly contribute to
disease phenotype contain many linked genes that may con-
tribute independently to disease penetrance and severity.17,18
The Sle1 locus on chromosome 1 derived from the NZW
background emerged as a major determinant of disease phe-
notype in the NZB/NZW mouse model of SLE. Generation
of congenic strains with portions of the Sle1 locus back-
crossed to a nonautoimmune background revealed, how-
ever, at least four segments of this locus, designated Sle1a
through Sle1d, which could independently modify the
susceptibility to autoantibody formation. Within Sle1b,
mutations that affect the level of expression of members
of the SLAM family of surface adhesion receptors were
identified that may contribute to autoimmunity by alter-
ing the threshold for deletion of autoreactive B cells.19,20
Within a separate NZB-derived locus on chromosome 1,
 264 SIEGEL  |  Autoimmunity
Nba2, the gene interferon-inducible 202 (ifi 202) may be
another immunoregulatory susceptibility gene.21 ifi 202
was found to be expressed at higher levels in autoimmune
progeny, and this correlated with resistance to experimen-
tally induced B cell apoptosis.21 As in the authentic human
diseases, the genetic contributions of each locus to dis-
ease pathogenesis and the interactions between loci have
proved to be exceedingly complex in polygenic models of
autoimmunity.
Another autoimmune susceptibility locus, the murine
Y chromosome–linked autoimmune accelerator yaa, origi-
nally described in the BXSB/MpJ autoimmune-prone strain,
has been shown to be caused by a translocated copy of an
X-chromosomal segment that results in expression of an
extra copy of TLR7.22,23 TLR7 recognizes single-stranded
RNA, and cells harboring the extra copy of TLR7, includ-
ing B cells, become hyperresponsive to stimulation by TLR7
agonists. Breeding TLR7 deficiency onto the MRL (lpr/lpr)
autoimmune-prone strain ameliorated many features of the
SLE-like disease found in these mice.24 These discoveries
illustrate how small changes in expression level of key regu-
latory proteins, particularly cell surface receptors, can sig-
nificantly alter the threshold for autoimmunity by changing
the intrinsic responsiveness of lymphocytes.
In contrast to most spontaneous autoimmune disease
models, single-gene mutations that lead to autoimmune dis-
ease reveal crucial genes that, if mutated, can lead with high
frequency to autoimmune disease. Background genes and
the environment also modify the phenotype of mice carry-
ing even these mutations, however. lpr/lpr mice, which are
homozygous for the lymphoproliferation (lpr) mutation in
the gene encoding the death receptor Fas, produce autoanti-
bodies including antinuclear antibody and anti-dsDNA. On
the MRL background, lpr/lpr mice develop fatal glomerulo-
nephritis and other autoimmune disease manifestations.25
A subset of MRL (lpr/lpr) mice also develop arthritis associ-
ated with rheumatoid factor production. The Fas mutation
in lpr mice abrogates expression of this TNF family receptor,
which induces cell death of numerous immune cell types.
A human genetic autoimmune disease, autoimmune lym-
phoproliferative syndrome (ALPS), was found to result from
heterozygous dominant negative mutations in the Fas gene,
causing a similar inhibition of Fas-induced apoptosis as in
lpr/lpr mice.26 Patients with ALPS can develop a variety
of autoantibody-mediated disease manifestations, chiefly
autoimmune hemolytic anemia and thrombocytopenia,
but rarely develop kidney disease, illustrating that effects
of genetic background also are important in human single-
gene autoimmune diseases.
Mutations in genes that control T cell repertoire selec-
tion and signaling have been identified in many single-gene
spontaneous models of autoimmunity. The autoimmune
regulator (AIRE) transcription factor promotes the expres-
sion of tissue-restricted genes in the ­thymus. When AIRE is
absent, deletion of T cells specific for these gene products
in the thymus is impaired, and T cell–driven autoimmune
destruction of numerous target organs results in AIRE-
deficient mice or the human disease autoimmune polyen-
docrinopathy syndrome, with candidiasis and ectodermal
dysplasia (APECED).27 Another spontaneous arthritis
model, known as the SKG mouse, has allowed insights into
mechanisms controlling the generation and activation of
arthritogenic CD4+ T cells.28 SKG mice develop macro-
scopic hallmarks of arthritis at 8 weeks of age and produce
high titers of rheumatoid factor and type II collagen–specific
autoantibodies.
Transfer of SKG CD4+ T cells into T cell–deficient
mice led to the development of arthritis, emphasizing that
arthritis in these mice is a T cell–intrinsic disease. Genetic
mapping experiments revealed that these mice have a muta-
tion in the SH2 domain of ZAP-70 that renders the T cells
hyporesponsive to TCR signals. It is thought that altered
signaling through the TCR leads to a selection shift dur-
ing thymic development, disrupting central T cell tolerance
and allowing arthritogenic cells to escape into the periphery.
The disease spectrum of SKG mice varies depending on the
mouse background strain and is lost when mice are bred in
a pathogen-free environment, indicating that other genetic
and extrinsic factors are important in producing this specific
disease phenotype.
ANIMAL MODELS IN WHICH DISEASE IS INDUCED
BY IMMUNOLOGIC MANIPULATION
A second category of experimental models depends on
induction of disease in susceptible strains, typically by immu-
nization with a self-antigen. These models usually rely on
complete Freund’s adjuvant, which is a mixture of mineral
oil and mycobacteria, to prime antigen-presenting cells and
overcome self-tolerance, and are better models for the effec-
tor phase of autoimmune disease rather than for the study of
immune tolerance. These types of models also have proved
useful for evaluating the effect of a specific gene or pathway
in a disease process through performing the experiment in
the presence of neutralizing antibodies against mediators of
that pathway or in gene knockout mice. Although most of
these immunization models use organ-specific antigens (e.g.,
myelin basic protein, acetylcholine receptor, thyroglobulin,
retinal antigens, type II collagen), some models have used
ubiquitous nuclear antigens to induce lupus-like autoanti-
bodies and disease.29
One well-studied induced disease model relevant to inflam-
matory arthritis in humans is collagen-induced arthritis (CIA),
in which mice or rats are usually immunized with type II col-
lagen isolated from bovine, chicken, or human cartilage.30,31
In susceptible mouse strains, cross-reactivity of immune
responses to murine type II collagen, involving autoreactive
T cells and autoantibodies, results in the development of
peripheral arthritis. This model has provided insight into the
effector mechanisms that lead to inflammation in patients
with arthritis. Other cartilage proteins also can be used to
induce arthritis in susceptible mice.32 Despite synovial pathol-
ogy reminiscent of RA, it is unclear whether these rodent
models of induced arthritis are ­ recapitulating the specific
immune responses in RA. None results in rheumatoid factor
production, although a more recent study showed induction of
antibodies against cyclic citrullinated peptides and suggested a
pathogenic role for these antibodies in CIA.33
Another class of animal models of induced autoimmu-
nity involves depletion of natural regulatory T cells, either
through neonatal thymectomy or through mutation of the
X-linked regulatory T cell–specific transcription factor
FoxP3 in the scurfy mouse. These mice develop multiorgan
autoimmunity (including gastritis, oophoritis/orchitis, and
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
thyroiditis). The human syndrome of immunodysregulation,
polyendocrinopathy, and enteropathy, X-linked, also has
been linked to loss-of-function FoxP3 mutations and results
in a similar constellation of findings, including autoantibody
production, but not arthritis or glomerulonephritis. When
T cells depleted of regulatory T cells are transferred into
immunodeficient hosts, extensive division and differentiation
of these T cells occurs, and autoimmune disease, principally
colitis, results. Colitis can be prevented by the simultaneous
transfer of regulatory T cells.34,35 Graft-versus-host disease
induced by the injection of allogeneic lymphocytes shares
many features with SLE, including autoantibody production,
and has been used as a model for understanding the steps
that lead to autoantibody production in the setting of strong
T cell stimulation by a pseudo–self-antigen.
ANIMAL MODELS WITH AUTOIMMUNE DISEASE
AFTER GENETIC MANIPULATION
A third category of experimental disease model involves
engineered manipulations of the mouse genome. Numer-
ous genetically engineered mouse strains with single-gene
gain or loss of function develop autoimmunity, with varied
underlying mechanisms. Lupus-like disease has resulted
from genetic deficiencies that cause defects in lymphocyte
apoptosis, unregulated B cell or T cell activation, defects
in the ability to clear apoptotic cells or cellular debris, and
altered cytokine production.36,37 Mice with gene knockouts
of IL-2, IL-2 receptor, IL-10, or depletion of various T cell
subsets have been shown to develop inflammatory colitis.37
In some cases, the background strain of the knockout
mouse can alter greatly the form of immunopathology that
ensues. IL-2 gene knockouts can lead to inflammatory colitis
or autoimmune hemolytic anemia, depending on the back-
ground strain. Knockout of the gene that encodes the type
IIB low-affinity receptor for IgG (FcRIIB) results in a severe
lupus-like syndrome when bred on one type of nonautoim-
mune (C57BL/6) background, but almost no pathology on
a different nonautoimmune (BALB/c) background.38 This
genetic deficiency results in unregulated B cell activation;
the reason for the different expression on the two different
genetic backgrounds is currently unclear. The single-gene
models do not recapitulate the complex genetics of human
autoimmune disease, but are useful to point to particular
genes and mechanistic pathways for study in human disease.
Knockout mice have provided at least a partial basis for
genetic studies in humans that have identified SLE patients
with deficient DNase I genes39,40 and with variant alleles
of the gene encoding programmed death-1 (PD-1), which
negatively regulates T cells and B cells after activation.41,42
Genetic manipulations have produced surprising systems
to study the development of autoimmune disease, including
arthritis. A model generating wide interest (referred to as the
K/B x N model) involves mice with transgenes encoding a
TCR that recognizes a foreign antigen (bovine ribonuclease)
in the context of the mouse MHC class II allele, I-Ak. As in
other TCR transgenics, nearly all of the T cells in these mice
express one type of TCR. When these TCR transgenic mice
are bred with mice expressing a different MHC class II mol-
ecule (I-Ag7 from the NOD strain), the progeny develop a
severe and destructive arthritis with histologic features rem-
iniscent of RA.38 These investigators subsequently showed
265
that in the context of I-Ag7, the transgenic TCR recognizes
a peptide derived from a glycolytic self-enzyme (glucose-
6-phosphate isomerase), and this unexpected cross-reactivity
allows for a T cell–dependent IgG antibody response to this
ubiquitous cytoplasmic antigen. Arthritis in these mice is
mediated by autoantibodies, and transfer of a small amount
of anti–glucose-6-phosphate isomerase autoantibody causes
severe arthritis in recipients.43,44 Although different in
pathogenesis from human RA, investigation of arthritis
caused by injection of anti–glucose-6-phosphate isomerase
antibodies has provided insights into the signaling pathways
and cells necessary for initiation of arthritis downstream of
autoantibody–self-antigen recognition.
Experimental systems frequently have used mice express-
ing transgenes that encode a TCR or BCR specific for one
autoantigen or for an exogenous antigen introduced geneti-
cally so as to become an autoantigen. In these mice, numer-
ous T cells or B cells with the same autoreactive receptor
can be easily identified and tracked, greatly facilitating their
characterization. One of the first, and most elegant, systems
of this kind is the H-Y TCR, which encodes a receptor spe-
cific for a peptide in the H-Y male-specific antigen gene.
In female TCR transgenic mice, predominantly CD8+ T
cells with high reactivity against H-Y develop, recapitulat-
ing the developmental fate of the CD8+ T cell from which
this TCR was derived. In male mice, most transgenic T
cells are deleted during thymic development, however, and
the remaining T cells downmodulate CD8 and are refrac-
tory to stimulation through the TCR, illustrating multiple
mechanisms for enforcing T cell self-tolerance.45 B cells in
BCR transgenic mice bearing self-reactive specificities also
tend to be deleted or anergized depending on the expres-
sion pattern or abundance of the self-antigen. When BCR
transgenic mice encoding anti-DNA or rheumatoid factor
specificities are bred onto autoimmune-prone mouse strains,
these same self-reactive B cells become activated and can
produce pathogenic autoantibodies.46,47
An alternative approach to discovering susceptibility
genes for autoimmunity has been through using “reverse
genetics,” in which the entire mouse genome is subjected
to mutagenesis, and mice are inbred and screened for auto-
antibody production or other phenotypes similar to human
autoimmune disease. Current mapping techniques can rap-
idly identify loci segregating with a disease phenotype, lead-
ing to identification of the culprit mutation. This approach
has identified many genes that predispose to autoimmunity
not discovered through other means. A previously unchar-
acterized Ring-type ubiquitin ligase named Roquin encoded
on chromosome 1 in mice and humans was identified by this
method. Roquin mutant mice develop severe autoimmune
disease.48 Although the pathogenesis of disease in Roquin
mutant mice is unclear, T cells producing the cytokine
IL-21, a B cell growth and differentiation factor, were found
in elevated levels in these mice. IL-21 also has been impli-
cated in other models of systemic autoimmunity.
GENETIC CONTRIBUTIONS
TO AUTOIMMUNE DISEASE
Considerable evidence indicates that most autoimmune
diseases have a strong genetic predisposition.17,18,49-51 Ini-
tially, this evidence came from genetic epidemiolog studies
 266 SIEGEL  |  Autoimmunity
of disease prevalence within families in which there is an
affected individual compared with the prevalence in the
general population and from analyses of disease prevalence
in monozygotic versus dizygotic twins. The concordance of
rheumatic autoimmune disease in monozygotic twins ranges
from 12% to 15% in RA to 24% to 57% in SLE (see Chapter
18).52 Concordances never approach 100%, however, indi-
cating a role for the environment or epigenetic factors. Com-
mon autoimmune diseases are complex genetic traits, which,
by definition, are not inherited in a simple mendelian way. By
contrast, many rare autoimmune disease syndromes are inher-
ited as single-gene diseases, and the mutations responsible for
these diseases identify a special class of genes that can strongly
predispose to autoimmune disease with a lesser contribution
by genetic variation within the human population.
For common autoimmune diseases, the low penetrance
of each contributing gene (i.e., the small increase in prob-
ability of disease expression given a particular disease allele)
is the main reason that autoimmune diseases are so complex
genetically. Overt autoimmune disease frequently does not
occur, however, even in the presence of a full complement of
susceptibility alleles, as in the identical twin of an affected
individual. Genetic complexity in autoimmunity also is
determined by genetic heterogeneity, in which the same
phenotype (e.g., anti-dsDNA autoantibody production or
lupus nephritis) is the result of a different set of genes. Gene
alleles that increase the risk of autoimmune disease also can
occur frequently in the general population, making their
identification as a disease allele difficult.
Initial genetic studies of human autoimmune diseases
have focused on the association of particular alleles with
disease, MHC genes, and other immunologically relevant
genes (see Chapter 18). Association studies are, however,
potentially subject to inherent biases such that spurious
associations are revealed because of the lack of appropriate
controls. The interpretation of associations may be compli-
cated further by the presence of linkage disequilibrium with
the actual causal gene.49 Association studies also require
an a priori hypothesis that a particular candidate gene is
involved in the disease process and are not suitable to inves-
tigate the many unknown non-MHC genetic contributions
to disease.
Because of the availability of high-density single-
nucleotide polymorphic markers covering the human
genome, and the discovery of “haplotype blocks,” which
reduce the number of single-nucleotide polymorphic mark-
ers that need to be tested,53,54 it is now possible to perform
genome-wide screens for susceptibility loci to complex
diseases.55 Screens done by numerous consortia have iden-
tified susceptibility genes for many common rheumatic dis-
eases, many of which would likely not have been identified
solely through the candidate gene approach.56 Some of the
same disease-associated genetic polymorphisms have been
identified in patients with different autoimmune diseases.
Loci identified in a genome-wide scan of patients with
RA from multiplex families also have been found to be
linked in separate cohorts to inflammatory bowel disease,
multiple sclerosis, and ankylosing spondylitis.57 It is well
known clinically that autoimmune diseases, such as type
1 diabetes, autoimmune thyroiditis, Addison’s disease,
autoimmune polyendocrine syndromes, vitiligo, and celiac
disease, may cluster in some families. These observations
have raised the possibility that common susceptibility
genes may contribute to multiple autoimmune diseases
among related individuals.
ASSOCIATION OF MAJOR HISTOCOMPATIBILITY
COMPLEX GENES WITH AUTOIMMUNE DISEASES
Polymorphisms in the mouse MHC or the analogous human
leukocyte antigen (HLA) locus have a powerful influence on
T cell and NK cell immune responses, and the HLA locus
has been linked to different autoimmune diseases in multiple
cohorts (see Chapter 18). The HLA region spans approxi-
mately 3.6 megabases on chromosome 6, encompasses more
than 200 genes, and is highly polymorphic. The HLA locus
consists of genes encoding classic class I (A, B, and C) and
class II (DR, DQ, and DP) HLA molecules that present pep-
tide antigens to CD8+ and CD4+ T cells. Many nonclassic
HLA molecules that function to present protein-based and
lipid-based antigens to NK/T cells bearing a restricted rep-
ertoire of αβ or γδ TCRs also reside in this locus. In addi-
tion, many other immunologically related genes, such as the
TNF family members TNF, LTα, and LTβ and complement
components C2, C4A, and C4B, lie within the HLA region.
Tight linkage between genes in this region has often made
it difficult to identify the specific HLA genes (and alleles)
that explain the association of a particular haplotype (usu-
ally identified by its HLA-DR and HLA-DQ genes) with
autoimmune disease. Within a given ethnic group, certain
HLA-DR alleles are frequently inherited with a similar set
of DQ alleles and other genes within the HLA region.
Genome-wide scans have confirmed a major role for the
HLA in determining the risk of developing RA, with most,
but not all, of that risk conferred by polymorphisms in the
DR beta chain gene (DRB) (see Chapters 18 and 65).57,58
DRB1*0401 and DRB1*0404 predominate in white
patients, but other DR4 subtypes (DR4*0405, DR4*0408)
and DRB1*0101, DRB1*1402, and DRB1*1001 also have
been implicated in disease susceptibility. Disease-associated
DR alleles all share a sequence motif at positions 67 to 74
(L-L-E-Q-R/K-R-A-A) in the third hypervariable region of
the DRB1 gene. This shared segment has been called the
“shared epitope.”49,58 The increased risk for disease develop-
ment in carriers of one copy of a shared epitope has been
estimated to be fivefold to sixfold for DRB1*0404 and
DRB1*0401, with absolute risks of 1 in 20 (DRB1*0404) and
1 in 35 (DRB1*0401). For individuals carrying both alleles
(i.e., DRB1*0401/*0404), the increased risk for developing
RA has been estimated to be about 30-fold, with an absolute
risk of 1 in 7.5. Individuals with two *04 alleles also seem to
have an increased risk of more severe destructive joint dis-
ease and extra-articular involvement.59 The association of
particular DR alleles with RA is frequently cited as the most
compelling evidence for the important role of CD4+ T cells
in this disease, although the exact peptides that are being
presented in the context of class II molecules that contain
the shared epitope are currently unknown.60
A clear association between particular MHC haplo-
types and development of SLE generally has been more
difficult to establish. Modest associations of HLA-DR3
(DRB1*0301) and DR2 (DRB1*1501) have been shown in
white SLE patients. The increased risk from haplotypes with
these HLA-DR alleles has been confirmed in transmission
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
­disequilibrium studies in families with SLE.61 Although it
has been suggested that the association of particular haplo-
types with SLE relates to genes distinct from DR and DQ,
such as those encoding complement component C4A and
TNF, studies using a dense map of markers across the HLA
region strongly suggested that it is the class II genes (e.g.,
DR and DQ) that underlie the increased risk of disease
from a particular HLA haplotype.59 A strong association
of SLE with particular class II alleles also has been shown
when patients are grouped according to the specific types of
autoantibodies produced. Autoantibodies to Ro/SS-A (plus
La/SS-B), U1RNP, Sm, phospholipids (i.e., anticardiolipin
antibodies or the lupus anticoagulant), and dsDNA have
been associated with particular DQ alleles. These data sug-
gest that the contribution of class II MHC molecules in SLE
is predominantly at the level of specific autoantibody pro-
duction, consistent with an impact on CD4+ T cell help.
A different mechanism may underlie the strong associa-
tion of spondyloarthropathies with individuals who carry
the HLA-B27 class I allele. A specific region of the peptide-
binding groove of the B27 molecule is conserved among
HLA subtypes linked to spondyloarthropathies, and it was
originally postulated that the structural features of HLA-B27
may favor binding of a particular set of peptides, perhaps
self-peptides cross-reacting with a pathogenic stimulus that
predisposes to spondyloarthropathies. In human reactive
arthritis and ankylosing spondylitis, HLA-B27-restricted
reactivity to bacterial and collagen self-peptides has been
identified.62-64 An alternative hypothesis builds on the
observation that HLA-B27 proteins exhibit inefficient fold-
ing and are exported to the cytoplasm and degraded more
rapidly than other HLA alleles,65 perhaps because of the
formation of abnormal disulfide-linked HLA-B27 dimers.66
Misfolded proteins may trigger a generalized cellular stress
response termed the unfolded protein response, which can
induce the transcription of proinflammatory genes under
some conditions.
These observations may explain how transgenic expres-
sion of HLA-B27 in rats results in colitis and spontaneous
inflammatory disease with some similarities to ankylosing
spondylitis.67 In these animals, restoring HLA-B27 fold-
ing with additional β2-microglobulin expression eliminated
colitis, but did not prevent arthritis and spondylitis,68 sug-
gesting that these manifestations may operate through
different mechanisms.
ASSOCIATION OF NON–MAJOR
HISTOCOMPATIBILITY COMPLEX
GENES WITH AUTOIMMUNE DISEASE
Multiple non-MHC candidate genes have been studied for
associations with autoimmune diseases. Studies of SLE have
addressed genes encoding complement genes, Fc recep-
tor alleles, mannose-binding protein, various cytokines
such as IL-10 and TNF, and gene segments of the TCR and
immunoglobulin gene complexes (see Chapter 74). Alleles
determining deficiencies of classic pathway complement
components have shown strong associations with SLE (see
Chapter 19).40,69,70 Most individuals with genetically deter-
mined complete deficiencies of complement C1q or C1r/C1s
develop a syndrome resembling SLE. C2 and C4, encoded
within MHC, also have alleles that result in deficiencies, and
267
complete deficiencies of these molecules markedly increase
the risk of developing SLE. The association of these deficiency
states and SLE is likely to be an etiologic one, but the mecha-
nism for the influence of genetic complement deficiencies
in SLE is unclear. Studies have suggested that complement
deficiencies may lead to SLE through defects in the clearance
of apoptotic cells and cellular debris, as in DNase I–deficient
humans and mice,39 or alternatively through defects in the
clearance of immune complexes.69 These genetic contribu-
tions may allow enhanced deposition or formation of immune
complexes in the kidney or possibly affect the inflammatory
response or end-organ response to complexes.
Associations also have been shown between SLE and
alleles of the genes encoding cell surface receptors for
IgG (i.e., Fcγ receptors).71,72 Investigators have shown an
association between SLE and alleles of the gene encoding
FcγRIIA (CD32), expressed by monocytes or macrophages
and neutrophils.71 Alleles of this gene differ substantially in
the ability to bind human IgG2, and studies found a decrease
in the prevalence of the high-affinity binding allele in SLE,
particularly in patients with lupus nephritis. Possibly similar
to complement deficiencies, the genetic contribution of this
Fcγ receptor deficiency may reflect a relatively late event in
the pathogeneisis of SLE-related immune complex damage,
such as in lupus nephritis.
Consistent with this hypothesis, other studies have
noted the underrepresentation of a high-binding FcγRIIIA
allele (expressed by NK cells and monocytes) in patients
with SLE, especially with lupus nephritis.72 Expression of
the inhibitory Fcγ receptor FcγRIIB seems to be extremely
important in controlling the generation of lupus-like disease
in mouse models. Deficiency of the gene encoding FcγRIIB,
which negatively regulates B cell activation, can cause
lupus-like disease in some otherwise normal mouse strains.38
Reduction in expression of the inhibitory FcγRIIB linked to
a promoter polymorphism has been associated with numer-
ous autoimmune-prone mouse strains.73 FcγRIIB can recruit
phosphatases that negatively regulate BCR signaling and
has been shown more recently to induce apoptosis in mouse
and human plasmablasts.74
Genome-wide linkage analyses of families with affected
siblings also have been reported in human type 1 diabetes,
autoimmune polyendocrine syndromes, autoimmune thy-
roid disease, multiple sclerosis, SLE, and RA.17,18,36,49,75,76
These studies are beginning to yield candidate autoimmune
disease–predisposing genes and have revealed an interest-
ing principle in autoimmune disease genetics: Often single
loci seem to predispose to multiple autoimmune diseases.
A chromosome 2q33 locus mapped in human families with
type 1 diabetes and autoimmune thyroid disease has led to
the identification of a variant allele of the gene encoding
CTLA4 and points to an important role for alternative
splice forms of this gene.77,78 In type 1 diabetes, other iden-
tified susceptibility genes based on linkage analyses include
the insulin gene in the human disease and the gene encod-
ing IL-2 in the NOD mouse model.36 Gene identification
after linkage studies in human SLE and mouse models of
lupus also has begun to provide new insight into the immu-
nopathogenesis of disease. One study of human families
has suggested that a locus on chromosome 2 is related to a
polymorphism in the gene encoding PD-1 (PDCD1), which
provides a negative signal to activated T cells.42
 268 SIEGEL  |  Autoimmunity
A candidate gene screening approach in a cohort of sib
pairs affected with RA resulted in the identification of a mis-
sense polymorphism in the tyrosine phosphatase PTPN22,
also known as lyp (858C > T, resulting in the amino acid
substitution R620W) strongly predisposing to RA,79 a find-
ing that has been replicated in multiple cohorts.80 PTPN22
polymorphisms predict RA independently of carriage of
“shared epitope” positive HLA class II genes. The same vari-
ant was identified as a susceptibility allele in patients with
type 1 diabetes,81 and carriers of the variant allele also have
been shown to be more susceptible to Graves’ disease, Addi-
son’s disease, and familial SLE. When studied biochemi-
cally, the PTPN22 R620W variant enhances the tyrosine
phosphatase activity of the enzyme, resulting in dominant
inhibition of T cell activation through the TCR.82 This
finding led to speculation that the R620W mutation predis-
poses to autoimmune disease by selectively impairing TCR-
induced apoptosis or regulatory T cell function.
Genome-wide scans to identify susceptibility genes are
under way in many autoimmune diseases and already have
identified novel candidate genes. Functional variants in
NOD2, a sensor for bacteria-induced inflammation, were
found to account for approximately 10% of the risk for
Crohn’s disease through mapping of a susceptibility locus
on chromosome 16.83 Another susceptibility gene more
recently identified in Crohn’s disease patients is the IL-23
receptor. This variant could function through enhancing
the action of the cytokine IL-23, which sustains a popula-
tion of proinflammatory IL-17-producing T cells in the gas-
trointestinal mucosa.84
HUMAN MENDELIAN AUTOIMMUNE DISEASES
In contrast to the polymorphisms discussed previously that
predispose to common autoimmune diseases and occur
with significant frequency in healthy human populations,
many rare autoimmune diseases are inherited in a mende-
lian fashion, and disease-causing mutations in these genes
carry a high risk of producing disease. These mutations give
insights into crucial checkpoints in self-tolerance. Develop-
ment of autoimmune disease even in carriers of these pow-
erful mutations can be influenced, however, by other genes
and the environment. Mutations in the TNF family recep-
tor Fas and its ligand FasL result in the lpr and gld recessive
autoimmune syndromes in mice, and dominant-negative
mutations in Fas underlie similar immunologic symptoms
in the autosomal dominant ALPS in humans.85 FasL trig-
gers apoptotic programmed cell death in chronically stimu-
lated T cells and B cells, and the disruption of this pathway
because of lpr and gld mutations results in the acceleration
of lupus-like autoimmunity and in a massive accumulation
of CD4−CD8− T cells.25
Although the mechanism by which mutations in Fas
lead to accelerated autoimmunity is not completely clear,
the most likely mechanism is that self-reactive T cells fail
to undergo apoptosis in the peripheral immune system after
chronic stimulation. Studies have shown that T cells and B
cells must carry the lpr mutation in Fas for maximal auto-
antibody production to occur. Surface expression of Fas
on B cells may be important in preventing inappropriate
CD4 T cell–dependent expansion of autoreactive B cells
in the periphery. Although lpr and gld mice can develop
nephritis and other hallmarks of SLE on the MRL genetic
background, humans with ALPS develop primarily hema-
topoietic autoimmune disease, such as autoimmune hemo-
lytic anemia or idiopathic thrombocytopenic purpura, and
rarely produce antinuclear antibodies or develop nephritis,
presumably because of the need for other disease-modifying
genes. In addition, Fas or FasL mutations have not been
identified in cohorts of patients with SLE despite extensive
efforts to do so.
Mutations in the AIRE gene affect central T cell toler-
ance and lead to autoimmune disease in mice and humans.
AIRE is a transcription factor that specifically upregulates
transcription of genes in medullary thymic epithelial cells
that are otherwise expressed only in peripheral tissues.
Without AIRE expression, T cells developing in the thy-
mus with TCRs recognizing peptides derived from these
proteins would not be deleted during negative selection in
the thymus. Mutations in AIRE deficiency lead to the reces-
sive autoimmune polyendocrine syndrome 1 (APECED),
in which patients develop autoimmune disease affecting
multiple endocrine organs. AIRE-deficient mice develop
similar abnormalities. These findings show that peripheral
tolerance mechanisms cannot always compensate for lack of
deletion of T cells in the thymus in preventing T cell–medi-
ated autoimmune disease.
ENVIRONMENTAL TRIGGERS
AND INFLUENCES
It seems likely that environmental triggers or influences
interact with susceptibility genes to result in autoimmune
disease (see Fig. 15-2). The effect of the environment in
autoimmune disease has been extremely difficult to define,
however. In some diseases, this difficulty may reflect the fact
that such a trigger does not exist, or that it is ubiquitous in
different populations. Relevant to rheumatic diseases, one
example of the latter may be infection with Epstein-Barr
virus (EBV), which is highly prevalent in most adult popu-
lations worldwide. Geographic clustering not explained by
genetic variation strongly suggests an environmental effect.
Case clustering has been documented in multiple sclerosis
and type 1 diabetes, but not in SLE or RA.86-88 Case reports
and epidemiologic studies suggest that infection with par-
ticular viruses, such as congenital rubella or enteroviruses, is
associated with an increased risk of developing type 1 diabe-
tes in susceptible individuals.
Studies of animal models also have emphasized that sto-
chastic events are important in the development of autoim-
munity and expression of autoimmune disease. The fact that
approximately 75% of monozygotic twins are discordant for
SLE has been used as evidence to support the existence of an
environmental trigger in this disease. Lupus-prone strains of
mice also can be created, however, such that 25% reproduc-
ibly develop disease or produce particular autoantibodies,
and careful examination of autoimmune mice has excluded
environmental effects as the major factor in variable expres-
sion.17,36 In SLE-prone mice, eliminating bacterial com-
mensal antigens also had no effect on the development of
systemic autoimmunity.89 In these mice, the probability of
disease seems to be genetically determined, with random
events, including perhaps the generation of the BCR and
TCR repertoire, contributing to disease incidence.
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
Because of their ability to trigger prolonged immune
responses, infectious microorganisms are obvious candi-
dates to consider in the triggering of autoimmune diseases.36
Infections of target tissues could result in the release or
expression of normally sequestered antigens or in the ability
to present self-antigens that are normally prevented from
being exposed to the immune system.90 Closely tied to this
effect may be the release of inflammatory cytokines at the
infected site and surrounding lymphoid tissues, which would
mobilize and activate inflammatory cells to allow effective
presentation of foreign antigens and self-antigens. Ligands
for TLRs in infectious organisms and numerous cytokines
produced during infections, such as type I (αβ) interferons,
interferon-γ, and IL-12, all are known to activate DCs and
potentiate their ability to present foreign antigens and tis-
sue-derived self-antigens. As discussed earlier, inflammatory
cytokines produced in response to infection may dampen
the effectiveness of regulatory T cells and potentiate the
differentiation of T helper type 1 (Th1) and Th17 T cell
subsets associated with tissue damage in autoimmune dis-
eases. Infections also may induce an immune response to the
pathogen that cross-reacts with some host antigen.
Possible infectious triggers of RA and SLE have been vigor-
ously pursued. Conclusive data linking the presence of an eti-
ologic agent to disease have been elusive, however. One agent
that continues to generate interest in RA and SLE is EBV.91,92
Studies have shown that RA patients generate increased and
qualitatively different antibody responses and different T cell
responses to the virus and its effects. Studies also have shown
that a subset of clonally expanded CD8+ T cells in the joints
of patients is directed to EBV proteins. One study showed that
children with SLE have a higher prevalence of EBV infec-
tion than control groups.93 Which condition is predisposing
to the other has yet to be clarified. Environmental influences
on the expression of disease manifestations also are seen in
autoimmune diseases. In SLE, exacerbation of rash or systemic
symptoms after sun exposure and exacerbations of disease after
viral or bacterial infections have been observed in patients. It
is possible that the latter associations are related to the exacer-
bating influence of interferons on disease activity in SLE.94
Most autoimmune diseases, including RA, SLE, and
autoimmune thyroiditis, are more common in women than
men, suggesting a role for steroid hormones in the patho-
genesis of some autoimmune diseases. Changes in disease
activity also have been noted after administration of exog-
enous hormones. Epidemiologic data and studies of animal
models support the contention that estrogens can increase
the risk of developing SLE or exacerbate disease. A ran-
domized trial of combined estrogen-progesterone hormone
replacement therapy in postmenopausal women with SLE
showed that hormone replacement therapy increased mild,
but not severe, flares of disease.95 Long-term treatment of
patients with certain drugs (e.g., procainamide, hydralazine)
can induce the production of antinuclear antibodies and a
lupus-like disease. Drug-induced lupus seems to be a differ-
ent disease from SLE, however, with a distinct natural his-
tory, autoantibody profile, and genetic predisposition.
Other environmental triggers being studied in the patho-
genesis of autoimmune diseases include quantifiable risks,
such as cigarette smoke. A history of smoking increases the
probability of rheumatoid factor–positive and anti–cyclic
citrullinated protein–positive RA, especially in ­individuals
269
harboring high-risk HLA-DR alleles.96 The mechanisms
of how cigarette smoke influences RA pathogenesis are
unclear, but may depend on polymorphisms in glutathione
S-transferases, which are believed to have an important role
in detoxifying carcinogens in tobacco smoke.97
CELLULAR MECHANISMS
OF AUTOIMMUNE DISEASE
ROLE OF T CELLS IN INITIATING
AND REGULATING AUTOIMMUNITY
The TCR repertoire of early immature T cells seems to
depend solely on the random rearrangement of TCR genes.
This process culminates in the generation of “double-
positive” T cells bearing moderate levels of the TCR and
CD4 and CD8. Subsequently, two major processes in the
thymus modify this repertoire (see Chapter 9). Positive
selection allows cells bearing a TCR with low affinity for
self-MHC/peptides to mature, which “tunes” the mature
T cell repertoire to recognize foreign antigens in the context
of self-MHC (i.e., self-MHC restriction). Cells that are not
positively selected undergo programmed cell death within
the thymus, sometimes termed death by neglect. The other
intrathymic process deletes T cells that have a high level
of interaction with self-MHC/peptide complexes, termed
negative selection. Thymocytes whose TCR and CD4 mol-
ecules engage MHC class II/peptide complexes generally
mature into CD4+ T cells, whereas cells that are positively
selected on class I MHC molecules become the CD8+ popu-
lation. Mature thymocytes subsequently migrate to periph-
eral lymphoid tissues, where they maintain these surface
characteristics.
There is little evidence to support the contention that
defects in central (intrathymic) tolerance lead to systemic
autoimmune disease, such as SLE.98 Studies in mouse models
of SLE have repeatedly shown that high-affinity responses
to self-antigens are tolerized normally in the thymus. In
contrast, accumulating data suggest that defects in central
tolerance may underlie disease in some individuals with
organ-specific autoimmunity, such as patients with autoim-
mune endocrine diseases. Clonal deletion in the thymus
may be the major process for eliminating T cells reactive
to self-antigens with high affinity, as long as the antigen is
present or expressed in the thymus during T cell develop-
ment. Studies have indicated that numerous antigens pre-
viously thought to be organ specific and organ sequestered
are expressed and presented by thymic epithelial cells under
control of the transcriptional activator AIRE. Organ-specific
autoimmune disease, primarily directed at endocrine organs
resulting from genetic deficiencies in AIRE, validates the
role of central tolerance through this mechanism in prevent-
ing autoimmune diseases.99,100
There is abundant evidence that potentially autoreac-
tive T cells can mature and reach the periphery in most
individuals. Numerous “loopholes” in self-tolerance may
allow this. Some organ-sequestered antigens are never pre-
sented adequately in the thymus. In addition, some self-
peptides may not be processed and presented efficiently in
the thymus. T cells that escape negative selection against
these peptides may be activated in the periphery when
these peptides are created by altered proteolysis during
 270 SIEGEL  |  Autoimmunity
Table 15-3  Mechanisms of Peripheral T cell   well-known ability to upregulate costimulatory molecules
Tolerance
Mechanism of
Peripheral T cell
Tolerance Modes of Tolerance Breakdown
Immune ignorance Release of sequestered antigens
Aberrant expression of MHC class II
Increased expression of autoantigen/
MHC class II
Molecular mimicry
Epitope spreading
Anergy Release of inflammatory mediators
Increased expression or function of
costimulatory molecules
Suppression of IDO
Regulatory T cells Release of inflammatory mediators
Apoptosis Defects in apoptosis signaling
Viral apoptosis inhibitors
IDO, indoleamine 2,3-dioxygenase; MHC, major histocompatibility complex.
inflammation and by post-translational modifications of
peptides, such as glycosylation or citrullination.101,102 As
discussed previously, several well-studied animal models
are generated by immunization of animals with peripheral
organ antigens, such as type II collagen in collagen-induced
arthritis and myelin-associated proteins in ­ Experimental
Allergic Encephalomyelitis (EAE), which can engen-
der robust T cell responses in the presence of appropriate
adjuvants. In addition, immunization of normal mice with
nuclear antigens or peptides derived from these antigens
can result in lupus-like autoantibody production.29,103,104
These experiments indicate that peripheral autoreactive
T cells exist and likely need to be kept under control to
prevent autoimmunity.
Many cellular mechanisms prevent peripheral self-
reactive T cells from mediating autoimmune responses
(Table 15-3). T cells specific for antigens that are physically
sequestered or not efficiently presented are effectively “igno-
rant” of self-antigen. This type of immune ignorance has
been seen in numerous TCR transgenic mouse models when
the antigen recognized by the TCR is specifically expressed
in a particular target organ, such as the pancreas, in which
T cells do not regularly traffic.105 If no other mechanisms
are active, this type of tolerance can be reversed easily in
animal models by the induction of inflammation in the
self-antigen–expressing tissue, which can reverse immune
ignorance by activating antigen-presenting cells to process
and present antigens from the tissue, and upregulation of
MHC class I and II molecules on tissue cells. Cross-reactive
peptides from infectious organisms also can reverse this type
of T cell tolerance.
Often, T cell tolerance is maintained through more
active processes. Activation of T cells without costimula-
tion through receptors such as CD28 can induce a reversible
state of unresponsiveness to TCR stimulation, termed T cell
anergy or abortive T cell activation, resulting in postactiva-
tion cell death through apoptosis.106,107 This state of unre-
sponsiveness may occur when self-antigens are presented to
T cells under noninflammatory conditions or by cells that
cannot make the CD28 ligands B7-1 (CD80) and B7-2
(CD86). Inflammatory cytokines, such as TNF and inter-
feron-γ, can reverse this type of tolerance through their
such as on antigen-presenting cells.
Other molecules in the CD28 costimulatory family,
such as CTLA4, bind B7-1 and B7-2 with higher affin-
ity than CD28, but deliver a negative signal to T cells.
This function of CTLA4 is required to keep autoreactive
T cells from becoming activated, as dramatically illustrated
by the T cell hyperactivation and infiltration of multiple
organs that results in death of CTLA4-deficient mice.108
Blockade of T cell costimulation can be effected with a
variety of agents, including a fusion protein of CTLA4 with
immunoglobulin (CTLA4-Ig). Additional costimulatory
­pathways that are relevant to autoimmunity include the
PD-1 interaction with its ligand, which also delivers a neg-
ative regulatory signal to chronically activated T cells.76-
78 Deficiencies of PD-1 have been implicated in lupus-like
disease in animals36 and as a possible gene contribution to
SLE.37 Members of the TNF family, including LIGHT, 4-
1BB, OX40L, and CD70, also have costimulatory activity
on T cells, which can regulate autoimmunity.109,110
Another mechanism that can induce proliferative arrest
and unresponsiveness in T cells is the secretion of the tryp-
tophan metabolizing enzyme indoleamine 2,3-dioxygen-
ase (IDO). Originally described as an enzyme secreted by
trophoblasts and macrophages that prevents rejection of
allogeneic fetuses,111 IDO has now been recognized to be
synthesized by plasmacytoid DCs in the mouse and in human
DCs after stimulation through B7 costimulatory molecules,
which can be ligated by CTLA4-Ig,112 although the corre-
lation between IDO expression and regulatory function of
DCs has been questioned.113 IDO catabolizes tryptophan,
which can induce local tryptophan deficiency and induce
proliferative arrest in T cells through the induction of the
stress-responsive kinase GCN2.114 A more permanent form
of T cell tolerance occurs in the setting of chronic exposure
to antigens, which leads to apoptosis through the action of
surface receptors such as Fas and likely underlies the predis-
position to autoimmunity that occurs in Fas-deficient ani-
mals and patients with the ALPS.115
Considerable evidence has emerged in recent years that
certain subsets of classic αβ TCR–expressing T cells have
the ability to suppress responder T cell proliferation and
in vivo T cell responses to self-antigens and foreign anti-
gens.116,117 NK cells and NK/T cells also possess intrinsic
self-reactivity and may participate in the regulation of auto-
immunity.118 Natural regulatory T cells are a subset of 5%
to 10% of peripheral CD4+ cells with a repertoire enriched
in self-reactive specificities. These T cells can be identified
by their constitutive expression of high levels of the T cell
activation marker CD25 and CTLA4. Natural regulatory
T cells express a unique transcription factor, FoxP3, which
confers many of their properties, such as poor proliferation
when activated and the ability to suppress proliferation and
cytokine secretion by other T cells activated in coculture
with natural regulatory T cells, in a cell contact–dependent,
but non–antigen-specific manner.119 Genetic deficiency in
regulatory T cells secondary to mutations in FoxP3 in the
scurfy mouse and the human familial syndrome IPEX results
in systemic autoimmune disease,120 and more recent experi-
ments in animal models show that experimental elimina-
tion of regulatory T cells in adult animals can lead quickly
to autoimmune disease.121
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
Regulatory T cells also may have a physiologic role in
dampening immune responses against pathogens. Other reg-
ulatory T cell populations that may be involved in control-
ling regulating peripheral anergy include IL-10 producing
“Tr1” cells and transforming growth factor (TGF)-β produc-
ing Th3 cells. These populations do not express FoxP3 and
suppress by means of their secreted cytokines.122 Given this
evidence, it has been tempting to propose that human auto-
immune diseases may be treated by increasing the produc-
tion of regulatory T cells, and such strategies have worked in
the mouse to reduce diseases induced by autoreactive T cells
in the setting of regulatory T cell deficiencies. Regulatory
T cells have been found in abundance, however, in the set-
ting of autoimmune inflammation, such as in the pancreatic
lymph nodes and islets of mice with autoimmune diabetes
and in the inflamed synovium,123,124 suggesting that simply
increasing their numbers may not be sufficient.
A large body of evidence suggests that T cells are required
for the full expression of most rheumatic and other autoim-
mune diseases.125 In particular, HLA associations, the pres-
ence of infiltrating CD4+ T cells at the sites of pathology in
various organ-specific autoimmune diseases, and evidence
for T cell help in the repertoire of autoantibodies all point
to a role for T cell help in autoimmune disease. Pathogenic
autoantibodies in SLE exhibit isotype switching and somatic
mutation that are hallmarks of T cell help.98 Most animal
models of lupus, type 1 diabetes, and antigen-induced auto-
immune disease can be prevented and in most cases ame-
liorated by T cell depletion. In humans, this has been more
difficult to accomplish, but antibodies against the TCR are
effective in treating acute graft rejection and new-onset
type 1 diabetes.126,127
The importance of CD4+ T cells in the initiation and
perpetuation of RA has been a controversial issue, mostly
related to studies showing that T cell–derived cytokines are
scarce in synovium compared with cytokines derived from
macrophage or fibroblast-like synoviocytes, and related to
the failure to define the antigens to which the synovial
T cells are responding.128 This situation led to the concept
that T cells may be important in initiating disease in RA, but
more dispensable in later stages of the disease.60 Although
small trials of CD4+ T cell depletion in RA showed limited
efficacy and significant toxicity, blockade of T costimulation
with CTLA4-Ig (abatacept) has proved to be efficacious in
RA.129,130 The success of costimulatory T cell blockade in
early and advanced RA validates the role of T cells in RA
pathogenesis.
ROLE OF B CELLS
B cell repertoire formation in the fetal liver and adult bone
marrow is random, and B cells with self-reactivity are gen-
erated. Negative selection of some autoreactive B cells,
involving deletion and anergy, is a normal part of B cell
development (see Chapter 10). A separate mechanism has
been described in which immature B cells with specificity
for self-antigens can modify their receptors through a pro-
cess termed receptor editing.131 In contrast to T cells, mature
B cells also have the ability to modify their BCRs in the
peripheral lymphoid tissues through a process of somatic
mutation (see Chapter 10). This process allows a secondary
immune response to generate antibodies with higher affinity
271
for the stimulating antigen, but offers additional opportu-
nity for the generation of self-reactive B cells.
Multiple checkpoints are involved in the prevention
of activation of autoreactive B cells in the peripheral lym-
phoid tissues.132 Autoreactive B cells are part of the normal
peripheral B cell repertoire, and defects in central B cell
­tolerance do not seem to be necessary to allow for patho-
genic autoantibody production.98 The escape of autoreac-
tive T cells secondary to intrathymic deficiency of AIRE is
sufficient for the subsequent development of autoantibod-
ies to multiple organs.100 The transfer of alloreactive CD4+
T cells and generation of chronic graft-versus-host disease
causes pathogenic lupus-like autoantibody production in
normal recipient animals.133 The ability of normal animals
to generate diverse antinuclear antibody responses after
immunization with one nuclear antigen is further support of
this conclusion.103,104 Similar to regulation of autoreactive
T cells, studies suggest that regulation of B cells in the periph-
eral lymphoid tissues may be important for the prevention
of B cell autoimmunity. B cells seem, however, to be more
important in the development of autoimmune disease than
just as a source of autoantibodies, with important functions
in cytokine production and antigen presentation.134,135
Studies in murine lupus have indicated that T cell acti-
vation in some circumstances may depend on the presence
of B cells136; this also may be a mechanism why B cells
are important for continued disease activity in RA. The
antigen-presenting function of B cells is likely to be impor-
tant in the broadening of autoantibody repertoire that
occurs during the progression of autoantibody disease that
is termed epitope spreading. Autoantigen-specific B cells
can promote epitope spreading because they can internal-
ize macromolecular self-antigen complexes through their
autoreactive BCR and efficiently process and present
linked autoantigen epitopes to T cells, allowing T cell help
to develop against “spread” epitopes.137 Epitope spreading
may explain how a response to one epitope can mature into
a full-blown autoimmune response.
INFLUENCE OF ANTIGEN PRESENTING AND
TISSUE ENVIRONMENT ON AUTOIMMUNITY
It has been increasingly evident that the manner and envi-
ronment in which T cells and B cells are activated can have
profound effects on their subsequent differentiation and
susceptibility to peripheral tolerance mechanisms.138,139
Stimuli derived from different pathogens can instruct DCs
to differentiate into different subtypes that prime T cells to
become different effector subtypes. Schistosome egg anti-
gen influences DCs to prime T cells to differentiate into
Th2 effector cells secreting IL-4, which is important for
immunity against this extracellular parasite, whereas DCs
exposed to toxoplasmosis-derived antigens strongly bias
T cells toward differentiation into Th1 cells that secrete
interferon-γ. DCs presenting self-antigen without acti-
vation or through alternative activation pathways can
induce T cell anergy or promote T cell differentiation into
IL-10-producing or FoxP3-positive regulatory T cells. The
cytokine TGF-β seems to be crucial for this alternative
activation pathway, at least in animal models.
Major differences between tissues in the responsiveness
and cytokine secretion patterns of immune and nonimmune
 272 SIEGEL  |  Autoimmunity
cells also are important in the control of immunity and auto-
immunity.140 Resident cells in the lung make large amounts
of the suppressive enzyme IDO when stimulated by micro-
bial DNA. By contrast, the spleen makes little IDO after
challenge with the same stimuli.141 These different patterns
of responsiveness most likely evolved to allow organ-specific
responses to pathogens by tissues that are exposed to com-
mensal bacteria in different ways, but also likely affect the
manifestations of autoimmunity in a tissue-specific way. The
influence of the synovium on immune responses is likely rel-
evant to inflammatory joint disease.
HELPER T CELL SUBSETS AND THE CYTOKINE
NETWORK
Although more sharply defined in mice than in humans,
naive T cells, after activation, may evolve into multiple sub-
types of helper T cells, distinguished by the cytokines that
they produce. The first two subsets to be defined, Th1 and
Th2, secrete different patterns of cytokines, with Th1 cells
producing predominantly interferon-γ, TNF, lymphotoxin,
and other inflammatory cytokines, and Th2 cells ­producing
IL-4, IL-5, IL-10, and IL-13. The DC-derived cytokine IL-
12 is crucial for the induction of Th1 cells. Th1 and Th2
cells reciprocally regulate each other, such that Th1-derived
cytokines repress Th2 differentiation and vice versa.142-144
Also, these two effector T cell subsets express specific tran-
scription factors that program and maintain these effector
cell subtypes, largely preventing conversion from one to
another.
In host defense, Th1 cells primarily enhance cell-mediated
inflammatory immune responses, such as delayed-type hyper-
sensitivity reactions, which frequently involve activation of
macrophages and effector T cells. The ability to mediate an
effective immune response against certain intracellular patho-
gens seems to depend strongly on the generation of a Th1
response. In contrast, Th2 cells mainly provide help for B cells
by promoting class switching and enhancing the production
of certain IgG isotypes and production of IgE, including in
allergic diseases. T cells producing the cytokine IL-21 also may
be important in promoting B cell functions.145 The Th2 cyto-
kines IL-4 and IL-10 also can function to limit macrophage
activity,146 and Th2 cells may negatively regulate inflamma-
tion in autoimmune disease.
Although Th1 cells were associated with organ-specific
autoimmune disease models such as CIA, EAE (multiple
sclerosis), and other induced diseases, more recent findings
in mouse models have shown that another T helper subset
that produces the cytokine IL-17 (Th17) is required for the
development of many of these diseases, including CIA and
EAE.147 IL-23, a cytokine that shares a common p40 subunit
with IL-12, but also uses a unique p19 subunit, is important
for the maintenance of Th17 cells, and blocking antibodies
against the IL-23-specific p19 subunit or genetic deletion
of p19 block the development of CIA, EAE, and T cell–
dependent models of inflammatory bowel disease.148-152 In
mice, there may be a reciprocal relationship between Th17
and regulatory T cells in that TGF-β can promote devel-
opment of FoxP3-positive T cells from naive T cells, but
addition of inflammatory cytokines such as IL-6 and TNF to
TGF-β promotes Th17 cell development.153,154 How these
different cytokines and T helper subsets influence human
autoimmune disease is just beginning to be worked out, but
it is notable that large amounts of IL-17 can be detected at
sites of inflammation, such as rheumatoid synovium.155,156
Blockade of p40, which makes up IL-12 and IL-23, has
proved to be effective in treating human inflammatory
bowel disease.157 In cases in which genetic lesions result in
overproduction of specific cytokines, such as IL-1 produc-
tion in the neonatal-onset multisystem inflammatory dis-
order, blocking IL-1 produces prompt and almost complete
remission of symptoms,158 but in sporadic autoimmune or
inflammatory disease, such treatments are unlikely to be as
universally effective. Therapies that modulate cytokine pro-
duction and action are potentially powerful immunostimu-
lants or suppressants and need to be tested with caution, but
could be an important addition to the armamentarium of
antirheumatic treatments.
CONCLUSION
The development of autoimmune diseases is a complex
process. Although knowledge regarding different aspects
of the immunopathogenesis of these diseases, especially
related to studies of animal models, has advanced dramati-
cally in recent years, major gaps in knowledge of human
autoimmune disease pathogenesis persist. Perhaps most
poorly understood are the mechanisms involved in the ini-
tial breakdown of self-tolerance. The cellular immunologic
abnormalities involved in the initiation and perpetuation of
disease also need much greater definition. The identification
and understanding of susceptibility genes and possible envi-
ronmental or infectious triggers should provide insights into
these questions. Understanding these processes also would
provide new directions for prophylaxis and treatment of dis-
ease. Much better understood are the later events in autoim-
mune disease pathogenesis, such as how autoantibodies or
cellular immune-mediated damage leads to disease manifes-
tations. Considering the remarkable increase in information
regarding the normal immune system, immunity, and the
genetic basis of complex traits, it is likely that our under-
standing of human autoimmunity will greatly increase in
the near future.
REFERENCES
  1. Medzhitov R, Janeway CA Jr: Innate immunity: The virtues of a
nonclonal system of recognition. Cell 91:295-298, 1997.
  2. Medzhitov R, Janeway CA Jr: Innate immune recognition and
­control of adaptive immune responses. Semin Immunol 10:351-353,
1998.
  3. Kamradt T, Lengl-Janssen B, Strauss AF, et al: Dominant recog-
nition of a Borrelia burgdorferi outer surface protein A peptide by
T helper cells in patients with treatment-resistant Lyme arthritis.
Infect Immun 64:1284-1289, 1996.
  4. Gross DM, Forsthuber T, Tary-Lehmann M, et al: Identification of
LFA-1 as a candidate autoantigen in treatment-resistant Lyme arthri-
tis. Science 281:703-706, 1998.
  5. Plotz PH: The autoantibody repertoire: Searching for order. Nat Rev
Immunol 3:73-78, 2003.
6. Martinon F, Petrilli V, Mayor A, et al: Gout-associated uric acid
crystals activate the NALP3 inflammasome. Nature 440:237-241,
2006.
  7. Martinon F, Agostini L, Meylan E, et al: Identification of bacterial
muramyl dipeptide as activator of the NALP3/cryopyrin inflamma-
some. Curr Biol 14:1929-1934, 2004.
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
  8. Allegretta M, Nicklas JA, Sriram S, et al: T cells responsive to
myelin basic protein in patients with multiple sclerosis. Science 247:
718-721, 1990.
  9. Zhang J, Markovic-Plese S, Lacet B, et al: Increased frequency of
interleukin 2-responsive T cells specific for myelin basic protein and
proteolipid protein in peripheral blood and cerebrospinal fluid of
patients with multiple sclerosis. J Exp Med 179:973-984, 1994.
10. McGonagle D, McDermott MF: A proposed classification of the
immunological diseases. PLoS Med 3:e297, 2006.
11. Arbuckle MR, McClain MT, Rubertone MV, et al: Development of
autoantibodies before the clinical onset of systemic lupus erythema-
tosus. N Engl J Med 349:1526-1533, 2003.
12. Linn-Rasker SP, van der Helm-van Mil AH, van Gaalen FA, et al:
Smoking is a risk factor for anti-CCP antibodies only in rheumatoid
arthritis patients who carry HLA-DRB1 shared epitope alleles. Ann
Rheum Dis 65:366-371, 2006.
13. Frisoni L, McPhie L, Colonna L, et al: Nuclear autoantigen translo-
cation and autoantibody opsonization lead to increased dendritic cell
phagocytosis and presentation of nuclear antigens: A novel patho-
genic pathway for autoimmunity? J Immunol 175:2692-2701, 2005.
14. Valencia X, Stephens G, Goldbach-Mansky R, et al: TNF downmod-
ulates the function of human CD4+CD25+ T-regulatory cells. Blood
108:253-261, 2006.
15. Pasare C, Medzhitov R: Toll pathway-dependent blockade of
CD4+CD25+ T cell-mediated suppression by dendritic cells. Science
299:1033-1036, 2003.
16. Anderson MS, Bluestone JA: The NOD mouse: A model of immune
dysregulation. Annu Rev Immunol 23:447-485, 2005.
17. Vyse TJ, Kotzin BL: Genetic susceptibility to systemic lupus ery-
thematosus. Annu Rev Immunol 16:261-292, 1998.
18. Wakeland EK, Liu K, Graham RR, et al: Delineating the genetic
basis of systemic lupus erythematosus. Immunity 15:397-408, 2001.
19. Kumar KR, Li L, Yan M, et al: Regulation of B cell tolerance by the
lupus susceptibility gene Ly108. Science 312:1665-1669, 2006.
20. Wandstrat AE, Nguyen C, Limaye N, et al: Association of extensive
polymorphisms in the SLAM/CD2 gene cluster with murine lupus.
Immunity 21:769-780, 2004.
21. Rozzo SJ, Allard JD, Choubey D, et al: Evidence for an ­interferon-
inducible gene, Ifi202, in the susceptibility to systemic lupus.
­Immunity 15:435-443, 2001.
22. Pisitkun P, Deane JA, Difilippantonio MJ, et al: Autoreactive
B cell responses to RNA-related antigens due to TLR7 gene dupli-
cation. Science 312:1669-1672, 2006.
23. Subramanian S, Tus K, Li QZ, et al: A Tlr7 translocation accelerates
systemic autoimmunity in murine lupus. Proc Natl Acad Sci U S A
103:9970-9975, 2006.
24. Christensen SR, Shupe J, Nickerson K, et al: Toll-like receptor 7 and
TLR9 dictate autoantibody specificity and have opposing inflam-
matory and regulatory roles in a murine model of lupus. Immunity
25:417-428, 2006.
25. Cohen PL, Eisenberg RA: Lpr and gld: single gene models of ­systemic
autoimmunity and lymphoproliferative disease. Annu Rev Immunol
9:243-269, 1991.
26. Fisher GH, Rosenberg FJ, Straus SE, et al: Dominant interfering Fas
gene mutations impair apoptosis in a human autoimmune lympho­
proliferative syndrome. Cell 81:935-946, 1995.
27. Ramsey C, Winqvist O, Puhakka L, et al: Aire deficient mice develop
multiple features of APECED phenotype and show altered immune
response. Hum Mol Genet 11:397-409, 2002.
28. Sakaguchi N, Takahashi T, Hata H, et al: Altered thymic T-cell
selection due to a mutation of the ZAP-70 gene causes autoimmune
arthritis in mice. Nature 426:454-460, 2003.
29. Topfer F, Gordon T, McCluskey J: Intra- and intermolecular spread-
ing of autoimmunity involving the nuclear self-antigens La (SS-B)
and Ro (SS-A). Proc Natl Acad Sci U S A 92:875-879, 1995.
30. Durie FH, Fava RA, Noelle RJ: Collagen-induced arthritis as a model
of rheumatoid arthritis. Clin Immunol Immunopathol 73:11-18, 1994.
31. Griffiths MM, Remmers EF: Genetic analysis of collagen-induced
arthritis in rats: A polygenic model for rheumatoid arthritis predicts
a common framework of cross-species inflammatory/autoimmune
disease loci. Immunol Rev 184:172-183, 2001.
32. Zhang Y, Guerassimov A, Leroux JY, et al: Arthritis induced by pro-
teoglycan aggrecan G1 domain in BALB/c mice. Evidence for T cell
involvement and the immunosuppressive influence of keratan sulfate
on recognition of T and B cell epitopes. J Clin Invest 101:1678-1686,
1998.
273
33. Kuhn KA, Kulik L, Tomooka B, et al: Antibodies against citrulli-
nated proteins enhance tissue injury in experimental autoimmune
arthritis. J Clin Invest 116:961-973, 2006.
34. Powrie F, Correa-Oliveira R, Mauze S, et al: Regulatory interactions
between CD45RBhigh and CD45RBlow CD4+ T cells are important
for the balance between protective and pathogenic cell-mediated
immunity. J Exp Med 179:589-600, 1994.
35. Izcue A, Coombes JL, Powrie F: Regulatory T cells suppress systemic
and mucosal immune activation to control intestinal inflammation.
Immunol Rev 212:256-271, 2006.
36. Marrack P, Kappler J, Kotzin BL: Autoimmune disease: Why and
where it occurs. Nat Med 7:899-905, 2001.
37. Davidson A, Diamond B: Autoimmune diseases. N Engl J Med
345:340-350, 2001.
38. Bolland S, Ravetch JV: Spontaneous autoimmune disease in
Fc(gamma)RIIB-deficient mice results from strain-specific epistasis.
Immunity 13:277-285, 2000.
39. Napirei M, Karsunky H, Zevnik B, et al: Features of systemic lupus
erythematosus in DNASE1-deficient mice. Nat Genet 25:177-181,
2000.
40. Yasutomo K, Horiuchi T, Kagami S, et al: Mutation of DNASE1 in
people with systemic lupus erythematosus. Nat Genet 28:313-314,
2001.
41. Nishimura H, Nose M, Hiai H, et al: Development of lupus-like
­autoimmune diseases by disruption of the PD-1 gene encoding an
ITIM motif-carrying immunoreceptor. Immunity 11:141-151, 1999.
42. Prokunina L, Castillejo-Lopez C, Oberg F, et al: A regulatory
­polymorphism in PDCD1 is associated with susceptibility to systemic
lupus erythematosus in humans. Nat Genet 32:666-669, 2002.
43. Matsumoto I, Staub A, Benoist C, et al: Arthritis provoked by
linked T and B cell recognition of a glycolytic enzyme. Science 286:
1732-1735, 1999.
44. Kouskoff V, Korganow AS, Duchatelle V, et al: Organ-specific
­disease provoked by systemic autoimmunity. Cell 87:811-822, 1996.
45. von Boehmer H, Teh HS, Kisielow P: The thymus selects the useful,
neglects the useless and destroys the harmful. Immunol Today 10:
57-61, 1989.
46. Wang H, Schlomchik MJ: Regulation of autoreactive anti-IgG
(­rheumatoid factor) B cells in normal and autoimmune mice.
­Immunol Res 19(2-3):259-270, 1999.
47. Erikson J, Mandik L, Bui A, et al: Self-reactive B cells in nonautoim-
mune and autoimmune mice. Immunol Res 17(1-2):49-61, 1998.
48. Vinuesa CG, Cook MC, Angelucci C, et al: A RING-type ubiquitin
ligase family member required to repress follicular helper T cells and
autoimmunity. Nature 435:452-458, 2005.
49. Gregersen PK: Teasing apart the complex genetics of human auto­
immunity: Lessons from rheumatoid arthritis. Clin Immunol 107:1-9,
2003.
50. Oksenberg JR, Barcellos LF, Hauser SL: Genetic aspects of multiple
sclerosis. Semin Neurol 19:281-288, 1999.
51. Onengut-Gumuscu S, Concannon P: Mapping genes for auto­
immunity in humans: Type 1 diabetes as a model. Immunol Rev 190:
182-194, 2002.
52. Wandstrat A, Wakeland E: The genetics of complex autoim-
mune diseases: Non-MHC susceptibility genes. Nat Immunol 2:
802-809, 2001.
53. Gabriel SB, Schaffner SF, Nguyen H, et al: The structure of haplo-
type blocks in the human genome. Science 296:2225-2229, 2002.
54. Daly MJ, Rioux JD, Schaffner SF, et al: High-resolution haplotype
structure in the human genome. Nat Genet 29:229-232, 2001.
55. Palmer LJ, Cardon LR: Shaking the tree: Mapping complex disease
genes with linkage disequilibrium. Lancet 366:1223-1234, 2005.
56. Spurkland A, Sollid LM: Mapping genes and pathways in auto­
immune disease. Trends Immunol 27:336-342, 2006.
57. Jawaheer D, Seldin MF, Amos CI, et al: A genomewide screen in
multiplex rheumatoid arthritis families suggests genetic overlap with
other autoimmune diseases. Am J Hum Genet 68:927-936, 2001.
58. Jawaheer D, Li W, Graham RR, et al: Dissecting the genetic
­complexity of the association between human leukocyte antigens
and rheumatoid arthritis. Am J Hum Genet 71:585-594, 2002.
59. Turesson C, Schaid DJ, Weyand CM, et al: The impact of HLA-
DRB1 genes on extra-articular disease manifestations in rheumatoid
arthritis. Arthritis Res Ther 7:R1386-R1393, 2005.
60. Firestein GS: Evolving concepts of rheumatoid arthritis. Nature
423:356-361, 2003.
 274 SIEGEL  |  Autoimmunity
61. Graham RR, Ortmann WA, Langefeld CD, et al: Visualizing human
leukocyte antigen class II risk haplotypes in human systemic lupus
erythematosus. Am J Hum Genet 71:543-553, 2002.
62. Atagunduz P, Appel H, Kuon W, et al: HLA-B27-restricted CD8+
T cell response to cartilage-derived self peptides in ankylosing spon-
dylitis. Arthritis Rheum 52:892-901, 2005.
63. Sieper J: Disease mechanisms in reactive arthritis. Curr Rheumatol
Rep 6:110-116, 2004.
64. Sieper J, Braun J, Rudwaleit M, et al: Ankylosing spondylitis: An
overview. Ann Rheum Dis 61(Suppl 3):iii8-iii18, 2002.
65. Mear JP, Schreiber KL, Munz C, et al: Misfolding of HLA-B27 as
a result of its B pocket suggests a novel mechanism for its role in
susceptibility to spondyloarthropathies. J Immunol 163:6665-6670,
1999.
66. Dangoria NS, DeLay ML, Kingsbury DJ, et al: HLA-B27 misfolding
is associated with aberrant intermolecular disulfide bond ­formation
(dimerization) in the endoplasmic reticulum. J Biol Chem 277:
23459-23468, 2002.
67. Tran TM, Satumtira N, Dorris ML, et al: HLA-B27 in transgenic
rats forms disulfide-linked heavy chain oligomers and multimers that
bind to the chaperone BiP. J Immunol 172:5110-5119, 2004.
68. Tran TM, Dorris ML, Satumtira N, et al: Additional human
beta2-microglobulin curbs HLA-B27 misfolding and promotes arthri-
tis and spondylitis without colitis in male HLA-B27-transgenic rats.
Arthritis Rheum 54:1317-1327, 2006.
69. Walport MJ: Complement: First of two parts. N Engl J Med 344:
1058-1066, 2001.
70. Tsukumo S, Yasutomo K: DNaseI in pathogenesis of systemic lupus
erythematosus. Clin Immunol 113:14-18, 2004.
71. Salmon JE, Millard S, Schachter LA, et al: Fc gamma RIIA alleles are
heritable risk factors for lupus nephritis in African Americans. J Clin
Invest 97:1348-1354, 1996.
72. Edberg JC, Langefeld CD, Wu J, et al: Genetic linkage and asso-
ciation of Fcgamma receptor IIIA (CD16A) on chromosome 1q23
with human systemic lupus erythematosus. Arthritis Rheum 46:
2132-2140, 2002.
73. Pritchard NR, Cutler AJ, Uribe S, et al: Autoimmune-prone mice
share a promoter haplotype associated with reduced expression and
function of the Fc receptor FcgammaRII. Curr Biol 10:227-230,
2000.
74. Xiang Z, Cutler AJ, Brownlie RJ, et al: FcgammaRIIb controls bone
marrow plasma cell persistence and apoptosis. Nat Immunol 8:
419-429, 2007.
75. Theofilopoulos AN: The basis of autoimmunity, Part II: Genetic
­predisposition. Immunol Today 16:150-159, 1995.
76. Kelly JA, Moser KL, Harley JB: The genetics of systemic lupus
­erythematosus: Putting the pieces together. Genes Immunity 3(Suppl
1):S71-S85, 2002.
77. Ueda H, Howson JM, Esposito L, et al: Association of the T-cell
regulatory gene CTLA4 with susceptibility to autoimmune disease.
Nature 423:506-511, 2003.
78. Lesage S, Goodnow CC: Organ-specific autoimmune disease: A
­deficiency of tolerogenic stimulation. J Exp Med 194:F31-F36, 2001.
79. Begovich AB, Carlton VE, Honigberg LA, et al: A missense single-
nucleotide polymorphism in a gene encoding a protein tyrosine
phosphatase (PTPN22) is associated with rheumatoid arthritis. Am J
Hum Genet 75:330-337, 2004.
80. Michou L, Lasbleiz S, Rat AC, et al: Linkage proof for PTPN22, a
rheumatoid arthritis susceptibility gene and a human autoimmunity
gene. Proc Natl Acad Sci U S A 104:1649-1654, 2007.
81. Bottini N, Musumeci L, Alonso A, et al: A functional variant of
­lymphoid tyrosine phosphatase is associated with type I diabetes. Nat
Genet 36:337-338, 2004.
82. Vang T, Congia M, Macis MD, et al: Autoimmune-associated
­lymphoid tyrosine phosphatase is a gain-of-function variant. Nat
Genet 37:1317-1319, 2005.
83. Ogura Y, Bonen DK, Inohara N, et al: A frameshift mutation in
NOD2 associated with susceptibility to Crohn’s disease. Nature
411:603-606, 2001.
84. Duerr RH, Taylor KD, Brant SR, et al: A genome-wide associa-
tion study identifies IL23R as an inflammatory bowel disease gene.
­Science 314:1461-1463, 2006.
85. Straus SE, Sneller M, Lenardo MJ, et al: An inherited disorder
of ­ lymphocyte apoptosis: The autoimmune lymphoproliferative
­syndrome. Ann Intern Med 130:591-601, 1999.
86. Notkins AL, Lernmark A: Autoimmune type 1 diabetes: Resolved
and unresolved issues. J Clin Invest 108:1247-1252, 2001.
87. Steinman L: Multiple sclerosis: A coordinated immunological attack
against myelin in the central nervous system. Cell 85:299-302,
1996.
88. Wynn DR, Rodriguez M, O’Fallon WM, et al: Update on the epide-
miology of multiple sclerosis. Mayo Clin Proc 64:808-817, 1989.
89. Maldonado MA, Kakkanaiah V, MacDonald GC, et al: The role of
environmental antigens in the spontaneous development of autoim-
munity in MRL-lpr mice. J Immunol 162:6322-6330, 1999.
90. Matzinger P: The danger model: A renewed sense of self. Science
296:301-305, 2002.
91. McClain MT, Heinlen LD, Dennis GJ, et al: Early events in lupus
humoral autoimmunity suggest initiation through molecular
mimicry. Nat Med 11:85-89, 2005.
92. James JA, Neas BR, Moser KL, et al: Systemic lupus erythematosus
in adults is associated with previous Epstein-Barr virus exposure.
­Arthritis Rheum 44:1122-1126, 2001.
93. McClain MT, Poole BD, Bruner BF, et al: An altered immune
response to Epstein-Barr nuclear antigen 1 in pediatric systemic lupus
erythematosus. Arthritis Rheum 54:360-368, 2006.
94. Blanco P, Palucka AK, Gill M, et al: Induction of dendritic cell
­differentiation by IFN-alpha in systemic lupus erythematosus.
­Science 294:1540-1543, 2001.
95. Buyon JP, Petri MA, Kim MY, et al: The effect of combined estrogen
and progesterone hormone replacement therapy on disease activity
in systemic lupus erythematosus: A randomized trial. Ann Intern
Med 142(12 Pt 1):953-962, 2005.
96. Mattey DL, Dawes PT, Clarke S, et al: Relationship among the
HLA-DRB1 shared epitope, smoking, and rheumatoid factor produc-
tion in rheumatoid arthritis. Arthritis Rheum 47:403-407, 2002.
97. Criswell LA, Saag KG, Mikuls TR, et al: Smoking interacts with
genetic risk factors in the development of rheumatoid arthritis among
older Caucasian women. Ann Rheum Dis 65:1163-1167, 2006.
98. Kotzin BL: Systemic lupus erythematosus. Cell 85:303-306, 1996.
99. Liston A, Lesage S, Wilson J, et al: Aire regulates negative selection
of organ-specific T cells. Nat Immunol 4:350-354, 2003.
100. Anderson MS, Venanzi ES, Klein L, et al: Projection of an immu-
nological self shadow within the thymus by the aire protein.
­Science 298:1395-1401, 2002.
101. Casciola-Rosen L, Andrade F, Ulanet D, et al: Cleavage by gran-
zyme B is strongly predictive of autoantigen status: Implications for
­initiation of autoimmunity. J Exp Med 190:815-826, 1999.
102. Backlund J, Carlsen S, Hoger T, et al: Predominant selection of
T cells specific for the glycosylated collagen type II epitope (263-270)
in humanized transgenic mice and in rheumatoid arthritis. Proc Natl
Acad Sci U S A 99:9960-9965, 2002.
103. Bockenstedt LK, Gee RJ, Mamula MJ: Self-peptides in the initiation
of lupus autoimmunity. J Immunol 154:3516-3524, 1995.
104. Craft J, Fatenejad S: Self antigens and epitope spreading in systemic
autoimmunity. Arthritis Rheum 40:1374-1382, 1997.
105. Ohashi PS, Oehen S, Buerki K, et al: Ablation of “tolerance” and
induction of diabetes by virus infection in viral antigen transgenic
mice. Cell 65:305-317, 1991.
106. Schwartz RH, Mueller DL, Jenkins MK, et al: T-cell clonal anergy.
Cold Spring Harbor Symp Quant Biol 54(Pt 2):605-610, 1989.
107. Van Parijs L, Ibraghimov A, Abbas AK: The roles of costimulation
and Fas in T cell apoptosis and peripheral tolerance. Immunity 4:
321-328, 1996.
108. Sharpe AH, Abbas AK: T-cell costimulation—biology, therapeutic
potential, and challenges. N Engl J Med 355:973-975, 2006.
109. Bansal-Pakala P, Jember AG, Croft M: Signaling through OX40
(CD134) breaks peripheral T-cell tolerance. Nat Med 7:907-912,
2001.
110. Croft M: Co-stimulatory members of the TNFR family: Keys to
­effective T-cell immunity? Nat Rev Immunol 3:609-620, 2003.
111. Munn DH, Zhou M, Attwood JT, et al: Prevention of allogeneic fetal
rejection by tryptophan catabolism. Science 281:1191-1193, 1998.
112. Munn DH, Sharma MD, Mellor AL: Ligation of B7-1/B7-2 by
human CD4+ T cells triggers indoleamine 2,3-dioxygenase activity
in ­dendritic cells. J Immunol 172:4100-4110, 2004.
113. Terness P, Chuang JJ, Bauer T, et al: Regulation of human auto- and
alloreactive T cells by indoleamine 2,3-dioxygenase (IDO)-­producing
dendritic cells: Too much ado about IDO? Blood 105:2480-2486,
2005.
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
114. Munn DH, Sharma MD, Baban B, et al: GCN2 kinase in T cells
mediates proliferative arrest and anergy induction in response to
indoleamine 2,3-dioxygenase. Immunity 22:633-642, 2005.
115. Siegel RM, Chan FK, Chun HJ, et al: The multifaceted role of
Fas ­ signaling in immune cell homeostasis and autoimmunity. Nat
­Immunol 1:469-474, 2000.
116. Piccirillo CA, Shevach EM: Naturally-occurring CD4+CD25+
immunoregulatory T cells: Central players in the arena of peripheral
tolerance. Semin Immunol 16:81-88, 2004.
117. Shevach EM: Regulatory/suppressor T cells in health and disease.
Arthritis Rheum 50:2721-2724, 2004.
118. Kronenberg M, Rudensky A: Regulation of immunity by self-reactive
T cells. Nature 435:598-604, 2005.
119. Zheng Y, Rudensky AY: Foxp3 in control of the regulatory T cell
lineage. Nat Immunol 8:457-462, 2007.
120. Bennett CL, Christie J, Ramsdell F, et al: The immune dysregula-
tion, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is
caused by mutations of FOXP3. Nat Genet 27:20-21, 2001.
121. Kim JM, Rasmussen JP, Rudensky AY: Regulatory T cells prevent
catastrophic autoimmunity throughout the lifespan of mice. Nat
Immunol 8:191-197, 2007.
122. Shevach EM: From vanilla to 28 flavors: Multiple varieties of T
­regulatory cells. Immunity 25:195-201, 2006.
123. Tang Q, Adams JY, Tooley AJ, et al: Visualizing regulatory T cell
control of autoimmune responses in nonobese diabetic mice. Nat
Immunol 7:83-92, 2006.
124. Nguyen LT, Jacobs J, Mathis D, et al: Where FoxP3-dependent
­regulatory T cells impinge on the development of inflammatory
arthritis. Arthritis Rheum 56:509-520, 2007.
125. Skapenko A, Lipsky PE, Schulze-Koops H: T cell activation as starter
and motor of rheumatic inflammation. Curr Top Microbiol Immunol
305:195-211, 2006.
126. Tesi RJ, Elkhammas EA, Henry ML, et al: OKT3 for primary therapy
of the first rejection episode in kidney transplants. Transplantation
55:1023-1029, 1993.
127. Herold KC, Gitelman SE, Masharani U, et al: A single course of
anti-CD3 monoclonal antibody hOKT3gamma1(Ala-Ala) results in
improvement in C-peptide responses and clinical parameters for at
least 2 years after onset of type 1 diabetes. Diabetes 54:1763-1769,
2005.
128. Bennett SR, Falta MT, Bill J, et al: Antigen-specific T cells in
­rheumatoid arthritis. Curr Rheumatol Rep 5:255-263, 2003.
129. Bluestone JA, St Clair EW, Turka LA: CTLA4Ig: Bridging the basic
immunology with clinical application. Immunity 24:233-238, 2006.
130. Kremer JM, Westhovens R, Leon M, et al: Treatment of rheuma-
toid arthritis by selective inhibition of T-cell activation with fusion
­protein CTLA4Ig. N Engl J Med 349:1907-1915, 2003.
131. Nussenzweig MC: Immune receptor editing: Revise and select.
Cell 95:875-878, 1998.
132. Goodnow CC, Cyster JG, Hartley SB, et al: Self-tolerance
checkpoints in B lymphocyte development. Adv Immunol 59:
279-368, 1995.
133. Via CS, Shearer GM: T-cell interactions in autoimmunity: Insights
from a murine model of graft-versus-host disease. Immunol Today
9(7-8):207-213, 1988.
134. Chan OT, Madaio MP, Shlomchik MJ: The central and multiple
roles of B cells in lupus pathogenesis. Immunol Rev 169:107-121,
1999.
135. Chan OT, Hannum LG, Haberman AM, et al: A novel mouse with
B cells but lacking serum antibody reveals an antibody-independent
role for B cells in murine lupus. J Exp Med 189:1639-1648, 1999.
136. Chan OT, Shlomchik MJ: Cutting edge: B cells promote CD8+ T cell
activation in MRL-Fas(lpr) mice independently of MHC class I anti-
gen presentation. J Immunol 164:1658-1662, 2000.
137. Mamula MJ, Fatenejad S, Craft J: B cells process and present lupus
autoantigens that initiate autoimmune T cell responses. J Immunol
152:1453-1461, 1994.
138.  Steinman RM: The control of immunity and tolerance by dendritic
cell. Pathol Biol 51:59-60, 2003.
275
139. Banchereau J, Steinman RM: Dendritic cells and the control of
immunity. Nature 392:245-252, 1998.
140. Raz E: Organ-specific regulation of innate immunity. Nat Immunol
8:3-4, 2007.
141. Hayashi T, Beck L, Rossetto C, et al: Inhibition of experimental
asthma by indoleamine 2,3-dioxygenase. J Clin Invest 114:270-279,
2004.
142. Coffman RL, Mocci S, O’Garra A: The stability and reversibility of
Th1 and Th2 populations. Curr Top Microbiol Immunol 238:1-12,
1999.
143. Mosmann TR, Coffman RL: TH1 and TH2 cells: Different patterns
of lymphokine secretion lead to different functional properties. Annu
Rev Immunol 7:145-173, 1989.
144. Agnello D, Lankford CS, Bream J, et al: Cytokines and transcription
factors that regulate T helper cell differentiation: New players and
new insights. J Clin Immunol 23:147-161, 2003.
145. Leonard WJ, Spolski R: Interleukin-21: A modulator of lymphoid
proliferation, apoptosis and differentiation. Nat Rev Immunol 5:
688-698, 2005.
146. Oswald IP, Gazzinelli RT, Sher A, et al: IL-10 synergizes with IL-4
and transforming growth factor-beta to inhibit macrophage cytotoxic
activity. J Immunol 148:3578-3582, 1992.
147. Dong C: Diversification of T-helper-cell lineages: Finding the family
root of IL-17-producing cells. Nat Rev Immunol 6:329-333, 2006.
148. Kastelein RA, Hunter CA, Cua DJ: Discovery and biology of IL-23
and IL-27: Related but functionally distinct regulators of inflamma-
tion. Annu Rev Immunol 25:221-242, 2007.
149. Oppmann B, Lesley R, Blom B, et al: Novel p19 protein engages
IL-12p40 to form a cytokine, IL-23, with biological activities similar
as well as distinct from IL-12. Immunity 13:715-725, 2000.
150. Langrish CL, Chen Y, Blumenschein WM, et al: IL-23 drives a patho-
genic T cell population that induces autoimmune ­ inflammation.
J Exp Med 201:233-240, 2005.
151. Langrish CL, McKenzie BS, Wilson NJ, et al: IL-12 and IL-23:
Master regulators of innate and adaptive immunity. Immunol Rev
202:96-105, 2004.
152. Lieberman LA, Cardillo F, Owyang AM, et al: IL-23 provides a
­limited mechanism of resistance to acute toxoplasmosis in the
absence of IL-12. J Immunol 173:1887-1893, 2004.
153. Bettelli E, Carrier Y, Gao W, et al: Reciprocal developmental
­pathways for the generation of pathogenic effector TH17 and regula-
tory T cells. Nature 441:235-238, 2006.
154. Mangan PR, Harrington LE, O’Quinn DB, et al: Transforming
growth factor-beta induces development of the T(H)17 lineage.
Nature 441:231-234, 2006.
155. Koenders MI, Joosten LA, van den Berg WB: Potential new targets
in arthritis therapy: Interleukin (IL)-17 and its relation to tumour
necrosis factor and IL-1 in experimental arthritis. Ann Rheum Dis
65(Suppl 3):iii29-iii33, 2006.
156. Lubberts E, Joosten LA, Oppers B, et al: IL-1-independent role of
IL-17 in synovial inflammation and joint destruction during colla-
gen-induced arthritis. J Immunol 167:1004-1013, 2001.
157. Mannon PJ, Fuss IJ, Mayer L, et al: Anti-interleukin-12 antibody for
active Crohn’s disease. N Engl J Med 351:2069-2079, 2004.
158. Goldbach-Mansky R, Dailey NJ, Canna SW, et al: Neonatal-onset
multisystem inflammatory disease responsive to interleukin-1beta
inhibition. N Engl J Med 355:581-592, 2006.
159. Watanabe-Fukunaga R, Brannan CI, Copeland NG, et al: Lympho­
proliferation disorder in mice explained by defects in Fasantigen
that mediates apoptosis. Nature 356:314-317, 1992.
160. Straus SE, Lenardo MJ, Puck JM, et al: Autoimmune lymphopro-
liferative syndrome: An inherited disorder of lymphocyte apoptosis.
Ann Intern Med, 130:591-601, 1998.
161. Chen Z, Koralov SB, Kelsoe G: Complement C4 inhibits systemic
autoimmunity through a mechanism independent of complement
receptors CR1 and CR2. J Exp Med 192:1339-1352, 2000.
 16
KEY POINTS
Innate immunity depends on recognition of conserved
­molecular patterns found in many microorganisms.
Several families of pattern-recognition receptors are
­responsible for triggering innate immune responses.
Toll-like receptors and other pattern-recognition receptors
with leucine-rich repeat domains play a key role in innate
­immune recognition.
Antimicrobial peptides are important effectors of innate
­immunity.
Phagocytic cells and several types of innate-like lymphocytes
are key cell types in mediating innate immunity.
Innate immune responses have a strong impact on the
­development of adaptive immunity.
Some defects in the innate immune system are associated
with a predisposition to infections or to autoimmune diseases.
It has become common practice in immunology to divide
the mechanisms involved in host defense into adaptive and
innate components, and this provides a useful framework
for classifying the numerous cells, receptors, and effector
molecules that combine to make up the vertebrate immune
system (Table 16-1). A specific immune response, such as
the production of antibodies or T cells against a particular
pathogen, is referred to as adaptive immunity because it rep-
resents an adaptation that occurs during the lifetime of an
individual as a result of exposure to that pathogen. Adaptive
immune responses involve the clonal expansion of T and B
lymphocytes bearing a large repertoire of somatically gen-
erated receptors that can be selected to recognize virtually
any pathogen. The adaptive immune system of any given
individual is profoundly molded by the immunologic chal-
lenges encountered by that individual during the course of
a lifetime. A hallmark of adaptive immune responses is that
they are highly specific for the triggering agent, and they
provide the basis for immunologic memory. This property
of memory endows the adaptive immune response with its
“anticipatory” property, which provides increased resistance
against future infection with the same pathogen and also
allows vaccination against future infectious threats.
Adaptive immunity is essential for the survival of all
­mammals and most other vertebrates, but a wide vari-
ety of other mechanisms that do not involve antigen-
specific lymphocyte responses are also involved in successful
immune protection. These diverse mechanisms are col-
lectively known as innate immunity because they are not
Innate Immunity
Steven A. Porcelli
dependent on prior exposure to specific pathogens for their
­amplification. Such responses are controlled by the products
of germline genes that are inherited and similarly expressed
by all normal individuals. Innate immune mechanisms
involve both constitutive and inducible components and
use a wide variety of recognition and effector mechanisms.
It has become clear in recent years that innate immune
responses have a profound influence on the generation and
outcome of adaptive immune responses. This ability of the
innate immune system to instruct the responses of the adap-
tive immune system suggests many ways in which innate
immunity can influence the development of both long-term
specific immunity and autoimmune diseases.
EVOLUTIONARY ORIGINS OF INNATE
IMMUNITY
In spite of its obvious importance for most vertebrate
­organisms, the adaptive immune system is a relatively recent
evolutionary development (Fig. 16-1). In the great major-
ity of present-day vertebrate species, the adaptive immune
system is based on the ability to generate large ­families of
variable lymphocyte receptors with immunoglobulin-like
structures. This ability has been conserved owing to the
acquisition of a specialized recombination system that
­mediates the assembly of gene segments in the T cell and
B cell receptor ­families, which most likely occurred through
the invasion of the genome of a primitive vertebrate by a
transposable element or virus carrying this machinery.1,2
This critical step in the evolution of the immune system
can be traced back to the emergence of the ancestors of
present-day jawed fish, which represent the most primitive
extant species that are known to have adaptive immune
systems based on the generation of large families of specific
immunoglobulin-type receptors.3 Recently, other ­ systems
of variable lymphocyte receptors that are unrelated to
immunoglobulins but also provide the basis for an adaptive
immune response have been discovered in primitive jaw-
less fish such as lampreys and hagfish.4,5 This finding shows
that at least two different strategies for the creation of an
adaptive immune system emerged at the dawn of vertebrate
evolution about 500 million years ago, and it emphasizes
the importance of adaptive immunity for the survival and
further evolution of the vertebrate lineages.6
Given this key role of adaptive immunity in the ­evolution
and survival of vertebrates, it is surprising that all inverte-
brate animals, and possibly some of the lowest vertebrate
species as well, completely lack the ability to generate
­lymphocyte populations bearing large families of clonally
diverse antigen receptors.7,8 In these animals, protection
against pathogen invasion depends entirely on innate
277
 278 PORCELLI  |  Innate Immunity
Table 16-1  Contrasting Features of the Innate and Adaptive Immune Systems
Property Innate Immune System
Receptors Relatively few (several hundred?)
Fixed in genome
Gene rearrangement not required
Distribution Nonclonal
All cells of a class identical
Targets Conserved molecular patterns
Lipopolysaccharides
Lipoteichoic acids
Glycans and peptidoglycans
Others
Self-nonself   Perfect: selected over evolutionary time
discrimination
Action time Immediate or rapid (seconds to hours)
Response Microbicidal effector molecules
Antimicrobial peptides
Superoxide
Nitric oxide
Cytokines (IL-1, IL-6, others)
Chemokines (IL-8, others)
IFN, interferon; IL, interleukin.
Adapted and modified from Medzhitov R, Janeway CA Jr: Innate immune recognition. Annu Rev Immunol 20:197, 2002.
immunity, elements of which appear to exist in all animals
and plants and must have evolved with the ­earliest multicel-
lular forms of life. In many cases, components of the innate
immune system are significantly conserved in structure and
function in animals from the lowliest invertebrates to the
most complex vertebrates.7 This preservation of innate
immune mechanisms, with their functions largely intact,
over such vast ­ evolutionary distances is a clear indication
of their importance, even in animals that have developed
sophisticated adaptive immune responses.
PATHOGEN RECOGNITION
BY THE INNATE IMMUNE SYSTEM
Some mechanisms of innate immunity are constitutive,
meaning that they are continuously expressed and not
­significantly modulated by the presence or absence of infec-
tion. Examples include the barrier functions provided by
epithelial surfaces that are continuously exposed to micro-
bial flora, such as those of the skin and intestinal and ­genital
tracts. In contrast, the inducible mechanisms of innate
immunity involve the increased production of mediators
and the upregulation of effector functions that eliminate
microorganisms. Induction occurs as a result of exposure
to a wide variety of microbes and represents a less specific
form of immune recognition than that associated with the
specific antibodies and T cells that mediate adaptive immu-
nity. The basic principle underlying this form of response is
a process known as pattern recognition. This recognition
strategy is based on the detection of commonly occurring
and conserved molecular patterns that are essential prod-
ucts or structural components of microbes.
PATHOGEN-ASSOCIATED MOLECULAR PATTERNS
The general name given to the targets of innate immune
recognition is pathogen-associated molecular patterns
(PAMPs). These are structural features or components
Adaptive Immune System
Many (potentially 1014 or more)
Encoded in gene segments
Gene rearrangement required
Clonal
All cells of a class distinct
Details of molecular structure
Proteins
Peptides
Carbohydrates
Imperfect: selected in individual somatic cells
Delayed (days to weeks)
Clonal expansion or anergy of specific T and B lymphocytes
Cytokines (IL-2, IL-4, IFN-γ, others)
Specific antibody production
Specific cytolytic T cell generation
­distinctive for microorganisms and are not normally found
in the animal host. The best-known example of a PAMP
is bacterial lipopolysaccharide (LPS), a ubiquitous glyco-
lipid constituent of the outer membranes of gram-negative
bacteria. Another important example is the peptidoglycan
structure present as the basic cell wall component in nearly
all bacteria. These structures may vary partially from one
bacterium to another, but the basic elements are conserved,
thus providing the possibility of recognizing a broad array of
pathogens by sensing a single or a relatively small number
of PAMPs. Many PAMPs that serve as targets of recognition
for the innate immune response are now known to be associ-
ated with bacteria, fungi, and viruses.
PATTERN-RECOGNITION RECEPTORS
The recognition of PAMPs is mediated by a collection of
germline-encoded molecules known collectively as pat-
tern-recognition receptors (PRRs) (Table 16-2). These
receptors are host proteins that have evolved, through
many millions of years of natural selection, to possess
defined specificities for particular PAMPs expressed by
microorganisms. The total number of PRRs present in
complex vertebrates such as humans is estimated to
be several hundred, a number limited by the size of the
genome of any animal and the number of genes it can
dedicate to immune protection. The human genome, for
example, is estimated to contain approximately 20,000
to 35,000 genes, most of which are not related directly
to the immune system. This demonstrates one of the
strong points of contrast between the innate and adap-
tive immune systems, because the latter can possess in
the range of 1014 different somatically generated recep-
tors for foreign antigens in the form of antibodies and T
cell receptors. With its much more limited array of recep-
tors, the innate immune system uses the strategy of target-
ing highly conserved PAMPs that are shared broadly by
large classes of microorganisms. Because most pathogens
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION 279

Variable lymphocyte receptors (lg type)

0 Mammalia Antibody, T cells
MHC
Thymus, spleen
Aves
Complement (CCP, ACP, LCP, C3, MAC)
Reptilia
Lectins and other PRRs
250 Antimicrobial peptides
Amphibia
Osteichthyes No antibody or T cells
(bony fish) MHC? Thymus? Spleen?
Chondrichthyes
(sharks, rays) Variable lymphocyte receptors (LRR type)
Complement (ACP, LCP, C3)
Agnatha Lectins and other PRRs
Time to present (million years)

450 (jawless fish) Antimicrobial peptides

Arthropoda
(insects)
Protochordates
(sea squirt) Annelida Mollusca
(earthworm)

No antibody or T cells
Echinodermata No variable lymphocyte receptors?
(sea urchin) MHC?
750
Lectins and other PRRs
Antimicrobial peptides
Deuterostomes Protostomes Complement in some (all?)

Coelomates
Porifera Acoelomates
(sponges)
900
Figure 16-1  Ancient evolutionary origin of the innate immune system. Studies of the immune systems of a wide range of vertebrates and inver-
tebrates have revealed that even the most primitive invertebrates possess many components of innate immunity (e.g., pattern-recognition receptors
of the lectin and Toll-like families, antimicrobial peptides, complement proteins). The innate immune system is thus extremely ancient, having arisen
early in the evolution of multicellular life. In contrast, the adaptive immune system is a much more recent development that did not appear until the
emergence of the ancestors of present-day sharks and rays, approximately 400 million years ago. The first species to acquire an adaptive immune sys-
tem based on immunoglobulin-type receptors must have arisen after the appearance of the direct ancestors of present-day jawless fish (lampreys and
hagfish), which are the most highly evolved living species that lack the ability to generate large families of variable immunoglobulin-type lymphocyte
receptors (arrow). ACP, alternative complement pathway; CCP, classic complement pathway; Ig, immunoglobulin; LCP, lectin-activated complement
pathway; LRR, leucine-rich repeat domain; MAC, membrane attack complex; MHC, major histocompatibility complex; PRR, pattern-recognition receptor.
(Adapted from Sunyer JO, Zarkadis IK, Lambris JD: Complement diversity: A mechanism for generating immune diversity? Immunol Today 19:519, 1998.)

c­ ontain PAMPs, this strategy allows the generation of at
least partial immunity against most infections.
PRRs are expressed by many cell types, some of which
are specialized effector cells of the immune system (e.g.,
neutrophils, macrophages, dendritic cells, lymphocytes),
and some of which are not generally regarded as part of
the immune system (e.g., epithelial and endothelial cells).
Unlike the T and B cell receptors used for adaptive immune
recognition, the expression of PRRs is not clonal, which
means that all the receptors displayed by a given cell type
(e.g., macrophages) have identical structure and ­specificity.
When PRRs are engaged by recognition of their associated
PAMPs, effector cells bearing the PRRs are triggered to per-
form their immune effector functions immediately, rather
than after undergoing proliferation and expansion, as in the
case of adaptive immune responses. This accounts for the
much more rapid onset of innate immune responses.
In recent years, considerable progress has been made
toward identifying many of the important PRRs involved
in the induction of innate immunity. These receptors can
be classified into three functional classes: secreted, endo-
cytic, and signaling PRRs (see Table 16-2). In addition,
many of the known PRRs can be classified into structurally
defined families on the basis of a few characteristic protein
domains. Among these, the best known include proteins
with calcium-dependent lectin domains, scavenger receptor
domains, and leucine-rich repeat domains.
Pattern-Recognition Receptors of the Lectin Family
Calcium-dependent lectin domains are common modules
of secreted and membrane-bound proteins involved in the
binding of carbohydrate structures. A well-characterized
PRR belonging to this class is the mannan-binding lectin
(MBL), also known as soluble mannose binding protein,
which represents a secreted PRR that functions in the initia-
tion of the complement cascade (Fig. 16-2).9,10 This protein
is synthesized primarily in the liver on a constitutive basis,
although its production can be increased as an acute-phase
reactant following many types of infection. MBL binds to
 280 PORCELLI  |  Innate Immunity
Table 16-2  Pattern-Recognition Receptors (PRRs)
Prominent Sites of
Receptor Class Examples Expression
Secreted PRRs Collectins Plasma
Mannan-binding lectin
Ficolins
Surfactant proteins (SP-A, SP-B)
Pentraxins
Short pentraxins (CRP, SAP)
Long pentraxins
Endocytic PRRs Lectin-family receptors Macrophages, dendritic Cell wall polysaccharides (­mannans Pathogen uptake by
Macrophage mannose receptor cells, some endothelia, and glucans), LPS, LTA, and
DEC-205 epithelia, and smooth
Dectin-1 muscle cells
Scavenger receptor A
MARCO
Complement receptors
CD11b/CD18 (CR3)
CD21/35 (CR2/1)
Signaling PRRs Toll-like receptors Macrophages, dendritic
CARD/NOD proteins cells, epithelia
PYRIN domain proteins
CARD, caspase activation and recruitment domain; CR, complement receptor; CRP, C-reactive protein; DEC-205, dendritic and epithelial cells, 205 kD; dsRNA,
double-stranded RNA; LPS, lipopolysaccharide; LTA, lipoteichoic acid; MARCO, macrophage receptor with collagenous structure; NOD, nucleotide-binding
oligomerization domain; SAP, serum amyloid P protein; SP, surfactant protein.
carbohydrates on the outer membranes and capsules of many
bacteria,11-13 as well as fungi,14 some viruses,15,16 and para-
sites.17 Although mannose and fucose sugars bound by MBL
can also be found on the surfaces of normal mammalian
cells, they are present at too low a density or in the wrong
orientation to efficiently engage the lectin domains of MBL.
In contrast, the coats of many microorganisms contain an
array of these sugars, which allows strong binding of MBL.
Thus, in this case, the spacing and orientation of specific
carbohydrate residues constitute the PAMP that triggers the
activation of innate immunity by MBL. MBL functions as
one of a small number of secreted PRRs that can initiate
the lectin pathway of complement activation. At least two
other soluble proteins with lectin activity in human plasma,
known as ficolins (ficolin/P35 and H-ficolin), can also acti-
vate this pathway following their interaction with bacterial
polysaccharides.18
Several of the soluble lectin-type PRRs also play an
important role in the opsonization of microbes by binding
to their surfaces and directing them to receptors on phago-
cytic cells. Among these are two pulmonary surfactant
proteins, SP-A and SP-D, which similarly recognize and
bind to the surface sugar codes of microbes in the respira-
tory tract.19-21 These molecules are similar in structure to
MBL, having both collagen-like and lectin domains, and
together they constitute a family of soluble PRRs known
as collectins. Another family of soluble PRRs that performs
a ­ similar function in plasma is the pentraxins, so called
because they are formed by the association of five identical
protein subunits.22,23 This family includes the acute-phase
reactants C-reactive protein (CRP) and serum amyloid P
protein (SAP), along with a number of so-called long pen-
traxins, which have an extended polypeptide structure with
Major Ligands Function
Carbohydrate arrays typical of Complement activation
­bacterial capsules, fungi, and   Opsonization
other microbes
Apoptotic cells and cellular debris,
including chromatin
­phagocytes
­opsonized cells and particles Delivery of ligands to
antigen-processing
­compartments
Clearance of cellular and
extracellular debris
Multiple conserved pathogen- Activation of inducible  
­associated molecular patterns innate immunity  
(LPS, LTA, dsRNA, lipoproteins, (antimicrobial peptides,
­flagellin, bacterial DNA, others) ­cytokines, reactive oxygen
or ­nitrogen intermediates)
Instruction of adaptive
­immune response
homology to the classic short pentraxins (i.e., CRP and
SAP) only at their carboxy-terminal domains. Long pen-
traxins are expressed in a ­variety of different tissues and cells,
and their specific functions are mostly unknown. However,
the long pentraxin PTX3 has been shown to play an impor-
tant, nonredundant role in resistance to fungal infections in
mice, and recent studies indicate that PTX3 is essentially a
functional ancestor of antibodies that recognizes microbes
and promotes their clearance through complement activa-
tion and phagocytosis.24
In addition to these soluble proteins, a large number of
membrane-bound glycoproteins with lectin domains are
known to exist, and some of them participate in innate
immunity by serving as endocytic PRRs for the uptake
of microbes or microbial products25 (Fig. 16-3). One of
the most extensively studied of these is the macrophage
mannose receptor (MMR).26 Although originally identi-
fied on alveolar macrophages and known to be expressed
on macrophage subsets throughout the body, this receptor
is also expressed on a variety of other cell types, includ-
ing certain endothelia, epithelia, and smooth muscle
cells. The MMR is a membrane-anchored, multilectin
domain-­containing protein that mediates the binding of
a broad range of pathogens, leading to their internaliza-
tion via ­endocytosis and phagocytosis.26-29 Although the
major function of the MMR appears to be directing the
uptake of its ligands, there is evidence that this receptor
may be capable of signaling to modify macrophage func-
tions following receptor ­engagement.30 Another member
of this receptor family, the ­ß-glucan binding cell surface
lectin known as Dectin-1, has a role in the modulation
of inflammation in a mouse model of infection-induced
arthritis.31
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION 281

MBL
C3

C4 MASP3
MASP2 sMAP MASP1
C2

C3
C2a Carbohydrates
C4b
C3b
Cysteine- Collagen-like Carbohydrate
rich region domain recognition
domain C3b
Bacterial surface
C3a

Figure 16-2  Structure and function of mannan-binding lectin (MBL), a soluble pattern-recognition receptor. Left, MBL is a multimer protein
s­ tructure with multiple carbohydrate-binding lectin domains. Three identical 32- kD polypeptides associate to form a subunit, which then oligomerize
to form functional complexes (the trimeric form consisting of three subunits is illustrated, which is one of several different oligomer sizes that has been
observed for MBL). Each polypeptide in the subunit contains an N-terminal cysteine-rich domain, a collagen-like domain, a neck region, and a C-terminal
carbohydrate-recognition domain. Right, Initiation of the lectin pathway for complement activation by MBL. The carbohydrate-recognition domains
of MBL bind to carbohydrates that are characteristic of bacterial surfaces. This leads to the recruitment of several other serum proteins, including
small MBL-associated protein (sMAP), and the three MBL-associated serine proteases (MASP1, MASP2, MASP3). The protease activity of MASP2 cleaves
complement C4 and C2 subunits, generating the C3 convertase (C4bC2a). MASP1 is able to cleave C3 directly. The deposition of C3 cleavage products
on the bacterial surface results in opsonization and phagocytosis of the bacterial cell.

C C C N
Pattern-Recognition Receptors of the Scavenger
N d Receptor Family
e The scavenger receptor family contains a broad range of
structurally diverse cell surface proteins that are expressed
C C C f most prominently on macrophages, dendritic cells, and
a C endothelial cells25,32 (see Fig. 16-3). Although they were
C C
originally defined by their ability to bind and take up modi-
fied serum lipoproteins, they also bind a wide range of other
b ligands, including bacteria and some of their associated
products. Two members of this family that have been impli-
cated as PRRs for innate immunity are the scavenger recep-
c
tor A (SR-A) and a related molecule called the macrophage
Extracellular receptor with collagenous structure (MARCO).33-36 Both
these molecules contain a scavenger receptor cysteine-rich
Cytoplasmic
domain in the distal ends of their membranes and a collagen-
NN N NN N NN N C C like stalk with a triple-helical structure. Both are known to
SR-A I SR-A II MARCO MR DEC-205 bind bacteria, and SR-A also binds well-known PAMPs such
as lipoteichoic acids and LPS.37,38 Mice that have been made
Scavenger receptor family Lectin family deficient in SR-A by targeted gene disruption show increased
Figure 16-3  Endocytic pattern-recognition receptors of the ­scavenger susceptibility to infections caused by a variety of bacteria,
receptor and lectin families. Left, Illustrations of three ­ members of thus providing strong evidence of scavenger receptors’ role
the scavenger receptor family. These are trimeric complexes of type II in protective immunity, most likely through the activation
­transmembrane polypeptides that have their N-terminals positioned
in the cytoplasm and their C-terminals in the extracellular space. Three
of innate immune mechanisms.39-41 Although these mem-
distinct extracellular structural domains are indicated: (a) the scavenger bers of the scavenger receptor family clearly function as
receptor cysteine-rich (SRCR) domain (absent in SR-A II), which has no cur- endocytic PRRs in the uptake of microbes, their potential to
rently known function; (b) the collagen-like domain, which is ­implicated serve as signaling receptors has not yet been established.
in the binding of polyanionic ligands; and (c) the α-helical coiled-coil
­domain (absent in MARCO), which is believed to assist in receptor
­trimerization. Right, Two examples of multilectin domain endocytic pat- Pattern-Recognition Receptors with Leucine-Rich
tern-­recognition receptors—macrophage mannose receptor (MMR) Repeat Domains
and DEC-205. Distinct extracellular domains in these receptors include
(d) a ­­cysteine-rich N ­terminal domain, (e) a fibronectin-like domain, and  Leucine-rich repeat domains (LRRs) are structural mod-
(f) multiple ­­calcium-dependent (C-type) lectin domains that bind various
carbohydrate ­ ligands. (Reproduced in part from Peiser L, Mukhopadhyay
ules found in many proteins, including PRRs involved in
S, Gordon S: Scavenger receptors in innate immunity. Curr Opin Immunol signaling the activation of innate immunity. Molecules in
14:123, 2002.) this class include, most notably, the family of mammalian
 282 PORCELLI  |  Innate Immunity

MD-2 b
LPS Ligand

CD14 CD14
c
Plasma membrane

TLR2
TLR6
TLR4
TLR4
TIRAP/MAL

Tollip Rac1 p85 Tollip

PI3

IRAK
IRAK
MyD88 Kinase MyD88
p110

PKR Akt
IRAK-2 MAPKs NF-κB MAPKs NF-κB
Figure 16-4  Toll-like receptors (TLRs) and associated proteins. Left, TLR4 is a transmembrane polypeptide present in the plasma membrane
as a homodimer. The TLR4 polypeptide has three distinct extracellular regions: (a) an N-terminal flanking domain; (b) the leucine-rich repeat (LRR)
region, which contains 21 leucine-rich motifs and is thought to be directly involved in binding to lipopolysaccharide (LPS) and other ligands; and
(c) a C-terminal flanking cysteine-rich domain. The cytoplasmic domains of TLR4 and all other TLRs have homology to the human interleukin (IL)-1
receptor and are designated Toll-IL-1 receptor (TIR) domains. The extracellular portion of TLR4 associates with at least two other proteins, CD14 and
MD-2, which are involved in ligand recognition. The intracellular TIR domains associate with multiple adapter proteins (MyD88, TIRAP/MAL, Tollip),
which link the receptor complex to kinases that activate signaling cascades. For TLR4, and probably for most other TLRs as well, activation of the
IL-1 receptor-associated kinase (IRAK) is an important step leading to the release of the active form of transcription factor nuclear factor B (NF B). In
addition, signaling through TLR4 leads to signal transduction through the activation of mitogen-activated protein kinases (MAPKs), double-stranded
RNA-binding protein kinase (PKR), and other members of the IRAK family such as IRAK-2. Right, A different set of ligands is recognized by TLR2, which
functions as part of a heterodimeric complex with other TLRs such as TLR6. The TLR2-TLR6 complex shares many features with the TLR4 complex
in terms of its associated proteins. However, the TIR domain of TLR2 also appears to recruit phosphatidyl-3-OH kinase (PI3 kinase, p85 and p110
subunits) and the membrane-associated GTPase Rac1, which allows the activation of other signaling molecules, such as the serine-threonine kinase
Akt. Thus, although the major signaling pathways activated by different TLRs are similar or identical (i.e., activation of NF B and MAPKs), it is likely
that each TLR complex has subtle differences in its secondary pathways of signal transduction. These differences may lead to partially overlapping
but distinct outcomes in response to ligands recognized by different TLR complexes. (Adapted from Underhill DM, Ozinsky A: Toll-like receptors: Key
mediators of microbe detection. Curr Opin Immunol 14:103, 2002.)

Toll-like receptors (TLRs), which are membrane-bound
signal-transducing molecules that play a central role in
the recognition of extracellular and vacuolar pathogens.42
Two families of cytoplasmic LRR-containing receptors have
also been identified, and they play a prominent role in the
innate immune recognition of PAMPS expressed by intra-
cellular pathogens. These are the family of caspase activa-
tion and recruitment domain (CARD) proteins and the
PYRIN domain proteins.43 These molecules are closely
related in structure and function to proteins found in inver-
tebrates and plants that are involved in pathogen resistance,
highlighting the ancient origin of these pathways for host
defense, which appear to have been recognizably conserved
through approximately 1 billion years of evolution.44,45
Toll-Like Receptors. The first member of the Toll family
to be discovered was the Drosophila Toll protein, which was
identified as a component of a signaling pathway control-
ling dorsoventral polarity during the development of the
fly embryo.46 The sequence of Toll showed it to be a trans-
membrane protein with a large extracellular domain con-
taining multiple tandemly repeated LRRs at the N-terminal
end, followed by a cysteine-rich domain and an intracel-
lular signaling domain (Fig. 16-4). A role for Toll in im-
mune responses was suggested by the observation that its
intracellular domain shows homology to the mammalian
interleukin-1 receptor (IL-1R) cytoplasmic domain.47 This
association was later confirmed in studies showing that Toll
was critical for the antifungal response in the fly, linking this
pathway for the first time to innate immunity.48 The iden-
tification of Drosophila Toll eventually led to a search for
similar proteins in mammals, and this effort has been richly
rewarded, yielding 11 Toll-like receptors (TLRs) in mice
and humans.42 Among these, TLR1 through TLR9 are con-
served between mouse and human; TLR10 is present only in
humans, and TLR11 is expressed only in mice.43 All these
molecules contain large extracellular domains with multiple
LRRs, as well as intracellular signaling domains known as
Toll/IL-1R, or TIR, domains.43 Many of these TLRs have
now been linked to the innate immune responses against
various PAMPs of different microorganisms.49
Toll-Like Receptor 4 and the Response to Lipopolysac-
charide. The first human TLR to be identified was the mol-
ecule now designated TLR4, which is a major component
in the response to one of the most common of all PAMPs,
bacterial LPS.50 Earlier studies on the response to LPS had
identified two proteins, CD14 and LPS-binding protein,
as molecules involved in the binding of LPS to the surface
of LPS-responsive cells. However, these molecules did not
­possess any potential for transducing signals into the cell,
so it was unclear how LPS binding would lead to the acti-
vation of cellular responses associated with gram-negative
bacterial infection. The answer was provided by positional
cloning studies of the LPS gene in the LPS-hyporesponsive
C3H/HeJ mouse.51,52 This revealed a single amino acid
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
s­ ubstitution in the signaling domain of TLR4. Specific dele-
tion of the TLR4 gene by targeted gene disruption in mice
subsequently confirmed the essential role of this molecule
in the response to LPS: these TLR4 knockout mice have
almost no response to LPS and are highly resistant to en-
dotoxic shock.53-55 Biochemical studies provide further sup-
port for TLR4 as a component of the LPS receptor; they
show that LPS bound to the surface of cells is in close con-
tact with both CD14 and TLR4, as well as another protein
called MD-2 that appears to perform an accessory function
in the binding of LPS to the receptor complex.56 Further
studies have elucidated many of the downstream elements
in the signaling pathways that connect TLR4 to the activa-
tion of genes associated with inducible innate immunity57-61
(see Fig. 16-4). Studies of Toll signaling pathways in Dro-
sophila identified the transcription factor nuclear factor B
(NF B) as one of the key effectors of gene activation follow-
ing the engagement of Toll, and this basic pathway in the fly
appears to be largely conserved in TLR signaling in higher
animals, including mammals.62
Other Pathogen-Associated Molecular Patterns Recog-
nized by Toll-Like Receptors. The search for ligands that
lead to signaling through ­ various TLRs has demonstrated
that this family of PRRs is ­ collectively responsible for in-
nate immune responses to an extraordinary array of PAMPs.
In addition to its central role in the signaling of responses
to LPS, TLR4 is involved in responses to multiple differ-
ent self- and nonself-ligands.42 The antimitotic agent Taxol
has been shown to mimic LPS-induced signaling in mouse
cells through a pathway that requires both TLR4 and MD-
2.63-65 Other foreign ligands of TLR4 are the fusion protein
(F protein) of respiratory syncytial virus66,67 and heat-shock
protein 60 (HSP60) of chlamydia.68,69 Interestingly, TLR4
can also signal in response to mammalian HSP60, a pro-
tein expressed at increased levels and most likely released
by stressed or damaged cells.70 This represents a variation
of the pattern-recognition principle in which the pattern
is not a PAMP produced directly by a pathogen but rather
a structural feature associated with infected or physically
damaged cells of the host. Other examples of TLR4’s recog-
nition of self-components include responses to oligosaccha-
ride breakdown products of tissue hyaluronans and respon­
ses to the extra domain A region of fibronectin produced
by alternative RNA splicing in response to tissue injury or
inflammation.71,72
The range of PAMPs recognized through TLR2 is prob-
ably even greater than for TLR4. TLR2 is known to be
involved in signaling in response to multiple PAMPs of
gram-­negative and gram-positive bacteria, including such
structures as bacterial glycolipids, bacterial lipoproteins,
parasite-derived glycolipids, and fungal cell wall polysac-
charides.54,73-76 TLR2 does not function independently in
responding to these PAMPs; it forms heterodimers with
either TLR1 or TLR6. This ability to pair with other TLRs
appears to be unique to TLR2, because other TLRs that
have been studied carefully (e.g., TLR4, TLR5) most likely
function only as monomers or homodimers. Other TLRs
with currently defined ligands are TLR5 (involved in the
response to bacterial flagellin),77 TLR3 (double-stranded
RNA),78 TLR7 (single-stranded RNA),79 and TLR9
(unmethylated bacterial DNA).80 It is apparent that most,
283
if not all, microbes contain multiple PAMPs that are recog-
nized by different TLRs. For example, a typical bacterium
expressing LPS also contains unmethylated DNA and thus
generates signals not only through TLR4 but potentially
through TLR9 as well. Because different TLRs are capable
of activating distinct signaling cascades (see Fig. 16-3), the
ability of a single cell to detect several different features of a
pathogen simultaneously with multiple TLRs may help the
innate immune response be more finely tuned to respond to
a particular challenge.81
CARD and PYRIN Domain Proteins. A large number of
­cytosolic proteins that have structural similarities to the
membrane-bound TLRs and also function as sensors for
PAMPs of intracellular pathogens and regulators of innate
immune responses have been identified. Many of these pro-
teins contain LRR domains and have been classified based
on their incorporation of a CARD or PYRIN domain. The
nomenclature and classification schemes for this growing
family of innate immune sensors and regulators are still
evolving, and it has been proposed that they all be grouped
and classified as members of a single family designated the
CATERPILLER (CARD, R [purine]-binding, pyrin, lots of
leucine repeats) family.82 The first intracellular microbial sen-
sors in this family to be described were the Nod1 and Nod2
proteins, which contain LRR domains linked to a central
nucleotide binding and oligomerizaion (NOD or NACHT)
domain and an N-terminal CARD domain.83 As in the case
of TLRs, the LRR domains of these proteins appear to be
involved in the recognition of pathogen-derived molecules,
and their CARD domains are linked to downstream signal-
ing for the activation of innate immunity. Although origi-
nally implicated in responses to bacterial LPS, it is now well
accepted that both Nod1 and Nod2 are primarily involved
in the recognition of muropeptide monomers released from
bacterial cell wall peptidoglycans.43 Signals resulting from
the recognition of peptidoglycan components by Nod1 and
Nod2 lead to activation of the NF B pathway, as in the case
of TLR signaling. However, other signaling pathways also
appear to be engaged, such as the activation of procaspase-1
and caspase-9 by CARD domain interactions, leading to in-
creased production of IL-1β and cell death by apoptosis.84
The PYRIN domain–containing proteins are believed to
signal in response to microbial invasion or cellular stress.
The prototype member of this family is pyrin, which is the
product of the gene that is mutated in those with familial
Mediterranean fever.82 Although pyrin itself lacks an LRR
domain, there are numerous other members of this family
that contain an LRR linked to a central NOD domain and
an N-terminal PYRIN domain. These include cryopyrin,
which is mutated in patients with a range of hereditary
inflammatory diseases now referred to collectively as cryo-
pyrin-associated periodic syndromes. These multiple related
proteins constitute the Nalp family (NACHT-LRR-PYD-
containing proteins).43,82,85,86 The human genome contains
14 genes encoding Nalp proteins, the precise functions of
which are mostly unknown. However, two of these (Nalp6
and Nalp12) have been shown to activate or regulate the
NF B pathway.43 In addition, several Nalp proteins have
been identified as key components in the formation of
intracellular complexes known as “inflammasomes,” which
are involved in the activation of caspases required for the
 284 PORCELLI  |  Innate Immunity
processing of inflammatory cytokines, including IL-1β
and IL-18.87 The direct recognition of specific PAMPs by
Nalp proteins remains to be established, although initial
studies implicate these proteins as either direct or indirect
­sensors of various stimuli, including constituents of bacteria
(­peptidoglycan, bacterial RNA, exotoxins), viruses (double-
stranded RNA), and uric acid crystals.88-91
EFFECTOR MECHANISMS OF INNATE
IMMUNE RESPONSES
The ability to recognize pathogens through PRRs allows
numerous antimicrobial effector mechanisms to be activated
by the innate immune response. These responses lead to
the killing of pathogens through the production of ­effector
molecules with direct microbicidal activities, including
the membrane attack complex of complement, a variety of
antimicrobial peptides, and the caustic reactive oxygen and
reactive nitrogen intermediates generated within phagocytic
cells. In invertebrates, these mechanisms represent virtually
the entire protective response against microbial invaders.
However, in most vertebrates, including mammals, innate
immune recognition also has profound effects on trigger-
ing and programming the adaptive immune response that
follows somewhat later. This ability of the innate immune
system to instruct the adaptive response has major implica-
tions for the development of long-term protective immunity
to infections and may also play a critical role in mechanisms
leading to autoimmunity.
CELL TYPES MEDIATING INNATE
IMMUNITY
Many types of cells have the ability to mount at least a limited
response to PAMPs, but the most effective cell types in this
regard are the specialized phagocytes, such as macrophages,
neutrophils, and dendritic cells. Upon recognition of micro-
bial stimuli, these cells have the ability to upregulate NADPH
oxidase by assembling the components of this enzyme com-
plex on phagosomal membranes, leading to an oxidative burst
that produces microbicidal superoxide ions.92 Many phago-
cytic cells also increase their expression of inducible nitric
oxide synthase (iNOS, or NOS2) upon contact with various
PAMPs.93,94 This leads to the production of reactive nitro-
gen intermediates, including nitric oxide and peroxynitrite,
which have potent direct antimicrobicidal activities. These
responses are synergistic because the antimicrobial activity of
the phagocyte oxidase system is frequently enhanced by the
expression of reactive nitrogen intermediates.
INNATE-LIKE LYMPHOCYTES
A number of distinct lymphocyte subsets also play important
roles in innate immune responses. One group of such lym-
phocytes, the natural killer (NK) cells, appears to be a true
member of the innate immune system. These lymphocytes
do not express receptors generated by somatic ­recombination
and thus depend on germline-encoded receptors for signal-
ing their responses against pathogen-infected cells.95 NK
cells participate in the early innate response against virally,
and probably bacterially, infected cells through the expres-
sion of cytotoxic activity and the secretion of cytokines.96
Several other subsets of lymphocytes belonging to the T
and B cell lineages have been identified as participants in
the rapid response against pathogens to which the host has
not previously been exposed. Although these cells express
clonally variable, somatically rearranged antigen receptors
(T cell antigen receptors or membrane immunoglobulins)
and thus could be classified as components of the adaptive
immune system, their manner of functioning is much more
characteristic of innate than adaptive immunity. These
innate-like lymphocytes (ILLs) may represent the remnants
of the earliest primitive adaptive immune system, and they
appear to have been conserved to varying degrees because
they ­ continue to make specialized contributions to host
immunity.97
Among the currently recognized ILLs are two B cell
populations, known as the B1 and marginal zone B cell sub-
sets.98,99 These are involved in the spontaneous production
of natural antibodies, which are largely germline-encoded
immunoglobulins that are reactive to commonly expressed
microbial determinants. In addition, both these B cell pop-
ulations generate rapid T cell–independent responses fol-
lowing bacterial challenges and thus contribute to the first
line of immune defense that precedes the onset of adaptive
immunity.
Among the T cells, two populations of ILLs have been
identified and characterized in detail: γδ T cells and NK T
cells. The γδ T cells express somatically rearranging recep-
tors that use a limited number of variable region genes and
are thought to recognize a narrow spectrum of foreign or
self-ligands.100 In humans, the specificities of two subsets
of γδ T cells have been at least partially defined. One of
these, the major circulating population expressing the Vδ2
gene product, responds rapidly and without prior immuni-
zation to a variety of small alkyl phosphate and alkyl amine
compounds that are produced by many bacteria. Another
subset, characterized by its expression of the Vδ1 gene
product, responds to major histocompatibility complex
(MHC) class I–related self-molecules of the MHC class I
chain–related A and B (MICA/B) and CD1 families.101,102
These molecules may serve as markers of cellular stress
and are upregulated on cells in the context of infection or
inflammation, leading to the activation of Vδ1-bearing γδ
T cells.
A similar principle appears to be involved in the
­functioning of NK T cells, which are so named because of
their coexpression of an αβ T cell antigen receptor and a
variety of receptor molecules that are typically associated
with NK cells.103 Like γδ T cells, NK T cells have somati-
cally rearranged antigen receptors that use a limited array of
V genes and most likely recognize a narrow range of foreign
or self-antigens. A major population of NK T cells is reactive
with the MHC class I–like CD1d molecule, and these ILLs
appear to be activated by recognition of a variety of lipid or
glycolipid ligands that can be presented by CD1d. Recently,
several bacterial glycolipids have been identified as specific
antigens that stimulate NK T cells, suggesting that these cells
may be rapid responders that contribute to innate antibacte-
rial immunity.104-106 A wide variety of mouse disease models
have shown that NK T cells also make significant contribu-
tions to the development of adaptive immune responses and
may play a particularly important role in immunoregulation
to prevent autoimmunity.103
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
ANTIMICROBIAL PEPTIDES
Antimicrobial peptides are the key effector molecules of
inducible innate immunity in many invertebrates and are
now being increasingly recognized as important elements of
innate immunity in higher animal species, including mam-
mals.107 They are evolutionarily ancient components of host
defense that are widely distributed throughout all ­multicellular
organisms in the animal and plant kingdoms. More than
800 such peptides have been identified (for a current list,
see  h ttp://www.bbcm.univ.trieste.it/∼tossi/amsdb.html),
and their diversity is so great that it is difficult to catego-
rize them. However, at a structural and mechanistic level,
most of these peptides share several basic features. They
are generally composed of amino acids arranged to create
an amphipathic structure with hydrophobic and cationic
regions. The cationic regions target a fundamental differ-
ence in ­membrane design between microbes and multicel-
lular animals, which is the abundance of negatively charged
phospholipid headgroups on the outer leaflet of the lipid
bilayer. The preferential association of antimicrobial pep-
tides with microbial membranes leads to a membrane-dis-
rupting activity, most likely involving the interaction of
the hydrophobic regions of the peptide with membrane
lipids.108-110
Antimicrobial peptides produced in response to engage-
ment of various PRRs account for the majority of the
in­d­ucible immunity against microbes in many invertebrate
animals and plants. Although these peptides are probably
less central to host immunity in most vertebrates, there is
evidence that they make important contributions to immu-
nity in more highly evolved animals, including mammals.111
In humans, active antimicrobial peptides, such as the α- and
β-defensins, are either constitutively or inducibly produced
in skin and in epithelia of the gastrointestinal and respira-
tory tracts.112-116 These molecules most likely act as natural
preservatives of epithelia that are colonized or frequently
exposed to microbial flora. Because the acquisition of resis-
tance against these agents by sensitive microbial strains is
extremely unusual, antimicrobial peptides are of great inter-
est as templates for the development of new antimicrobial
pharmaceuticals.117
INFLUENCE OF INNATE MECHANISMS
ON ADAPTIVE IMMUNITY
In addition to functioning as a first line of defense against
invading pathogens, another critical feature of the innate
immune system in higher animals such as mammals is its
effect on activating the adaptive immune system. In fact, it
is now clear that in most situations, the adaptive immune
system mounts a response to a pathogen only after the
pathogen has generated signals via PRRs of the innate
immune system. This principle is the basis for the adjuvant
effect, which is the observation that antibody and T cell
responses are efficiently generated against protein anti-
gens only if these are introduced together with a nonspe-
cific ­ activator of the immune system, which is generically
known as an adjuvant. Most adjuvants are in fact extracts or
­products of bacteria, and it is now clear that in most or all
cases, ­adjuvant effects result from activation of the innate
immune response.118
285
PAMPs
(e.g., LPS) Signaling PPR
e.g., TLR4
Cytokines
IL-1,-6,-12, etc.
Pathogen
Endocytic PPR NF-kB B7 CD28
(e.g., MR)
+ +
TCR +
MHC
+
+
Endo/Lys
T cell
Antigen
processing
APC
Figure 16-5  Instruction of the adaptive immune response by the
innate immune system. When an antigen presenting cell (APC) comes
into contact with pathogen-bearing pathogen-associated molecular pat-
terns (PAMPs), responses are triggered via innate immune mechanisms
that dramatically alter the ability of the APC to stimulate an adaptive 
(T cell–mediated) immune response. For example, signals generated by
contact with PAMPs such as lipopolysaccharide (LPS) with Toll-like recep-
tor 4 (TLR4) lead to the activation of transcription factor nuclear factor B
(NF B), which enters the nucleus of the APC and assists in switching on
genes for cytokines (e.g., interleukin [IL]-1, -6, and -12 and a variety of
chemokines) and costimulatory molecules (e.g., the B7 family members
CD80 and CD86). In addition, binding of the pathogen to endocytic pat-
tern-recognition receptors (PRRs) such as the mannose receptor leads to
the delivery of the pathogen to endosomes (Endo) and lysosomes (Lys).
There, the protein antigens of the pathogen are partially degraded to
generate antigenic peptides that can be presented by major histocom-
patibility complex (MHC) class II molecules for recognition by the T cell
antigen receptors (TCRs) of specific T cells. These effects of pattern rec-
ognition by the innate immune system lead to expression of the signals
required for the activation of quiescent antigen-specific T cells and the
subsequent generation of specific antibodies. (Adapted from Medzhitov
R, Janeway C Jr: Innate immunity. N Engl J Med 343:338, 2000.)
There are many ways in which innate immune responses
can prime or potentiate the adaptive immune response
(Fig. 16-5). In the case of T cell responses, one extremely
­important and well-recognized mechanism involves the
upregulation of costimulatory molecules. T cells require at
least two signals to become activated from a naive, resting
state. One signal is provided through the T cell antigen
receptor by its binding to a specific peptide ligand ­presented
by an MHC class I or II molecule. The second signal is
provided by one of several costimulatory ligands that are
expressed by specialized antigen presenting cells such as
dendritic cells. The best studied of these are the molecules
of the B7 family—B7-1 (CD80) and B7-2 (CD86)—which
engage the activating receptor CD28 on the surface of the
T cell. The expression of B7 family costimulatory molecules
on the surface of antigen presenting cells is controlled by
the innate immune system, such that these molecules are
induced to appear at functional levels only after PRRs,
such as members of the TLR family, have been activated by
­recognition of their cognate PAMPs.50
Recent studies have shown that innate immune ­signaling
through TLRs has a major impact on the responses of
­phagocytic antigen presenting cells, and it also provides
an important second signal for immunoglobulin produc-
tion by B cells. In the case of phagocytic cells, the uptake
 286 PORCELLI  |  Innate Immunity
of microbes by phagocytosis and the subsequent matura-
tion of the phagosome are stimulated by concurrent TLR
signaling.119 In dendritic cells, which are the major antigen
presenting cells for the priming of T cell responses, TLR
signaling has a major impact on whether antigens from
­phagocytosed microbes are effectively presented on MHC
class II molecules.120 For B cells responding to foreign anti-
gens, it has been demonstrated that concurrent ­ signaling
through TLRs is necessary for the efficient stimulation of
T cell–dependent differentiation into plasma cells and sub-
sequent antibody secretion.121 This concept is also relevant
for T cell responses to ­autoantigens, including ­several prom-
inent nuclear antigens that are targets of autoantibodies in
rheumatic diseases.122-124
Innate immune responses also trigger the production
of many cytokines and chemokines, which enhance the
development of adaptive immune responses and change
the nature of the adaptive response generated. For example,
contact between dendritic cells and PAMPs such as LPS or
bacterial lipoproteins leads to the production of IL-12 as a
result of signaling through TLRs.74,118,125 This cytokine acts
on ­antigen-specific T cells to promote their differentiation
into T helper type 1 cells, which are associated with the pro-
duction of interferon-γ and other effector mechanisms that
favor the clearance of bacterial pathogens.126 In the case of
myeloid-lineage dendritic cells, signaling through TLRs (and
potentially other PRRs) induces a process known as matura-
tion, which is associated with the increased ­ expression of
antigen presenting and costimulatory molecules that enables
the efficient priming of naive antigen-specific T cells.118,127
This requirement for the innate immune response to
“switch on” the expression of molecules required for the prim-
ing and differentiation of T cell responses helps ensure that
proinflammatory adaptive immune responses occur mainly
in the setting of a relevant infectious challenge. After acti-
vation, helper T cells control other components of adap-
tive immunity, such as the activation of cytotoxic T cells,
B cells, and macrophages. Innate immune recognition, there­
fore, appears to control all the major aspects of the adaptive
immune response through the initial recognition of infectious
microbes by PRRs. Recently, this paradigm has been extended
and slightly reinterpreted by the observation that certain self-
molecules upregulated by cellular stress or damage can substi-
tute for PAMPs of infectious microbes in activating adaptive
immune responses.70,72 This more extended view, sometimes
referred to as the “danger model,” helps explain why certain
self-ligands produced or released in the setting of infection or
tissue damage can function in essentially the same manner as
the PAMPs associated with microorganisms.128-130
DISEASE ASSOCIATIONS INVOLVING
INNATE IMMUNITY
Given the obvious role of the innate immune response in
virtually all types of infectious diseases, one might expect
that gross defects in the mechanisms of innate immunity
occur relatively rarely and in association with clinical
immunodeficiency. In fact, there is increasing evidence
that mutations that inactivate various innate immune path-
ways can lead to increased pathogen sensitivity in both
laboratory mice and humans.12,40,111,131-133 Because many
of the pathways leading to innate immunity are amplified
during recurrent or prolonged activation of the immune
system, they must also participate in mediating tissue dam-
age in chronic inflammatory diseases. In addition, certain ­
­self-molecules that are produced or released at increased
levels as a result of inflammation, including heat-shock pro-
teins, nucleic acids, and microcrystals of monosodium urate
or calcium pyrophosphate, may act in a manner analogous
to PAMPs.70,122,124,134,135 These may signal through TLRs or
other PRRs to stimulate adjuvant-like effects that increase
the potential for autoreactive lymphocytes to be activated.
Perhaps a more surprising finding has been that some defects
in innate immunity are associated with a markedly increased
predisposition to autoimmune disease. Several different mech-
anisms have been proposed to explain this paradoxical asso-
ciation. Mechanisms of the innate immune response play an
important role in the clearance of self-antigens released from
necrotic or apoptotic cells, resulting in a noninflammatory
clearance of self-antigens that tends to favor tolerance rather
than the stimulation of immune responses.136 Failure of such
clearance may lead to excessive exposure to self-antigens,
triggering normally silent autoreactive lymphocyte clones to
expand and differentiate into effector cells. This may account
for the development of lupus-like autoimmunity in mice with
targeted deletion of the gene for the short pentraxin SAP,
which, along with other components of the innate immune
system, appears to play a significant role in the clearance of
DNA-chromatin complexes.22,137 Reduced levels of serum
mannose-binding lectin in humans also appears to be a risk
factor for the development of systemic lupus erythematosus,
possibly because of the role of this soluble PRR in facilitating
the clearance of apoptotic cells.138
Deficiencies of the early components of the classic
­pathway of complement activation have been strongly asso-
ciated with lupus-like autoimmunity in both humans and
mouse models.139-143 This may also be the result of alterations
in the clearance of apoptotic cells or other sources of self-
antigens, resulting in the increased stimulation of normally
silent autoreactive lymphocytes.144-146 An alternative, but
nonexclusive, mechanism relates to the ­involvement of the
complement system, particularly the early components C1
and C4, in facilitating the induction of self-­tolerance by the
adaptive immune system by increasing the localization of
autoantigens such as double-stranded DNA and nucleopro-
teins within the primary lymphoid compartment.140,147,148
Thus, a deficiency of C1 or C4 appears to result in a fail-
ure to delete or functionally inactivate autoreactive B
cell clones as they arise during lymphopoiesis in the bone
marrow.147,149 Studies carried out in mouse ­models ­suggest
that this ­ tolerance-inducing mechanism is also ­ partially
disrupted in animals that are deficient in a variety of other
components of innate immunity, including SAP and the
complement receptors CD21/CD35.137,147
Multiple examples of links between defects in signaling
receptors of the innate immune system and chronic inflam-
matory diseases have emerged from studies of the CARD
and PYRIN families of cytosolic PRRs. The first associa-
tion of this type was provided by genetic mapping studies
that identified the Nod2 protein as the product of the IBD1
locus, which contributes to disease susceptibility in a sub-
set of patients with Crohn’s disease.150-153 This soluble PRR
of the CARD family normally functions by inducing cyto-
kine production in response to bacterial peptidoglycan, but
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
mutant alleles associated with an increased risk of Crohn’s
disease are defective in this function.150 In this case, it
may be a failure of innate immunity to adequately control
bacterial colonization or infection in the intestine that
leads to the final expression of disease. Consistent with this
view, a recent study has demonstrated diminished expres-
sion of a class of antimicrobial peptides (β-defensins known
as ­cryptdins) in the Paneth cells from the ileum of patients
with Crohn’s disease and Nod2 mutations.154 Other stud-
ies have established links between various members of the
PYRIN family and specific chronic inflammatory disorders.
These include the causative association of mutations in
pyrin with familial Mediterranean fever and of cryopyrin
with the cryopyrin-associated periodic syndromes.82 These
diseases and other chronic inflammatory or autoimmune
disorders associated with specific deficiencies in innate
immune mechanisms are now frequently considered together
as autoinflammatory diseases.155 The recognition that these
diseases are frequently associated with a dysregulation of
inflammatory cytokine production, in particular IL-1β, has
led to some striking therapeutic advances in the treatment
of selected patients using the systemic administration of the
IL-1 receptor antagonist anakinra.156-158
Deficiencies in at least two populations of ILLs—NK cells
and NK T cells—have also been associated with multiple
autoimmune syndromes in both humans and mice.103,159-161
This is believed to reflect a significant role for these ILLs in
regulating adaptive immune responses, although the precise
mechanisms by which they act are still not fully understood.
Given the complex interplay between innate and adaptive
immunity, it is extremely likely that associations between
alterations in innate immunity and autoimmune diseases
will continue to emerge. As for some of the examples cited
here, a fuller understanding of these associations is likely to
lead to new and successful therapies for autoimmune and
autoinflammatory diseases.
REFERENCES
  1. Agrawal A, Eastman QM, Schatz DG: Transposition mediated
by RAG1 and RAG2 and its implications for the evolution of the
immune system. Nature 394:744, 1998.
  2. Schatz DG: Transposition mediated by RAG1 and RAG2 and
the evolution of the adaptive immune system. Immunol Res 19:
169, 1999.
  3. Flajnik MF: Churchill and the immune system of ectothermic verte-
brates. Immunol Rev 166:5, 1998.
  4. Alder MN, Rogozin IB, Iyer LM, et al: Diversity and function of
adaptive immune receptors in a jawless vertebrate. Science 310:
1970, 2005.
  5. Pancer Z, Saha NR, Kasamatsu J, et al: Variable lymphocyte receptors
in hagfish. Proc Natl Acad Sci U S A 102:9224, 2005.
  6. Pancer Z, Cooper MD: The evolution of adaptive immunity. Annu
Rev Immunol 24:497, 2006.
  7. Mushegian A, Medzhitov R: Evolutionary perspective on innate
immune recognition. J Cell Biol 155:705, 2001.
  8. Hoffmann JA, Reichhart JM: Drosophila innate immunity: An evolu-
tionary perspective. Nat Immunol 3:121, 2002.
  9. Fraser IP, Koziel H, Ezekowitz RA: The serum mannose-binding pro-
tein and the macrophage mannose receptor are pattern recognition
molecules that link innate and adaptive immunity. Semin Immunol
10:363, 1998.
10. Stover C, Endo Y, Takahashi M, et al: The human gene for mannan-
binding lectin-associated serine protease-2 (MASP-2), the effector
component of the lectin route of complement activation, is part of a
tightly linked gene cluster on chromosome 1p36.2-3. Genes Immun
2:119, 2001.
287
11. Jack DL, Jarvis GA, Booth CL, et al: Mannose-binding lectin accel-
erates complement activation and increases serum killing of Neisseria
meningitidis serogroup C. J Infect Dis 184:836, 2001.
12. Jack DL, Klein NJ, Turner MW: Mannose-binding lectin: Targeting
the microbial world for complement attack and opsonophagocytosis.
Immunol Rev 180:86, 2001.
13. Polotsky VY, Belisle JT, Mikusova K, et-al: Interaction of human
mannose-binding protein with Mycobacterium avium. J Infect Dis
175(5):1159-1168:1997.
14. Fraser IP, Takahashi K, Koziel H, et al: Pneumocystis carinii enhances
soluble mannose receptor production by macrophages. Microbes
Infect 2:1305, 2000.
15. Kase T, Suzuki Y, Kawai T, et al: Human mannan-binding lectin
inhibits the infection of influenza A virus without complement.
Immunology 97:385, 1999.
16. Thielens NM, Tacnet-Delorme P, Arlaud GJ: Interaction of C1q and
mannan-binding lectin with viruses. Immunobiology 205:563, 2002.
17. Klabunde J, Uhlemann AC, Tebo AE, et al: Recognition of plasmo-
dium falciparum proteins by mannan-binding lectin, a component of
the human innate immune system. Parasitol Res 88:113, 2002.
18. Matsushita M, Kuraya M, Hamasaki N, et al: Activation of the lec-
tin complement pathway by H-ficolin (Hakata antigen). J Immunol
168:3502, 2002.
19. Lu J, Teh C, Kishore U, et al: Collectins and ficolins: Sugar pattern
recognition molecules of the mammalian innate immune system.
Biochim Biophys Acta 1572:387, 2002.
20. Reid KB, Colomb M, Petry F, et al: Complement component C1 and
the collectins—first-line defense molecules in innate and acquired
immunity. Trends Immunol 23:115, 2002.
21. McCormack FX, Whitsett JA: The pulmonary collectins, SP-A and
SP-D, orchestrate innate immunity in the lung. J Clin Invest 109:
707, 2002.
22. Garlanda C, Bottazzi B, Bastone A, et al: Pentraxins at the crossroads
between innate immunity, inflammation, matrix deposition, and
female fertility. Annu Rev Immunol 23:337, 2005.
23. Bottazzi B, Garlanda C, Salvatori G, et al: Pentraxins as a key com-
ponent of innate immunity. Curr Opin Immunol 18:10, 2006.
24. Garlanda C, Hirsch E, Bozza S, et al: Non-redundant role of the long
pentraxin PTX3 in anti-fungal innate immune response. Nature
420:182, 2002.
25. Taylor PR, Martinez-Pomares L, Stacey M, et al: Macrophage recep-
tors and immune recognition. Annu Rev Immunol 23:901, 2005.
26. Taylor PR, Gordon S, Martinez-Pomares L: The mannose recep-
tor: Linking homeostasis and immunity through sugar recognition.
Trends Immunol 26:104, 2005.
27. Zamze S, Martinez-Pomares L, Jones H, et al: Recognition of bacte-
rial capsular polysaccharides and lipopolysaccharides by the macro-
phage mannose receptor. J Biol Chem 277:41613, 2002.
28. Apostolopoulos V, McKenzie IF: Role of the mannose receptor in the
immune response. Curr Mol Med 1:469, 2001.
29. Martinez-Pomares L, Linehan SA, Taylor PR, et al: Binding proper-
ties of the mannose receptor. Immunobiology 204:527, 2001.
30. Nigou J, Zelle-Rieser C, Gilleron M, et al: Mannosylated lipoarabi-
nomannans inhibit IL-12 production by human dendritic cells: Evi-
dence for a negative signal delivered through the mannose receptor.
J Immunol 166:7477, 2001.
31. Yoshitomi H, Sakaguchi N, Kobayashi K, et al: A role for fungal
β-glucans and their receptor Dectin-1 in the induction of autoim-
mune arthritis in genetically susceptible mice. J Exp Med 201:949,
2005.
32. Peiser L, Mukhopadhyay S, Gordon S: Scavenger receptors in innate
immunity. Curr Opin Immunol 14:123, 2002.
33. Krieger M: The other side of scavenger receptors: Pattern recognition
for host defense. Curr Opin Lipidol 8:275, 1997.
34. Peiser L, De Winther MP, Makepeace K, et al: The class A macro-
phage scavenger receptor is a major pattern recognition receptor for
Neisseria meningitidis which is independent of lipopolysaccharide and
not required for secretory responses. Infect Immun 70:5346, 2002.
35. Kraal G, van der Laan LJ, Elomaa O, et al: The macrophage receptor
MARCO. Microbes Infect 2:313, 2000.
36. Elshourbagy NA, Li X, Terrett J, et al: Molecular characterization of
a human scavenger receptor, human MARCO. Eur J Biochem 267:
919, 2000.
37. Hampton RY, Golenbock DT, Penman M, et al: Recognition and
plasma clearance of endotoxin by scavenger receptors. Nature
352:342, 1991.
 288 PORCELLI  |  Innate Immunity
38. Dunne DW, Resnick D, Greenberg J, et al: The type I macrophage
scavenger receptor binds to gram-positive bacteria and recognizes
lipoteichoic acid. Proc Natl Acad Sci U S A 91:1863, 1994.
39. Suzuki H, Kurihara Y, Takeya M, et al: A role for macrophage scaven-
ger receptors in atherosclerosis and susceptibility to infection. Nature
386:292, 1997.
40. Thomas CA, Li Y, Kodama T, et al: Protection from lethal gram-
positive infection by macrophage scavenger receptor-dependent
phagocytosis. J Exp Med 191:147, 2000.
41. Haworth R, Platt N, Keshav S, et al: The macrophage scavenger
receptor type A is expressed by activated macrophages and protects
the host against lethal endotoxic shock. J Exp Med 186:1431, 1997.
42. Takeda K, Kaisho T, Akira S: Toll-like receptors. Annu Rev
Immunol 21:335, 2003.
43. Werts C, Girardin SE, Philpott DJ: TIR, CARD and PYRIN: Three
domains for an antimicrobial triad. Cell Death Differ 13:798, 2006.
44. Dangl JL, Jones JD: Plant pathogens and integrated defence responses
to infection. Nature 411:826, 2001.
45. Medzhitov R, Janeway C Jr: Innate immune recognition: Mecha-
nisms and pathways. Immunol Rev 173:89, 2000.
46. Hashimoto C, Hudson KL, Anderson KV: The Toll gene of Drosoph-
ila, required for dorsal-ventral embryonic polarity, appears to encode
a transmembrane protein. Cell 52:269, 1988.
47. Gay NJ, Keith FJ: Drosophila Toll and IL-1 receptor. Nature
351:355, 1991.
48. Lemaitre B, Nicolas E, Michaut L, et al: The dorsoventral regula-
tory gene cassette spatzle/Toll/cactus controls the potent antifungal
response in Drosophila adults. Cell 86:973, 1996.
49. Kawai T, Akira S: Pathogen recognition with Toll-like receptors.
Curr Opin Immunol 17:338, 2005.
50. Medzhitov R, Preston-Hurlburt P, Janeway CA Jr: A human
homologue of the Drosophila Toll protein signals activation of adap­
tive immunity. Nature 388:394, 1997.
51. Poltorak A, He X, Smirnova I, et al: Defective LPS signaling in
C3H/HeJ and C57BL/10ScCr mice: Mutations in TLR4 gene. Sci-
ence 282:2085, 1998.
52. Qureshi ST, Lariviere L, Leveque G, et al: Endotoxin-tolerant mice
have mutations in Toll-like receptor 4 (TLR4). J Exp Med 189:615,
1999.
53. Hoshino K, Takeuchi O, Kawai T, et al: Cutting edge: Toll-like
receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopoly-
saccharide: Evidence for TLR4 as the LPS gene product. J Immunol
162:3749, 1999.
54. Takeuchi O, Hoshino K, Kawai T, et al: Differential roles of TLR2
and TLR4 in recognition of gram-negative and gram-positive bacte-
rial cell wall components. Immunity 11:443, 1999.
55. Takeuchi O, Akira S: Genetic approaches to the study of Toll-like
receptor function. Microbes Infect 4:887, 2002.
56. da Silva CJ, Soldau K, Christen U, et al: Lipopolysaccharide is in close
proximity to each of the proteins in its membrane receptor complex
transfer from CD14 to TLR4 and MD-2. J Biol Chem 276:21129,
2001.
57. Medzhitov R, Preston-Hurlburt P, Kopp E, et al: MyD88 is an adap-
tor protein in the hToll/IL-1 receptor family signaling pathways. Mol
Cell 2:253, 1998.
58. Fitzgerald KA, Chen ZJ: Sorting out Toll signals. Cell 125:834,
2006.
59. Horng T, Barton GM, Flavell RA, et al: The adaptor molecule
TIRAP provides signalling specificity for Toll-like receptors. Nature
420:329, 2002.
60. Yamamoto M, Sato S, Hemmi H, et al: Essential role for TIRAP
in activation of the signalling cascade shared by TLR2 and TLR4.
Nature 420:324, 2002.
61. Kawai T, Akira S: TLR signaling. Cell Death Differ 13:816, 2006.
62. Belvin MP, Anderson KV: A conserved signaling pathway: The Dro-
sophila Toll-dorsal pathway. Annu Rev Cell Dev Biol 12:393, 1996.
63. Kawasaki K, Akashi S, Shimazu R, et al: Involvement of TLR4/MD-2
complex in species-specific lipopolysaccharide-mimetic signal trans-
duction by Taxol. J Endotoxin Res 7:232, 2001.
64. Kawasaki K, Gomi K, Nishijima M: Cutting edge: Gln22 of mouse
MD-2 is essential for species-specific lipopolysaccharide mimetic
action of Taxol. J Immunol 166:11, 2001.
65. Kawasaki K, Akashi S, Shimazu R, et al: Mouse Toll-like receptor
4.MD-2 complex mediates lipopolysaccharide-mimetic signal trans-
duction by Taxol. J Biol Chem 275:2251, 2000.
66. Haynes LM, Moore DD, Kurt-Jones EA, et al: Involvement of Toll-
like receptor 4 in innate immunity to respiratory syncytial virus.
J Virol 75:10730, 2001.
67. Kurt-Jones EA, Popova L, Kwinn L, et al: Pattern recognition recep-
tors TLR4 and CD14 mediate response to respiratory syncytial virus.
Nat Immunol 1:398, 2000.
68. Sasu S, LaVerda D, Qureshi N, et al: Chlamydia pneumoniae and
chlamydial heat shock protein 60 stimulate proliferation of human
vascular smooth muscle cells via Toll-like receptor 4 and p44/
p42 mitogen-activated protein kinase activation. Circ Res 89:
244, 2001.
69. Vabulas RM, Ahmad-Nejad P, da Costa C, et al: Endocytosed HSP60s
use Toll-like receptor 2 (TLR2) and TLR4 to activate the Toll/inter-
leukin-1 receptor signaling pathway in innate immune cells. J Biol
Chem 276:31332, 2001.
70. Ohashi K, Burkart V, Flohe S, et al: Cutting edge: Heat shock protein
60 is a putative endogenous ligand of the Toll-like receptor-4 com-
plex. J Immunol 164:558, 2000.
71. Jiang D, Liang J, Fan J, et al: Regulation of lung injury and repair by
Toll-like receptors and hyaluronan. Nat Med 11:1173, 2005.
72. Okamura Y, Watari M, Jerud ES, et al: The extra domain A of fibro-
nectin activates Toll-like receptor 4. J Biol Chem 276:10229, 2001.
73. Aliprantis AO, Yang RB, Mark MR, et al: Cell activation and apop-
tosis by bacterial lipoproteins through Toll-like receptor-2. Science
285:736, 1999.
74. Brightbill HD, Libraty DH, Krutzik SR, et al: Host defense mecha-
nisms triggered by microbial lipoproteins through Toll-like receptors.
Science 285:732, 1999.
75. Campos MA, Almeida IC, Takeuchi O, et al: Activation of Toll-like
receptor-2 by glycosylphosphatidylinositol anchors from a protozoan
parasite. J Immunol 167:416, 2001.
76. Akira S, Takeda K, Kaisho T: Toll-like receptors: Critical proteins
linking innate and acquired immunity. Nat Immunol 2:675, 2001.
77. Hayashi F, Smith KD, Ozinsky A, et al: The innate immune response
to bacterial flagellin is mediated by Toll-like receptor 5. Nature
410:1099, 2001.
78. Alexopoulou L, Holt AC, Medzhitov R, et al: Recognition of double-
stranded RNA and activation of NF-kappaB by Toll-like receptor 3.
Nature 413:732, 2001.
79. Diebold SS, Kaisho T, Hemmi H, et al: Innate antiviral responses by
means of TLR7-mediated recognition of single-stranded RNA. Sci-
ence 303:1529, 2004.
80. Hemmi H, Takeuchi O, Kawai T, et al: A Toll-like receptor recog-
nizes bacterial DNA. Nature 408:740, 2000.
81. Underhill DM, Ozinsky A: Toll-like receptors: Key mediators of
microbe detection. Curr Opin Immunol 14:103, 2002.
82. Ting JP, Kastner DL, Hoffman HM: CATERPILLERs, pyrin and
hereditary immunological disorders. Nat Rev Immunol 6:183,
2006.
83. Kufer TA, Banks DJ, Philpott DJ: Innate immune sensing of microbes
by Nod proteins. Ann N Y Acad Sci 1072:19, 2006.
84. Kufer TA, Fritz JH, Philpott DJ: NACHT-LRR proteins (NLRs) in
bacterial infection and immunity. Trends Microbiol 13:381, 2005.
85. Neven B, Callebaut I, Prieur AM, et al: Molecular basis of the spec-
tral expression of CIAS1 mutations associated with phagocytic cell-
mediated autoinflammatory disorders CINCA/NOMID, MWS, and
FCU. Blood 103:2809, 2004.
86. Tschopp J, Martinon F, Burns K: NALPs: A novel protein family
involved in inflammation. Nat Rev Mol Cell Biol 4:95, 2003.
87. Martinon F, Burns K, Tschopp J: The inflammasome: A molecular
platform triggering activation of inflammatory caspases and process-
ing of proIL-beta. Mol Cell 10:417, 2002.
88. Martinon F, Petrilli V, Mayor A, et al: Gout-associated uric acid crys-
tals activate the NALP3 inflammasome. Nature 440:237, 2006.
89. Kanneganti TD, Ozoren N, Body-Malapel M, et al: Bacterial RNA
and small antiviral compounds activate caspase-1 through cryopyrin/
Nalp3. Nature 440:233, 2006.
90. Kanneganti TD, Body-Malapel M, Amer A, et al: Critical role for
cryopyrin/Nalp3 in activation of caspase-1 in response to viral infec-
tion and double-stranded RNA. J Biol Chem 281(48):36560-36568,
2006.
91. Boyden ED, Dietrich WF: Nalp1b controls mouse macrophage sus-
ceptibility to anthrax lethal toxin. Nat Genet 38:240, 2006.
92. Babior BM, Lambeth JD, Nauseef W: The neutrophil NADPH oxi-
dase. Arch Biochem Biophys 397:342, 2002.
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
93. Xie QW, Cho HJ, Calaycay J, et al: Cloning and characterization
of inducible nitric oxide synthase from mouse macrophages. Science
256:225, 1992.
94. Nathan C: Inducible nitric oxide synthase in the tuberculous human
lung. Am J Respir Crit Care Med 166:130, 2002.
95. Vilches C, Parham P: KIR: Diverse, rapidly evolving receptors
of innate and adaptive immunity. Annu Rev Immunol 20:217,
2002.
96. Biron CA, Nguyen KB, Pien GC, et al: Natural killer cells in antivi-
ral defense: Function and regulation by innate cytokines. Annu Rev
Immunol 17:189, 1999.
97. Bendelac A, Bonneville M, Kearney JF: Autoreactivity by design:
Innate B and T lymphocytes. Nat Rev Immunol 1:177, 2001.
98. Berland R, Wortis HH: Origins and functions of B-1 cells with notes
on the role of CD5. Annu Rev Immunol 20:253, 2002.
99. Martin F, Kearney JF: Marginal-zone B cells. Nat Rev Immunol
2:323, 2002.
100. Carding SR, Egan PJ: Gamma delta T cells: Functional plasticity and
heterogeneity. Nat Rev Immunol 2:336, 2002.
101. Wu J, Groh V, Spies T: T cell antigen receptor engagement and
specificity in the recognition of stress-inducible MHC class I-related
chains by human epithelial gamma delta T cells. J Immunol 169:
1236, 2002.
102. Dutronc Y, Porcelli SA: The CD1 family and T cell recognition of
lipid antigens. Tissue Antigens 60:337, 2002.
103. Yu KO, Porcelli SA: The diverse functions of CD1d-restricted NKT
cells and their potential for immunotherapy. Immunol Lett 100:
42, 2005.
104. Mattner J, Debord KL, Ismail N, et al: Exogenous and endogenous
glycolipid antigens activate NKT cells during microbial infections.
Nature 434:525, 2005.
105. Kinjo Y, Wu D, Kim G, et al: Recognition of bacterial glycosphingo-
lipids by natural killer T cells. Nature 434:520, 2005.
106. Kinjo Y, Tupin E, Wu D, et al: Natural killer T cells recognize dia-
cylglycerol antigens from pathogenic bacteria. Nat Immunol 7:
978, 2006.
107. Zasloff M: Antimicrobial peptides of multicellular organisms.
Nature 415:389, 2002.
108. Shai Y: Mechanism of the binding, insertion and destabilization of
phospholipid bilayer membranes by alpha-helical antimicrobial and
cell non-selective membrane-lytic peptides. Biochim Biophys Acta
1462:55, 1999.
109. Yang L, Weiss TM, Lehrer RI, et al: Crystallization of antimicro-
bial pores in membranes: Magainin and protegrin. Biophys J 79:
2000, 2002.
110. Matsuzaki K: Why and how are peptide-lipid interactions utilized for
self-defense? Magainins and tachyplesins as archetypes. Biochim Bio-
phys Acta 1462:1, 1999.
111. Nizet V, Ohtake T, Lauth X, et al: Innate antimicrobial peptide
protects the skin from invasive bacterial infection. Nature 414:
454, 2001.
112. Harder J, Bartels J, Christophers E, et al: Isolation and character-
ization of human beta-defensin-3, a novel human inducible peptide
antibiotic. J Biol Chem 276:5707, 2001.
113. Harder J, Bartels J, Christophers E, et al: A peptide antibiotic from
human skin. Nature 387:861, 1997.
114. Singh PK, Jia HP, Wiles K, et al: Production of beta-defensins by
human airway epithelia. Proc Natl Acad Sci U S A 95:14961, 1998.
115. Cunliffe RN, Mahida YR: Expression and regulation of antimicrobial
peptides in the gastrointestinal tract. J Leukoc Biol 75:49, 2004.
116. Cunliffe RN: Alpha-defensins in the gastrointestinal tract. Mol
Immunol 40:463, 2003.
117. Shai Y: From innate immunity to de-novo designed antimicrobial
peptides. Curr Pharm Des 8:715, 2002.
118. Schnare M, Barton GM, Holt AC, et al: Toll-like receptors con-
trol activation of adaptive immune responses. Nat Immunol 2:
947, 2001.
119. Blander JM, Medzhitov R: Regulation of phagosome maturation by
signals from Toll-like receptors. Science 304:1014, 2004.
120. Blander JM, Medzhitov R: Toll-dependent selection of microbial
antigens for presentation by dendritic cells. Nature 440:808, 2006.
121. Pasare C, Medzhitov R: Control of B-cell responses by Toll-like
receptors. Nature 438:364, 2005.
122. Leadbetter EA, Rifkin IR, Hohlbaum AM, et al: Chromatin-IgG
complexes activate B cells by dual engagement of IgM and Toll-like
receptors. Nature 416:603, 2002.
289
123. Christensen SR, Kashgarian M, Alexopoulou L, et al: Toll-like recep-
tor 9 controls anti-DNA autoantibody production in murine lupus.
J Exp Med 202:321, 2005.
124. Lau CM, Broughton C, Tabor AS, et al: RNA-associated autoanti-
gens activate B cells by combined B cell antigen receptor/Toll-like
receptor 7 engagement. J Exp Med 202:1171, 2005.
125. Barton GM, Medzhitov R: Control of adaptive immune responses by
Toll-like receptors. Curr Opin Immunol 14:380, 2002.
126. Constant SL, Bottomly K: Induction of Th1 and Th2 CD4+ T cell
responses: The alternative approaches. Annu Rev Immunol 15:
297, 1997.
127. Banchereau J, Steinman RM: Dendritic cells and the control of
immunity. Nature 392:245, 1998.
128. Matzinger P: The danger model: A renewed sense of self. Science
296:301, 2002.
129. Seong SY, Matzinger P: Hydrophobicity: An ancient damage-
associated molecular pattern that initiates innate immune responses.
Nat Rev Immunol 4:469, 2004.
130. Shi Y, Evans JE, Rock KL: Molecular identification of a danger signal
that alerts the immune system to dying cells. Nature 425:516, 2003.
131. Mockenhaupt FP, Cramer JP, Hamann L, et al: Toll-like receptor
(TLR) polymorphisms in African children: Common TLR-4 vari-
ants predispose to severe malaria. Proc Natl Acad Sci U S A 103:
177, 2006.
132. Schroder NW, Diterich I, Zinke A, et al: Heterozygous Arg753Gln
polymorphism of human TLR-2 impairs immune activation by Bor-
relia burgdorferi and protects from late stage Lyme disease. J Immunol
175:2534, 2005.
133. Schroder NW, Schumann RR: Single nucleotide polymorphisms of
Toll-like receptors and susceptibility to infectious disease. Lancet
Infect Dis 5:156, 2005.
134. Liu-Bryan R, Scott P, Sydlaske A, et al: Innate immunity conferred
by Toll-like receptors 2 and 4 and myeloid differentiation factor 88
expression is pivotal to monosodium urate monohydrate crystal-
induced inflammation. Arthritis Rheum 52:2936, 2005.
135. Liu-Bryan R, Pritzker K, Firestein GS, et al: TLR2 signaling in
chondrocytes drives calcium pyrophosphate dihydrate and monoso-
dium urate crystal-induced nitric oxide generation. J Immunol 174:
5016, 2005.
136. Gershov D, Kim S, Brot N, et al: C-reactive protein binds to apop-
totic cells, protects the cells from assembly of the terminal comple-
ment components, and sustains an antiinflammatory innate immune
response: Implications for systemic autoimmunity. J Exp Med
192:1353, 2000.
137. Bickerstaff MC, Botto M, Hutchinson WL, et al: Serum amyloid P
component controls chromatin degradation and prevents antinuclear
autoimmunity. Nat Med 5:694, 1999.
138. Tsutsumi A, Takahashi R, Sumida T: Mannose binding lectin: Genet-
ics and autoimmune disease. Autoimmun Rev 4:364, 2005.
139. Walport MJ: Complement and systemic lupus erythematosus. Arthri-
tis Res 4(Suppl 3):S279, 2002.
140. Einav S, Pozdnyakova OO, Ma M, et al: Complement C4 is pro-
tective for lupus disease independent of C3. J Immunol 168:
1036, 2002.
141. Paul E, Pozdnyakova OO, Mitchell E, et al: Anti-DNA autoreactiv-
ity in C4-deficient mice. Eur J Immunol 32:2672, 2002.
142. Mitchell DA, Pickering MC, Warren J, et al: C1q deficiency and
autoimmunity: The effects of genetic background on disease expres-
sion. J Immunol 168:2538, 2002.
143. Chen Z, Koralov SB, Kelsoe G: Complement C4 inhibits systemic
autoimmunity through a mechanism independent of complement
receptors CR1 and CR2. J Exp Med 192:1339, 2000.
144. Korb LC, Ahearn JM: C1q binds directly and specifically to surface
blebs of apoptotic human keratinocytes: Complement deficiency
and systemic lupus erythematosus revisited. J Immunol 158:4525,
1997.
145. Cutler AJ, Botto M, van Essen D, et al: T cell-dependent immune
response in C1q-deficient mice: Defective interferon gamma produc-
tion by antigen-specific T cells. J Exp Med 187:1789, 1998.
146. Mitchell DA, Taylor PR, Cook HT, et al: Cutting edge: C1q protects
against the development of glomerulonephritis independently of C3
activation. J Immunol 162:5676, 1999.
147. Prodeus AP, Goerg S, Shen LM, et al: A critical role for complement
in maintenance of self-tolerance. Immunity 9:721, 1998.
148. Paul E, Carroll MC: SAP-less chromatin triggers systemic lupus ery-
thematosus. Nat Med 5:607, 1999.
 290 PORCELLI  |  Innate Immunity
149. Goodnow CC, Cyster JG, Hartley SB, et al: Self-tolerance check-
points in B lymphocyte development. Adv Immunol 59:279, 1995.
150. Ogura Y, Bonen DK, Inohara N, et al: A frameshift mutation in
NOD2 associated with susceptibility to Crohn’s disease. Nature
411:603, 2001.
151. Hugot JP, Chamaillard M, Zouali H, et al: Association of NOD2
leucine-rich repeat variants with susceptibility to Crohn’s disease.
Nature 411:599, 2001.
152. Cuthbert AP, Fisher SA, Mirza MM, et al: The contribution of
NOD2 gene mutations to the risk and site of disease in inflammatory
bowel disease. Gastroenterology 122:867, 2002.
153. Hampe J, Cuthbert A, Croucher PJ, et al: Association between inser-
tion mutation in NOD2 gene and Crohn’s disease in German and
British populations. Lancet 357:1925, 2001.
154. Wehkamp J, Harder J, Weichenthal M, et al: NOD2 (CARD15)
mutations in Crohn’s disease are associated with diminished mucosal
alpha-defensin expression. Gut 53:1658, 2004.
155. Brydges S, Kastner DL: The systemic autoinflammatory diseases:
Inborn errors of the innate immune system. Curr Top Microbiol
Immunol 305:127, 2006.
156. Metyas SK, Hoffman HM: Anakinra prevents symptoms of familial
cold autoinflammatory syndrome and Raynaud’s disease. J Rheuma-
tol 33:2085, 2006.
157. Goldbach-Mansky R, Dailey NJ, Canna SW, et al: Neonatal-onset
multisystem inflammatory disease responsive to interleukin-1ß inhi-
bition. N Engl J Med 355:581, 2006.
158. Mirault T, Launay D, Cuisset L, et al: Recovery from deafness in a
patient with Muckle-Wells syndrome treated with anakinra. Arthri-
tis Rheum 54:1697, 2006.
159. van der Vliet HJ, von Blomberg BM, Nishi N, et al: Circulating
Va24+ Vß11+ NKT cell numbers are decreased in a wide variety of
diseases that are characterized by autoreactive tissue damage. Clin
Immunol 100:144, 2001.
160. Fort MM, Leach MW, Rennick DM: A role for NK cells as regula-
tors of CD4+ T cells in a transfer model of colitis. J Immunol 161:
3256, 1998.
161. Shi FD, Wang HB, Li H, et al: Natural killer cells determine the out-
come of B cell-mediated autoimmunity. Nat Immunol 1:245, 2000.
 17
KEY POINTS
Lymphocytes are born and many self-reactive cells deleted in
primary lymphoid tissues.
Immune responses are usually initiated in secondary
­lymphoid tissues.
Tertiary lymphoid tissue can be generated at sites of
­inflammation and may promote tissue-specific autoimmunity.
Self-tolerance is established by antigen recognition in
primary lymphoid tissues or elsewhere in the absence of
­inflammation.
Generation of an adaptive immune response depends on
­innate immune system activation.
Dendritic cells sense tissue-specific factors and innate
­immune stimuli in shaping T cell responses to antigens.
The adaptive immune system is so named because it can
adapt to virtually any pathogen or toxin that enters the
body. Although invertebrates defend themselves through
innate immunity alone,1 vertebrates all have developed
some form of adaptive immunity—the ability to generate
novel receptors by genetic recombination mechanisms that
can be selected to recognize diverse macromolecules associ-
ated with rapidly evolving pathogens.2 A molecule that can
be recognized by the adaptive immune system is known as
an antigen (Table 17-1).
The ability to produce molecules and cells that can
attack any biologic structure is a double-edged sword.2
Pathologic autoimmunity is not described in invertebrates,
which evolved with a hard-wired immune system, whereas
it is a common problem in vertebrates.3-6 Self-recogni-
tion must be offset by redundant mechanisms to produce
self-tolerance. This chapter discusses some mechanisms
involved in this process and the anatomy behind these.
In addition to self-antigen, animals with adaptive immu-
nity also are exposed to many harmless environmental
antigens that have the potential to induce allergic reac-
tions.7 Mechanisms to distinguish self from foreign, and
benign foreign from harmful foreign, macromolecules are
crucial processes in successful adaptive immunity. At the
core of these mechanisms is the essential partnership of
adaptive and innate immunity, first formulated by Jane-
way.8 Although the adaptive immune system can recog-
nize any foe and focus powerful effector mechanisms to
destroy this foe, its ability to calculate the relative risks of
mounting an immune response or becoming tolerant to a
given recognized structure is guided by innate recognition
of pathogen-associated molecule patterns or inflammation
associated with tissue destruction.
Adaptive Immunity
Including Organization
of Lymphoid Tissues
Michael L. Dustin
The function of the adaptive immune system is tightly
linked to its anatomy.9-11 T lymphocytes are cells without
boundaries that can be found in almost any tissue at any
time. T lymphocytes and B lymphocytes are readily moni-
tored in patients because they are reliably found in the blood
of normal individuals. Most T lymphocytes and B lym-
phocytes are located in secondary lymphoid tissues, how-
ever, where they search for antigens. Three types of tissues
concentrate lymphocytes—the primary lymphoid tissues,
where these cells are born; the secondary lymphoid tissues,
where they search for antigens; and tertiary lymphoid tis-
sues, which form at sites of chronic inflammation. Second-
ary and tertiary lymphoid tissues have a characteristic and
functionally important organization into B cell and T cell
zones. All of the above-mentioned histologic findings are
directly related to functional goals of the adaptive immune
system, as is discussed subsequently. This chapter addresses
the basics of adaptive immunity and its partnership with
innate immunity in the anatomic contexts in which these
responses occur.
LYMPHOCYTE MIGRATION PARADIGMS
FOR HOMING, INTERSTITIAL
NAVIGATION, AND EGRESS
Because adaptive immunity is discussed in the context of sec-
ondary lymphoid tissues, this is a good time to review what
is known about lymphocyte migration. It has been appre-
ciated since the early 1960s that lymphocytes “recirculate”
between secondary lymphoid tissues and the blood, and that
on activation they take on different tissue-specific homing
properties that are important for function.12 If one arbitrarily
begins this circuit with a naive T cell in the blood, three dis-
tinct transitions can be considered: (1) interaction with the
vessel wall and extravasation; (2) interstitial locomotion in
the tissue parenchyma; and (3) egress from the tissue paren-
chyma back into the blood, sometimes via lymph. General
features of these three steps are addressed here, and relevant
details are commented on in different functional contexts in
relation to adaptive immunity.
MULTISTEP PARADIGM FOR EXTRAVASATION
The movement of lymphocytes from blood to tissues is com-
plicated by the high flow rates in blood vessels. Springer and
Butcher established the current paradigm called the “multi-
step model” for T cell extravasation.13,14
The first step is the initial tethering of the free-flowing
leukocyte to the vessel wall. A special class of adhesion
molecules known as selectins and their carbohydrate ligands
mediates this step (see Table 17-1).15,16 There are three
291
 292 DUSTIN  |  ADAPTIVE IMMUNITY INCLUDING ORGANIZATION OF LYMPHOID TISSUES
Table 17-1  Terms and Definitions
Antigen Any molecular structure recog-
nized by the adaptive immune
system. The ligand for B cell
receptor/antibody or T cell
receptor
Chemokine Family of small secreted or shed
proteins (typically 8 kD) that
bind to G protein–coupled
­receptors and activate or at-
tract cells. A chemical (chemo-)
that induces movement (-kine)
Integrin Family of adhesion molecules
that are specialized for stable
adhesion and locomotion. Non-
covalent heterodimers that are
regulated rapidly by signaling
from chemokine ­receptors and
antigen receptors
Selectin Family of adhesion molecules that
are specialized for ­mediating
initial attachment of leukocytes
from flowing blood to the
vessel wall. Have N-terminal
C-type lectin domains and
interact with carbohydrate
ligands that can incorporate
protein determinants
­ embers of the selectin family, called L-selectin, E-selectin,
m are the major ICAMs expressed on endothelial cells, with
and P-selectin.17 L-selectin is expressed on naive T cells and
B cells and is essential for the entry of these cells into lymph
nodes, but not the spleen.18,19 The ligand for L-selectin is
a structure of sulfated sialic acid bearing complex carbohy-
drates linked to different protein backbones expressed on
high endothelial cells in postcapillary venules in primary,
secondary, and tertiary lymphoid tissues.15 E-selectin and P-
selectin are expressed on activated endothelial cells at sites
of inflammation in diverse tissues.20 They bind to glycopro-
tein ligands expressed on leukocytes, which have terminal
sialyl-Lewisx blood group antigens, and can incorporate
other structural modification.16 The high-affinity ligand for
P-selectin is the protein backbone PSGL-1 with a stretch of
sulfated tyrosines that compose part of the ligand.21,22 PSGL-
1 can be expressed by lymphocytes without the necessary
secondary modification to make it a ligand for P-selectin. A
fusion of P-selectin to immunoglobulin Fc that can be puri-
fied and fluorescently tagged is the best probe to determine
if a leukocyte is competent to bind to P-selectin express-
ing endothelial cells.23 The genetic basis of forming selec-
tin ligands is complex with requirements for expression of
core proteins, specific sialotransferases, fucosyltransferases,
and sulfotransferases.16 Defects in fucose metabolism are the
basis for a rare genetic immunodeficiency/mental retarda-
tion syndrome called leukocyte adhesion deficiency type
II.24 Selectins mediate only leukocyte tethering and rolling,
not arrest and extravasation.
Selectin-mediated tethering allows the leukocyte to bring
G protein–coupled receptors close enough to the vascular
wall to bind ligands either attached to glycoproteins or dif-
fusing in the thin unstirred layer near the endothelial sur-
face.25,26 Chemokine receptor signaling is crucial to activate
closely linked integrin family members to bind their ligands.27
This chemokine signal is pertussis toxin ­sensitive, indicating
that it involves Giα coupled receptors. With respect to lym-
phocyte recirculation, there are three important chemokine
receptors—CCR7, CXCR4, and CXCR5. CCR7, binding
to ligands CCL19 and CCL21, is the most important che-
mokine system for entry of T cells into secondary lymphoid
tissues.28 CXCR4 binding to its ligand CXCL12 also con-
tributes to entry of T cells and B cells.29,30 CXCR5, binding
to its ligand CXCL13, is the major system controlling entry
of B cells into B cell follicles.31,32 After activation, CCR7 is
downregulated on many effector T cells, and other chemo-
kine receptors are upregulated allowing these cells to home
to peripheral sites of inflammation.33,34 Activated endo-
thelial cells in these tissues express ligands that selectively
recruit subsets of activated T cells. In contrast, activated B
cells either become memory cells that retain CXCR5 and
CCR7 expression35,36 or differentiate into plasma cells that
downregulate CXCR5 and CCR7 and upregulate CXCR4,
targeting these cells to medullary cords and bone marrow.37
Integrin family members are the close recipients of che-
mokine signals to produce immediate arrest of rolling leuko-
cytes and to initiate the extravasation process.27 The major
integrin that mediates homing to secondary lymphoid tissues
is LFA-1, which is composed of the αL and β2 subunits.38
LFA-1 is expressed only on leukocytes and binds to five dif-
ferent intercellular adhesion molecules (ICAMs) named
ICAM-1 through ICAM-5, all of which are members of
the immunoglobulin superfamily.39,40 ICAM-1 and ICAM-2
ICAM-1 displaying regulated expression in response to
inflammatory mediators, such as tumor necrosis factor and
interferon-γ.41,42
The deficiency of the integrin β2 subunit is the basis of
a rare genetic syndrome known as leukocyte adhesion defi-
ciency type I.43 In this disease, leukocyte extravasation at
sites of inflammation is defective, and patients are highly
susceptible to bacterial infections of the skin and mucous
membranes. Patients with leukocyte adhesion deficiency
type I are developmentally normal and can be treated by
bone marrow transplantation with a high success rate.44
A leukocyte adhesion deficiency type III also has been
described, in which multiple leukocyte integrins show
defects in regulation of Rap1, a small G protein important
in LFA-1 regulation.45
The structure of integrins reveals remarkable machinery
for regulated adhesion.46-48 The inactive form is folded into
a compact globular structure, in which the ligand-binding
domain points toward the leukocyte surface.47 After activa-
tion by chemokines, the integrin extends to twice its origi-
nal height and projects the ligand binding site to greater
than 20 nm from the leukocyte membrane, with orienta-
tion toward the endothelial cell surface.47,49 This dramatic
change is closely coupled to cytoskeletal association, pro-
viding anchorage needed for arrest and cell spreading after
ligand binding.50,51 A second integrin expressed on naive
lymphocytes called VLA-4 is composed of the α4 and
β1 subunits and can play a small role in entry into lymph
nodes, but a major role in entry into inflamed sites.52 Its
ligand is VCAM, also a member of the immunoglobulin
superfamily regulated by inflammatory cytokines.53 When T
lymphocytes are activated to home to mucosal effector sites,
they upregulate expression of the integrin β7 subunit, which
also ­ associates with α4 to form the gut homing ­ integrin
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
α4β7,54 and bind a ­ different immunoglobulin superfamily
ligand called MAdCAM (for mucosal addressin) expressed
on endothelial cells in the gut.55
The extravasation process involves the movement of
lymphocytes between or through endothelial cells.56 The
endothelial junctional complexes include special adhesion
molecules that need to be transiently disengaged to allow
lymphocyte passage between endothelial cells.57 Although
the observation that lymphocytes seem to move through
endothelial cells by a transcellular route was first made by
Marchesi and Gowans in the early 1960s,58 the molecu-
lar basis of this transcellular route and its acceptance as a
legitimate pathway have been more recent. The transcel-
lular pathway may be dominant in situations in which the
endothelial junctions are particularly sturdy, as in the brain,
thymus, or lymph nodes. Junctional and transcellular routes
involve active processes in the leukocyte and endothelial
cells, but this step is not thought to be regulated to control
homing decisions.
Three types of receptor ligand pairs define the key regu-
lated steps in lymphocyte homing. The compatibility of all
three is required to gain entry into the tissue. If a compatible
selectin-ligand pair is not available, the leukocyte is unable
to initiate adhesion to the endothelial wall and flows past.
If a chemokine receptor–chemokine pair is unavailable, it is
impossible to activate integrins, even if the selectin-ligand
pair mediates rolling or tethering, and the cell eventually
releases and remains in the blood. If the integrin-ligand pair
is incompatible, the cells are unable to arrest and extrava-
sate, even if the selectin-ligand pair and chemokine-ligand
systems are engaged. These molecular pairs can be thought
of as a hierarchic area code for lymphocyte homing—the
digits defined by the compatible interactions, each of which
must be correctly engaged to allow entry.59
TISSUE ORGANIZATION AND INTERSTITIAL
MIGRATION
Classic histology and mouse genetics have been used to
establish the molecular mechanisms that account for the
segregation of T cells and B cells within secondary lym-
phoid tissues. The T cell zone is defined by the production
of CCL19 and CCL21 by stromal cells and the expression
of CCR7 on T cells.28,60 These are the same signals that
trigger the arrest of T cells on endothelial cells for tis-
sue entry. T cell zones also are amply populated by con-
ventional dendritic cells (DCs), which express CCR7 as
they mature. DCs seem to form a network on a scaffold
of reticular fibers.61 The parenchyma of lymph nodes and
splenic white pulp nodules are crisscrossed by thick col-
lagen bundles sheathed in fibroblastic reticular cells.62-65
The inner compartment of these fibers forms a network of
conduits in the lymph node and spleen that allows DCs in
the parenchyma access to the afferent lymph or blood. The
B cell follicles depend on CXCL13 expressed by follicular
stromal cells and CXCR5 expressed on B cells.60 The bal-
ance of CXCR5 and CCR7 expression by B cells controls
their proximity to the boundary between T cell zone and B
cell zone, where B cells can encounter helper T cells.35,36 B
cell zones also are populated by variable numbers of follicu-
lar DCs, which are differentiated stromal cells, rather than
cells of hematopoietic origin.66,67
293
The dynamics of immune cells in secondary lymphoid
tissues has been revealed by intravital fluorescence micros-
copy. The leading model for T cell interaction with DCs in
the lymph node was based on thinking of the reticular fiber
network as defining corridors along which the T cells would
migrate to make contact with lined-up DCs, a “receiv-
ing line” model.9 Two-photon laser scanning fluorescence
microscopy of fluorescently labeled T cells in explanted
lymph nodes in short-term cultures and later in live mice
revealed a totally different reality.68-71 The T cells did not
migrate in lines at all, but appeared to migrate randomly
within the T cell zones. The boundaries for confinement
in the T cell zone were large compared with the imaged
areas such that quantitative analysis revealed a true random
walk with a short persistence length of a few micrometers
between changes in direction. The speed of migration was
up to 30 μm/min with an average speed of approximately
12 μm/min, which is fast for ameboid locomotion. B cells
in follicles move about 30% more slowly, but in a similar
random pattern. When mice in which all DCs were fluo-
rescently labeled were imaged, it appeared that the antigen
presenting cells were largely sessile.61 The process by which
a DC that has brought antigen from the periphery can show
this antigen to many T cells is based on forming random
contacts with thousands of T cells per hour. Because of its
random nature, this model has been referred to as “stochas-
tic repertoire scanning.”
The signals that stimulate the rapid and random migra-
tion in lymph nodes are unknown. The speed of B lympho-
cyte migration is significantly reduced in mice lacking Giα2
subunit of G protein–coupled receptors, suggesting that che-
mokines may have a role in this process.72 Chemokines can
act as chemoattractants, but also can stimulate migration in
a random fashion—called chemokinesis—under some con-
ditions. It is possible that the conditions in the lymph node
are highly favorable for chemokine-induced chemokinesis,
and that this is the signal for the rapid, random migration of
T cells and B cells in the steady state.
Although the initial scanning process is random, when
antigen bearing DCs begin to interact with antigen-spe-
cific T cells, many nonrandom elements of migration are
established. B cells that recognize antigen upregulate CCR7
and downregulate CXCR5 such that they are attracted to
the boundary between the T cell and B cell zones.35 DCs
that have been in contact with CD4+ T cells produce CCL3
and CCL4, which attract CD8+ T cells under inflammatory
conditions.73 This directed migration of CD8+ T cells biases
the scanning of the CD8+ T cell repertoire toward DCs that
have received T cell help and have come into contact with
foreign antigens. This process likely increases the efficiency
of repertoire scanning.
IMMUNOLOGIC SYNAPSES MAINTAIN
ANTIGEN-SPECIFIC INTERACTIONS WITH
DENDRITIC CELLS
The most extreme change in lymphocyte migration during
an immune response is the near full arrest referred to as an
immunologic synapse.74,75 In vitro analysis has revealed elab-
orately organized structures underlying the arrest of T cell
migration in contact with DCs bearing appropriate major
histocompatibility complex (MHC)–peptide complexes.76,77
 294 DUSTIN  |  ADAPTIVE IMMUNITY INCLUDING ORGANIZATION OF LYMPHOID TISSUES
In vivo imaging analysis has repeatedly revealed stable
T cell–DC interactions as a common feature of tolerance
and immunity induced by high-affinity ligands.78-80 This
ability of antigen presenting cells to stop T cells seems to
be more related to MHC-peptide strength than the level of
innate stimulation, although this may differ for CD8+ versus
CD4+ T cells.80-82
EGRESS FROM LYMPH NODES AND THE
THYMUS—SPHINGOSINE-1-PHOSPHATE
The recirculation of lymphocytes required that they peri-
odically cease their scanning activity in the relevant sec-
ondary lymphoid tissue zones and exit the tissue to the
lymph or blood. Such egress processes also are important
for recently matured lymphocytes to leave the bone mar-
row or thymus to enter into the recirculating pool. Insights
into the molecular processes controlling egress from lymph
nodes and the thymus were provided through investigation
of the fungal metabolite FTY720, which is in clinical tri-
als as an immunosuppressive drug.83 FTY720 administra-
tion rapidly induced reduction in T cells and B cells in the
peripheral blood. FTY720 was found to be phosphorylated
by sphingosine kinase and to act as an agonist for many
sphingosine-1-phosphate receptors that are expressed on
lymphocytes and endothelial cells.84 Further insight was
provided by the study of bone marrow chimeric mice in
which fetal liver cells from embyronic lethal sphingosine-
1-phosphate receptor 1 (S1P1) knockout mice were used to
reconstitute lethally irradiated syngeneic wild-type mice.85
The S1P1-deficient single positive thymocytes were unable
to exit the thymus. When single positive thymocytes were
transferred into wild-type recipients, they were able to enter
lymph nodes normally, but could not exit. These results sug-
gested a T cell autonomous defect in egress from the thy-
mus and lymph node. Although FTY-720-P is an agonist of
S1P1, it has the effect of downregulating expression of the
receptor such that the compound recapitulates the knock-
out phenotype. A parallel study with reversible S1P1 ago-
nists and antagonists suggested that S1P1 also has a role in
controlling lymphatic endothelium permeability to T cell
transmigration.86
PRIMARY LYMPHOID TISSUES: SITES
WHERE T CELLS AND B CELLS ARE
GENERATED, AND SELF-TOLERANCE
MECHANISMS ARE INITIATED
The critical event in the birth of T cells and B cells is the
rearrangement of the antigen receptor genes to generate T
cell antigen receptors (TCR) and B cell antigen receptors
(BCR) that are expressed on the cell surface. Each T cell
and B cell has a single antigen receptor as birth certificate
and identification card. The life, death, or expansion of
each of these cells controls the availability of this antigen
binding structure to the adaptive immune system. This is
the heart of the clonal selection theory first proposed by
Burnett in the 1950s. Self-reactive cells need to be deleted
or inactivated shortly after birth. This process occurs in the
bone marrow for B cells and the thymus for T cells. The
anatomy of these two sites is very different in keeping with
the dramatically different recognition mode employed by
B cells and T cells. The bone marrow space is discussed first
followed by the functional anatomy of the thymus.
B CELL DEVELOPMENT IN THE BONE MARROW
Immature B cells express surface BCR composed of sur-
face immunoglobulin noncovalently complexed to the
signal transducing subunits Igα and Igβ.87 Self-tolerance
can be established if these immature B cells are exposed
to self-­antigens leading to apoptosis or inactivation of the
B cells.88,89 Three major sources for this antigen can be iden-
tified anatomically.
The first source is the bone marrow stroma and other
developing hematopoietic cells. If BCR encounter surface
immobilized antigens on these cells, they undergo apop-
tosis.90 The second source is the blood. The bone marrow
parenchyma is bathed in blood plasma antigens owing to
fenestrated endothelial cells lining bone marrow sinusoids.
When soluble antigens bind to BCR, the B cells may undergo
apoptosis if the antigen is multivalent and cross-links the
BCR inducing strong signaling. If the antigen is monova-
lent, the B cell may become anergic—a form of nonrespon-
siveness that is induced by weaker signaling through the
BCR.90 The third source is mature DCs that populate the
bone marrow parenchyma. These cells seem to migrate from
secondary lymphoid tissues via the efferent lymph such that
these cells may be responsible for bringing tissue-specific
antigens to the bone marrow to allow negative selection
of immature B cells specific for these antigens.91 The idea
that conventional DCs (distinct follicular DCs are discussed
later) present intact antigens to B cells has gained strength
in recent years.92 The identification of a migration pathway
for mature DCs to the bone marrow has been described only
recently, and the implications for tolerance induction have
not been fully investigated.
T CELL DEVELOPMENT IN THE THYMUS
T cell precursors arrive in the thymus and initiate TCR
gene rearrangement in this microenvironment. The thymus
is an epithelial organ formed during development from the
third pharyngeal arch. Histologically, the tissue has a clearly
distinguishable cortex, medulla, and vascular corticome-
dullary junction.11 Figure 17-1 shows fluorescence staining
with Ulex europeus antigen-1 lectin, a marker for the thymic
medulla, on the left side and MHC class II antigen in the
thymic cortex on the left side.93 The stepwise path of thy-
mocyte migration is outlined on the left.
Early CD4− and CD8− progenitors enter via postcapil-
lary venules at the corticomedullary junction (step 1) and
migrate to the subcapsular region of the cortex where TCR
gene rearrangement occurs, and CD4 and CD8 become
expressed on the surface of immature T cells, also called
thymocytes (step 2). These cells migrate randomly among
thymic epithelial cells in the cortex sampling self-MHC–
peptide complexes (step 3). If they express a TCR that rec-
ognizes self-MHC–peptide complexes with low affinity, they
undergo “positive selection.” Positively selected thymocytes
mature into CD4+ or CD8+ cells that migrate to the medulla
under control of CCR7 ligands (step 4).94,95 Medullary thy-
mic epithelial cells express a transcription factor called AIRE
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
Cortex
Medulla
5
1, 6 4
2
3 Capsule
1 mm
UEA-1 lectin
Figure 17-1  Section of mouse thymus stained with Ulex europeus antigen-1 lectin (left) or anti–major histocompatibility complex (MHC) class II (right)
visualized with immunofluorescence microscopy. The Ulex europeus antigen-1 staining is strongest in the medulla, but also highlights the capsule and
stroma of the cortex. The cortical thymic epithelial cells are strongly positive for MHC molecules, which allow negative and positive selection of thy-
mocytes. (Courtesy of Richard Lewis, Stanford University. From Bhakta NR, Oh DY, Lewis RS: Calcium oscillations regulate thymocyte motility during positive
selection in the three-dimensional thymic environment. Nat Immunol 6:143-151, 2005.)
that mediates expression of numerous tissue-specific genes.96
Rare patients lacking expression of AIRE have a complex
autoimmune syndrome characterized by polyendocrinopathy
and other tissue-specific autoimmune diseases. The hypoth-
esis is that AIRE-mediated transcription of tissue-specific
genes in thymic epithelial cells promotes negative selection
of some autoreactive T cells and may promote generation of
tissue antigen–specific regulatory T cells (step 5). The small
percentage of thymocytes that express a TCR that pass posi-
tive and negative selection downregulate CD69 and S1P1
and exit the lymph nodes as naive mature T cells (step 6).
SECONDARY LYMPHOID TISSUES: SITES
WHERE ANTIGEN FINDS RARE SPECIFIC
T CELLS AND B CELLS
The frequency of naive T cells that recognize any particular
antigen is so low that it has been challenging to estimate.
Enrichment methods using antibody-coated magnetic beads
have been developed that facilitate direct measurement of
precursors by flow cytometry with high-avidity MHC-pep-
tide–based probes known as tetramers.97 The number of cells
specific for commonly used model antigens is approximately
500 in most mouse strains when pooling cells from all sec-
ondary lymphoid tissues (spleen and lymph nodes). There
are approximately 500 million total CD4+ T cells in these
tissues, such that antigen presenting DCs need to make
contact with 1 million irrelevant T cells to find one spe-
cific T cell. A few antigen-positive DCs early in an infec-
tion need to have access to highly concentrated swarms of T
cells in secondary lymphoid tissues to sample enough T cells
to find a few specific precursors to activate and expand. As
described earlier, this search process is initially random, but
may become more directed and efficient as a response pro-
gresses. Although naive T cells recirculate in an unbiased
manner through secondary lymphoid tissues, the antigen
carrying cells, primarily DCs, have a high degree of regional
bias in their movement, such that they are thought to relay
information about innate immune stimulation and tissue of
origin of antigens.
When these antigen-specific cells come into contact
with DCs, they form immunologic synapses; integrate sig-
nals through the TCR and costimulatory ligands, which
represent part of the innate immune system contribution to
T cell activation; and divide more than 20 times with very
295
MHC class II on cortical thymic
epithelial cells
0.1 mm
short cell cycle times of approximately 6 hours.98,99 Expan-
sion of CD8+ T cells, which give rise to cytotoxic effector
cells, is greater in general than expansion of CD4+ T cells,
which give rise to various types of helper T cells. After
expansion, which peaks around 7 to 10 days, the infectious
agent is often eradicated, and most of the effector T cells
undergo apoptosis. The flulike symptoms associated with
viral infections are due to cytokines produced by the divid-
ing and differentiating T cells.
Because primary adaptive responses take a week or lon-
ger to develop, the host depends on innate immune mecha-
nisms, such as natural antibodies, neutrophils, interferons,
and natural killer cells, to control the infection until suf-
ficient numbers of effector T cells are generated. Thousands
of the expanded T cells that survive after the pathogen is
destroyed become memory cells.100,101 There are two subsets
of memory T cells: Central memory cells express L-selectin,
CCR7, and LFA-1 and recirculate via secondary lymphoid
tissues, and effector memory cells lack L-selectin and CCR7,
but may express P-selectin and E-selectin ligands and other
chemokine receptors, such as CXCR4, CCR5, CCR4, and
CCR9.33,102 These effector memory cells migrate to periph-
eral sites of inflammation and are equipped for rapid effector
function if they encounter antigen. Memory cells respond
rapidly to recurrence of the same infection and together
with antibodies rapidly eradicate that same agent if it is
encountered a second time. These memory cells also may
cross-react with other pathogens, and depending on the
degree of cross-reaction, this type of response can result in
rapid clearance of a new pathogen or sometimes an impaired
response.103
ANTIGENS FROM BLOOD ARE DETECTED MOST
EFFICIENTLY IN SPLEEN AND LIVER (PORTAL
SYSTEM)
The spleen is a large visceral organ that filters approximately
5% of the cardiac output. The red pulp is an important loca-
tion for removal of aged red blood cells from the circulation.
The red pulp also contains many DCs that come into direct
contact with naive T cells that are in the blood. The func-
tion of these red pulp DCs is unknown.
Most attention has been focused on the white pulp nod-
ules and the marginal zone as sites of T cell–DC interac-
tion and antigen capture (Fig. 17-2, left). Blood flows into
 296 DUSTIN  |  ADAPTIVE IMMUNITY INCLUDING ORGANIZATION OF LYMPHOID TISSUES

Red pulp Lymphocytes

Marginal zone sinus

Marginal zone
Capsule White pulp
macrophages
B
B

Central arteriole
O
T B
White pulp arteriole

B
Red pulp White pulp
Red pulp
sinus
100 µm
200 µm
Figure 17-2  Organization of the spleen. Left, Tissue section from a mouse injected with low-molecular-weight (blue) and high-molecular-weight (red)
fluorescent dextrans. The low-molecular-weight dextran labels the red pulp (blue) and the high-molecular-weight dextran labels macrophages in the
marginal zone (red) such that the white pulp nodule containing approximately 109 lymphocytes per cm3 is dark. The white pulp nodule forms around a
central arteriole from which smaller arterioles branch to the red pulp sinuses. B and T cells are segregated into follicles (B cells) and the periarteriolar lym-
phoid sheath (T cells). (Scale bar 0.2 mm.) Right, Image of a live mouse spleen during injection of low-molecular-weight fluorescent dextran. The blood
carrying the dextran passes through the white pulp in small blood vessels (arrows) emanating from the central arteriole (not shown) and connected to
red pulp sinuses that rapidly fill with blood, which fills the marginal sinus, but not the white pulp itself. In this image, approximately 0.3% of the T cells
in the periarteriolar lymphoid sheath are labeled with a fluorescent dye. These areas are packed with T and B cells. Marginal zone macrophages and 
B cells have direct access to blood to capture pathogens. White pulp lymphocytes are not in direct contact with blood and need antigen presentation
by cells that migrate from the marginal zone or antigens that are delivered to dendritic cells via reticular fibers.65 (Scale bar 0.1 mm.) (Courtesy of Janelle
Waite, New York University.)

the spleen via an artery that splits into arterioles, which
empty into venous sinuses in the marginal zone and red
pulp (Fig. 17-2, right). The blood is then recollected into a
venous system that drains to the liver, joining with the por-
tal tract. The arterioles are surrounded by sheaths of T cells
(periarterial lymphoid sheath [PALS]) that make up the T
cell zones of the white pulp. Bridging channels connect the
red pulp, where lymphocytes leave the blood, to the PALS,
where they migrate in a pertussis toxin–sensitive man-
ner.104 B cell follicles and the marginal zone surround the
PALS. Macrophages, DCs, and marginal zone B cells line
the marginal zone where they have direct access to blood
antigens. DCs that pick up antigens in the marginal zone
migrate to the PALS.105 A reticular fiber network connects
the marginal zone to the PALS and allows soluble antigens
from the blood to reach resident DCs in the PALS.65 The
spleen provides multiple opportunities to mount primary
and recall responses to particulate or soluble antigens in
the blood.
As mentioned previously, the spleen drains into the
liver. Immune responses in the liver are poorly under-
stood, but this is an important site because many patho-
gens colonize the liver.103,106 Two blood circulations supply
the liver—the portal vein with deoxygenated blood from
the gut and spleen and the hepatic artery with oxygen-
ated blood. These circulations mix in the liver sinusoids,
a low-pressure network of blood spaces between sheets of
hepatocytes connecting the portal tract and the central
collecting vein. Most of the liver parenchyma appears as
plates of hepatocytes alternating with blood-carrying sinu-
soids (Fig. 17-3, left). The liver is rich in a type of sessile
macrophage called a Kupffer cell and patrolling lympho-
cytes, particularly natural killer T cells (Fig. 17-3, right).107
The major antigen presenting cell in the liver is not the
Kupffer cell, but the perivascular Ito cells.108 The liver also
plays an important role in the clearance of B cells mediated
by therapeutic anti-CD20 antibodies that are used to treat
patients with rheumatoid arthritis.109
ANTIGENS FROM MUCOSAL SURFACES ARE
DETECTED MOST EFFICIENTLY IN PEYER’S
PATCHES AND MESENTERIC LYMPH NODES
The mucosal-associated lymphoid tissues include the tonsils,
Peyer’s patches, lamina propria, cryptopatches, and appen-
dix. Populations of Peyer’s patch and lamina propria DCs
have different mechanisms for sampling the contents of the
gut lumen. Peyer’s patches are composed of large B cell fol-
licles with smaller T cell zones (Fig. 17-4, left). They have
high endothelial venules that allow efficient entry of naive
T cells and B cells and memory cells with gut homing phe-
notypes.110 The large size of the follicles in Peyer’s patches
causes a domelike effect with the epithelium protruding into
the lumen. Some of the microvilli-laden absorptive epithe-
lial cells in this dome region are replaced by smooth-surfaced
M cells. These cells act as relay points for entry of enteric
pathogens into the dome region of Peyer’s patches where
a population of CCR6+ DCs efficiently presents pathogen-
related antigens to induce rapid local T cell responses (Fig.
17-4, bottom left).111
In contrast to Peyer’s patches, the core of the villi in
the terminal ilium is populated by activated T cells, plasma
cells, and CX3CR1+ DCs. These DCs, project thin processes
through the epithelial sheet directly into the gut lumen (Fig.
17-4, right).112,113 The function of these cells is unknown, but
they seem to actively sample the gut contents and then could
migrate to mesenteric lymph nodes via the large lymphatic
ducts. Because the mesenteric lymph nodes are sites at which
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION 297

NKT cells

Figure 17-3  Microcirculation and

Kupffer cells
immune cells in liver. Left, Intravital
Blood Blood imaging of blood space in mouse liver.
Confocal imaging of hepatocytes
(green autofluorescence) and blood
Hepatocytes
space (red fluorescent dextran). The
Hepatocytes flow pattern is toward the center
of the image panel. Right, Intravital
imaging of NKT cells (bright green)
and SIGN R1+ Kupffer cells in liver
sinusoids (dark spaces) between
hepatocytes (faint green autofluo-
rescence). The Ito cells also would
be lining the sinusoids, but on the
other side of the endothelial cells in
the space of Disse. (Courtesy of Tom
Cameron, New York University.)

50 µm
DCs
Villus

Intestinal
lumen Intestinal lumen

M cells
B
Figure 17-4  Gut-associated lym-
phoid tissues. Top left, Immuno­
fluorescence image of Peyer’s
patches with B cells in green and T
cells in red.31 Bottom left, A similarly
T ­oriented view of Peyer’s patches
0.2 mm from mouse in which CCR6+
10 µm ­dendritic cells also express GFP ow-
ing to ­targeting of green fluorescent
protein (GFP) to CCR6 locus.111 Right,
Images of CD11c+ ­ dendritic cells in
the tips of microvilli in the terminal
CCR6+ DC ilium after exposure to Salmonella
bacteria. The dendritic cells extend
processes through the epithelial
layer to facilitate bacterial uptake.113
The CCR6+ dendritic cells, rather
than the CX3CR1+ lamina propria
dendritic cells, seem to be the most
important for the T cell ­response to
Salmonella antigens.

tolerance to food antigens is established, it is possible that
this more direct sampling of the gut lumen has a role in oral
tolerance to food antigens.82
ANTIGENS FROM OTHER TISSUES AND SOLID
ORGANS ARE DETECTED IN PERIPHERAL
LYMPH NODES
An extensive network of peripheral lymph nodes filters
lymph from the skin, organs, and nervous system. Lymph
is composed of fluid that leaves blood vessels and then
must be collected from the tissues in afferent lymphatic
vessels, passes through at least one lymph node, exits
the lymph node as efferent lymph, and is returned to the
blood via conduits such as the thoracic duct. The affer-
ent lymphatics of a lymph node connect to the capsule
of the node and drain into the subcapsular sinus, which
contains many macrophages (Fig. 17-5). The floor of the
subcapsular sinus covers the lymph node parenchyma and
is the point of origin of the reticular fiber network that
connects to the high endothelial venules and medullary
cords, where cells move from the parenchyma into the
efferent lymph. Lymph does not come into direct con-
tact with cells in the parenchyma, but cells lining the
reticular fibers and a space between the high endothelial
venules and the reticular fibroblast sheath are exposed to
lymph fluid.9,62,63
The parenchyma is divided into T cell and B cell zones
defined by CCL19/21 and CXCL13 producing stroma (see
Fig. 17-5). DCs migrate from peripheral tissues in a CCR7-
dependent manner and join networks of DCs in the T cell
zones with scattered cells in the follicles.61,114 Emigrant DCs
 298 DUSTIN  |  ADAPTIVE IMMUNITY INCLUDING ORGANIZATION OF LYMPHOID TISSUES

Capsule

Migrating DC
Sessile DC

SIGN R1+ macrophages

T cells

Subcapsular DCs T-zone DCs Perifollicular DCs

Capsule Afferent
DC cluster lymphatics

B B cells

Lymphocytes T
M

Migrating DC
Efferent
T-B interface DCs Anatomic locations lymphatics B-zone DCs
Figure 17-5  Lymph node schematic and dendritic cell morphologies. Schematic: B, B cell follicles; M, medullary cords; T, T cell zone. Other structures
are labeled. Lymph enters via the afferent lymphatics and exits via the efferent lymphatics. T cells enter the lymph nodes via the high endothelial
­venules and exit via medulla to the efferent lymphatics. Intravital microscopy–based images of dendritic cells in lymph nodes of CD11c-YFP marker
mice are linked to a schematic of the lymph node. (All scale bars 50 μm.) (From Lindquist RL, Shakhar G, Dudziak D, et al: Visualizing dendritic cell networks
in vivo. Nat Immunol 5:1243-1250, 2004.)

have been reported to array around high endothelial venules
where they are efficiently encountered by newly extravasated
T cells and B cells. Antigen-positive DCs have been shown
in experimental models to stop B cell and T cell migration
during antigen encounter.78,92 These ­immunologic synapses,
discussed earlier, play an important role in antigen transfer
for B cells and for priming of proliferative response in T
cells. Different populations of DCs, such as dermal DCs and
Langerhans cells, actually populate different subregions of
the T cell zones, but the significance of this is unclear.
PERIPHERAL TOLERANCE INDUCTION UNDER
STEADY-STATE CONDITIONS
Lymph nodes are associated with peripheral tolerance
induction. DC presentation of antigens in the steady state
induces proliferation of T cells, which are then deleted or
anergic by 7 days.115-117 This process may be particularly
important to tissue-specific antigens that are not present in
the thymus in the steady state.118 Low-affinity self-antigens
can escape peripheral tolerance induction by this mecha-
nism.118,119 Low-affinity, self-antigen–specific T cells that are
activated in the context of infection by a pathogen with
strong innate stimulation may induce tissue-specific auto-
immunity.118
REGULATORY T CELLS REDUCE AUTOREACTIVITY
BY INHIBITING IMMUNOLOGIC SYNAPSE
FORMATION
Regulatory T cells are CD25+, IL-2-dependent T cells
that express the transcription factor FoxP3 and have
the ability to suppress immune responses to tissue-
­specific self-antigens.120,121 These cells represent another
layer of regulation to prevent autoimmune attack by
the adaptive immunity. One way in which regulatory
T cells function is to block immunologic synapse forma-
tion between T cells and autoantigen presenting DCs.122,123
This mechanism seems to operate on low-affinity self-
antigens that are the most likely type of self-reactive
T cell to escape peripheral tolerance mechanisms. Pre-
vention of long-lived T cell–DC interactions may reduce
proliferation and cytokine production by autoreactive
T cells.
CHANGES IN THE LYMPH NODE DURING
INFECTION OR VACCINATION
Infection or vaccination in tissues induces a strong reac-
tion in draining lymph nodes. Innate signals trigger produc-
tion of inflammatory cytokines, which leads to increased
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
blood flow and increased adhesion molecule and chemo-
kine expression to increase T cell and B cell entry and
suppression of T cell and B cell exit by downregulation of
S1P1.124 DCs in reactive lymph nodes express higher lev-
els of costimulatory ligands such as CD80 and CD86 and
promote robust proliferation, survival, and differentiation
of antigen-specific T cells.80 T cells that are activated in
the lymph nodes regain expression of S1P1 after 3 to 4 days
and a new repertoire of homing molecules and migrate to
effector sites.
TISSUE ENVIRONMENT OF IMMATURE DENDRITIC
CELLS DETERMINES T CELL IMPRINTING
When T cells are activated in lymph nodes, the repertoire
of homing molecules expressed by the activated effector T
cells is determined partly by the origin of the DC.125 DCs
arise from a common monocyte-like precursor that migrates
into tissues via the blood. DCs that drain from the gut pro-
duce retinoic acid from vitamin A.126 On maturation and
migration to draining lymph nodes, these DCs secrete reti-
noic acid, which induces activated T cells to express gut
homing chemokine receptors such as CCR9 and gut-spe-
cific integrins such as α4β7. Because gut-associated post-
capillary venules express MAdCAM and present CCL25
(a CCR9 ligand), these effector T cells tend to home to the
gut. In the absence of retinoic acid produced by gut-derived
DCs, the signals induced by skin-derived DCs favor expres-
sion of ligands for E-selectin and P-selectin and CCR4 on
T cells.125 Because endothelial cells of skin-associated post-
capillary venules express P-selectin and present CCL17,
these T cells tend to home to inflamed skin.
It has been shown more recently that DCs in the skin
metabolize vitamin D to generate a signal for T cell expres-
sion of CCR10, which allows these cells to move to migrate
to the epidermis in response to CCL27.127 Although DCs
strongly skew T cells to home back to the sites from which
the DCs migrated, the expression of homing receptors and
chemokines on lymphocytes also has a stochastic compo-
nent, which means that these effector cells and memory cells
also show up in diverse peripheral sites scattered throughout
the animal—giving effector T cells their ability to appear in
any tissue at any time.34 Overall, these results show that DCs
sense their tissue environment and innate immune activa-
tion signals in the process of shaping T cell responses.
GERMINAL CENTER REACTIONS ARE SITES OF
ANTIBODY AFFINITY MATURATION AND CLASS
SWITCH RECOMBINATION
B cell activation by antigens in conjunction with costimula-
tion via complement receptor or other forms of innate immune
stimulation can lead to immediate proliferation of the B cells
and the formation of plasma cells producing IgM antibodies
from daughter cells.128 Specific T cell help may promote the
formation of germinal centers within the B cell zone of any
secondary lymphoid tissues.129-131 Germinal centers are roughly
spherical collections of hundreds of antigen-specific B cells that
undergo somatic mutation and class switching in the light zone
and proliferation and apoptosis in the dark zone (Fig. 17-6).132
The light zone is populated by stromal follicular DCs, which,
in contrast to conventional DCs, are of ­ nonhematopoietic
299
le
p su B cell follicle
Ca
LZ
Centrocytes
GC DZ
Centroblasts
T
zone
Plasma cells
FDC-M1+
Reticular fibers
Specific B cells Medulla
25 µm
Figure 17-6  Germinal center in living mouse lymph node. A germi-
nal center was induced by vaccination after introduction of fluorescent
­antigen-specific B cells and antigen. Follicular dendritic cells were la-
beled with FDC-M1 antibody conjugated to a red fluorescent dye. Re-
ticular fibers, which serve as antigen conduits into the follicle, are labeled
blue. The germinal center (GC) is outlined and divided into a dark zone
(DZ) containing aggregated centroblasts (green) and a light zone (LZ)
containing loosely aggregated centrocytes, follicular dendritic cells
(FDC-M1+) and follicular T cells (not shown). The surrounding follicle is
densely packed with bystander B cells (not shown), which often enter
and traverse the LZ. Green cells in the medullary region are plasma cells
produced in the germinal center reaction. (From Schwickert TA, Lindquist
RL, Shakhar G, et al: Germinal center imaging reveals a dynamic open struc-
ture. Nature 446:83-87, 2007.)
origin, and CXCR5+ follicular helper T cells. Follicular DCs
use complement receptors to hold immune complexes on their
surface for sampling by antigen-specific B cells. The follicular
helper T cells provide cytokine help to B cells that maintain
or increase their affinity for antigens and can provide cytokine
signals to promote class switching.133
Intravital microscopy studies show that germinal centers
are dynamic open structures in which antigen-specific B cells
are continuously in motion, and follicular DCs are accessible
to interaction with B cells having diverse receptor specific-
ity.134 Antigen-specific B cells can be recruited into germinal
center reactions at any time in the process and can compete
openly with B cells that were present earlier. Interactions
between follicular helper T cells and centrocytes in the light
zone also are highly dynamic.135 Because most somatic muta-
tions destroy the BCR or decrease its affinity, there is a large
amount of apoptosis in the germinal center in addition to
proliferation to provide substrates for mutations.
TERTIARY LYMPHOID TISSUES:
GENERATED AT SITES OF CHRONIC
INFLAMMATION
Tertiary lymphoid tissues resemble secondary lymphoid tis-
sues in many respects, but are formed in adults in response
to chronic inflammation in locations where such tissues do
 300 DUSTIN  |  ADAPTIVE IMMUNITY INCLUDING ORGANIZATION OF LYMPHOID TISSUES
not exist in steady-state conditions. The induction of ter-
tiary lymphoid tissues can be compared with the formation
of lymph nodes during normal development. Normal lymph
node development involves the colonization of connective
tissues in characteristic vascular nexus by RORγt-dependent,
CD4+, lymphotoxin-positive lymph node inducer cells.136
These cells use surface lymphotoxin and tumor necrosis
factor to induce local stromal cells to express ICAM-1 and
VCAM-1 and produce CXCL13 leading to development of
lymph node stroma with a reticular fiber conduit system that
is integrated into the developing lymphatic vessel system.137
Constitutive use of inflammatory cytokines plays a key role
in this process. This normal process also can be recapitu-
lated in adults as chronic inflammation induces production
of tumor necrosis factor and lymphotoxin in normal tissues
leading to the induction of stromal cells to form organized
follicles and T cell zones within the inflamed tissues.138
Induction of stromal cells to produce CCL19/21 or CXCL13
or both is probably important in this process because trans-
genic expression of CXCL13 in ectopic locations in mice
leads to formation of fully developed B cell follicles in these
tissues.139,140 B cells may have a particular capacity to induce
tertiary lymphoid tissues.
Tertiary lymphoid tissues often are associated with au-
toimmune diseases, including rheumatoid arthritis, and the
local infiltration of naive T cells in an area with high con-
centrations of tissue-specific self-antigens and innate stimu-
lation may promote progression of disease by recruiting new
T cell and B cell specificities into the autoimmune pro-
cess.141,142 Breaking this cycle may be a key target of anticy-
tokine and anti–B cell therapies that have been remarkably
effective; these therapies are discussed in later chapters.
FOUR MAJOR TYPES OF EFFECTOR
T CELLS
CD8+ effector T cells are generally cytotoxic and involved
in killing of infected cells. CD8+ T cell responses can be
initiated without CD4+ T cell help, but CD4+ T cell help
is required for effective CD8+ T cell memory.143 An impor-
tant transcription factor for effector CD8+ T cell differen-
tiation is Eomesodermin.144 Three major types of effector
CD4+ T cells are recognized. The first defined axis for CD4+
T cell differentiation was between interferon-γ-producing
T cells expressing the master transcription factor Tbet and
IL-4-producing T cells using the master transcription ­factor
GATA3.145 The interferon-γ-producing cells are referred to
as Th1 cells and help inflammatory cytotoxic T cell and
macrophage responses; the IL-4-producing cells are called
Th2 cells and help antiparasitic B cell responses leading
to IgE production and eosinophilic infiltration. The major
cytokines that initiate Th1 responses are interferon-γ and
IL-12 in the absence of IL-4, whereas IL-4 is the major cyto-
kine that initiates the Th2 program.146
It has been appreciated more recently that a third type
of effector T cell produces IL-17 and is important in many
inflammatory diseases. Differentiation of these Th17 cells
requires the orphaned nuclear hormone receptor RORγt.147
The cytokine conditions that lead to development of Th17
cells are transforming growth factor-β plus IL-6, with IL-
23 for maintenance.148 The role of transforming growth
factor-β places Th17 cells on the same axis with induced
regulatory T cells, with the deciding factor being the pres-
ence of IL-6 to favor Th17 over Treg induction. These are
highly proinflammatory T cells that recruit neutrophils, but
not CD8+ effectors.149 Although Th1, Th2, and Th17 cells
and memory T cells may have certain biases with respect
to homing molecules, it has been observed that imprinting
by DCs for tissue-specific homing and the cytokines that
control differentiation into Th1, Th2, and Th17 are inde-
pendent.100
SUMMARY
Adaptive immune responses have a degree of flexibility that
is unparalleled in molecular recognition systems. This flex-
ibility has a dangerous side that is constrained by anatomy
and strong coupling to innate immunity. Autoimmune dis-
ease may result when weak points in tolerance mechanisms
intersect with infection or tissue damage leading the adap-
tive system to attack self-organs in a self-amplifying process
of tissue destruction, inflammation, and inappropriate ana-
tomic adaptation, such as tertiary lymphoid tissue genesis.
These processes are highly relevant to rheumatoid diseases.
A mouse model for autoimmune rheumatic disease called
the KRN transgenic mouse develops a T cell– and B cell–
dependent joint disease.150-152 Strategies to break these cycles
by attacking innate or adaptive immune system components
nonspecifically have met with success, but more specific
strategies would reduce negative consequences of general
immunosuppression.
REFERENCES
  1. Hoffmann JA: The immune response of Drosophila. Nature 426:
33-38, 2003.
  2. Cooper MD, Alder MN: The evolution of adaptive immune sys-
tems. Cell 124:815-822, 2006.
   3. Nappi AJ, Vass E, Malagoli D, et al: The effects of parasite-derived
immune-suppressive factors on the cellular innate immune and auto-
immune responses of Drosophila melanogaster. J Parasitol 90:1139-
1149, 2004.
   4. Hafler DA, Slavik JM, Anderson DE, et al: Multiple sclerosis. Immu-
nol Rev 204:208-231, 2005.
   5. Jacobi AM, Diamond B: Balancing diversity and tolerance: Lessons
from patients with systemic lupus erythematosus. J Exp Med 202:341-
344, 2005.
   6. Marrack P, Kappler J, Kotzin BL: Autoimmune disease: Why and
where it occurs. Nat Med 7:899-905, 2001.
   7. Umetsu DT, DeKruyff RH: The regulation of allergy and asthma.
Immunol Rev 212:238-255, 2006.
  8. Janeway CA Jr, Medzhitov R: Innate immune recognition. Annu
Rev Immunol 20:197-216, 2002.
   9. Ebnet K, Kaldjian EP, Anderson AO, et al: Orchestrated informa-
tion transfer underlying leukocyte endothelial interactions. Annu
Rev Immunol 14:155-177, 1996.
10. von Andrian UH, Mackay CR: T-cell function and migration: Two
sides of the same coin. N Engl J Med 343:1020-1034, 2000.
  11. Petrie HT: Cell migration and the control of post-natal T-cell lym-
phopoiesis in the thymus. Nat Rev Immunol 3:859-866, 2003.
  12. Gowans JL, Knight EJ: The route of re-circulation of lymphocytes in
the rat. Proc Roy Soc 159:257-282, 1964.
  13. Lawrence MB, Springer TA: Leukocytes roll on a selectin at physi-
ologic flow rates: Distinction from and prerequisite for adhesion
through integrins. Cell 65:859-873, 1991.
  14. von Andrian UH, Chambers JD, McEvoy LM, et al: Two-step model
of leukocyte-endothelial cell interaction in inflammation: Distinct
roles for LECAM-1 and the leukocyte beta 2 integrins in vivo. Proc
Natl Acad Sci U S A 88:7538-7542, 1991.
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
  15. Rosen SD: Ligands for L-selectin: Homing, inflammation, and  
beyond. Annu Rev Immunol 22:129-156, 2004.
  16. Lowe JB: Glycosyltransferases and glycan structures contributing to
the adhesive activities of L-, E- and P-selectin counter-receptors.
Biochem Soc Symp 69:33-45, 2002.  
  17. Agarwal SK, Brenner MB: Role of adhesion molecules in synovial
inflammation. Curr Opin Rheumatol 18:268-276, 2006.
  18. Arbones ML, Ord DC, Ley K, et al: Lymphocyte homing and leuko-
cyte rolling and migration are impaired in L-selectin-deficient mice.  
Immunity 1:247-260, 1994.
  19. Nolte MA, Hamann A, Kraal G, et al: The strict regulation of lym-
phocyte migration to splenic white pulp does not involve common  
homing receptors. Immunology 106:299-307, 2002.
  20. Wong J, Johnston B, Lee SS, et al: A minimal role for selectins in the
recruitment of leukocytes into the inflamed liver microvasculature. J  
Clin Invest 99:2782-2790, 1997.
  21. Pouyani T, Seed B: PSGL-1 recognition of P-selectin is controlled by
a tyrosine sulfation consensus at the PSGL-1 amino terminus. Cell  
83:333-343, 1995.
  22. Wilkins PP, Moore KL, McEver RP, et al: Tyrosine sulfation of P-
selectin glycoprotein ligand-1 is required for high affinity binding to
P-selectin. J Biol Chem 270:22677-22680, 1995.  
  23. Borges E, Tietz W, Steegmaier M, et al: P-selectin glycoprotein
ligand-1 (PSGL-1) on T helper 1 but not on T helper 2 cells binds
to P-selectin and supports migration into inflamed skin. J Exp Med  
185:573-578, 1997.
  24. Wild MK, Luhn K, Marquardt T, et al: Leukocyte adhesion defi-
ciency, II: Therapy and genetic defect. Cells Tissues Organs 172:161-  
173, 2002.
  25. Bargatze RF, Butcher EC: Rapid G protein-regulated activation  
event involved in lymphocyte binding to high endothelial venules. J
Exp Med 178:367-372, 1993.
  26. Campbell JJ, Hedrick J, Zlotnik A, et al: Chemokines and the arrest  
of lymphocytes rolling under flow conditions. Science 279:381-384,
1998.
  27. Shamri R, Grabovsky V, Gauguet JM, et al: Lymphocyte arrest  
requires instantaneous induction of an extended LFA-1 conforma-
tion mediated by endothelium-bound chemokines. Nat Immunol
6:497-506, 2005.  
  28. Forster R, Schubel A, Breitfeld D, et al: CCR7 coordinates the pri-
mary immune response by establishing functional microenviron-
ments in secondary lymphoid organs. Cell 99:23-33, 1999.
  29. Zou YR, Kottmann AH, Kuroda M, et al: Function of the chemokine
receptor CXCR4 in haematopoiesis and in cerebellar development.  
Nature 393:595-599, 1998.
  30. Scimone ML, Felbinger TW, Mazo IB, et al: CXCL12 mediates
CCR7-independent homing of central memory cells, but not naive T  
cells, in peripheral lymph nodes. J Exp Med 199:1113-1120, 2004.
  31. Forster R, Mattis AE, Kremmer E, et al: A putative chemokine
receptor, BLR1, directs B cell migration to defined lymphoid
organs and specific anatomic compartments of the spleen. Cell 87:  
1037-1047, 1996.
  32. Okada T, Ngo VN, Ekland EH, et al: Chemokine requirements for B
cell entry to lymph nodes and Peyer’s patches. J Exp Med 196:65-75,  
2002.
  33. Sallusto F, Kremmer E, Palermo B, et al: Switch in chemokine recep-
tor expression upon TCR stimulation reveals novel homing potential  
for recently activated T cells. Eur J Immunol 29:2037-2045, 1999.
  34. Sallusto F, Lenig D, Mackay CR, et al: Flexible programs of chemo-
kine receptor expression on human polarized T helper 1 and 2 lym-  
phocytes. J Exp Med 187:875-883, 1998.
  35. Okada T, Miller MJ, Parker I, et al: Antigen-engaged B cells undergo
chemotaxis toward the T zone and form motile conjugates with  
helper T cells. PLoS Biol 3:e150, 2005.
  36. Reif K, Ekland EH, Ohl L, et al: Balanced responsiveness to che-
moattractants from adjacent zones determines B-cell position. Nature  
416:94-99, 2002.
  37. Hargreaves DC, Hyman PL, Lu TT, et al: A coordinated change in
chemokine responsiveness guides plasma cell movements. J Exp Med  
194:45-56, 2001.
38. Springer TA: Adhesion receptors of the immune system. Nature  
346:425-434, 1990.
  39. Dustin ML, Rothlein R, Bhan AK, et al: Induction by IL-1 and inter-
feron, tissue distribution, biochemistry, and function of a natural
adherence molecule (ICAM-1). J Immunol 137:245-254, 1986.
301
40. de Fougerolles AR, Qin X, Springer TA: Characterization of the
function of intercellular adhesion molecule (ICAM)-3 and com-
parison with ICAM-1 and ICAM-2 in immune responses. J Exp Med
179:619-629, 1994.
41. Dustin ML, Springer TA: Lymphocyte function-associated antigen-1
(LFA-1) interaction with intercellular adhesion molecule-1 (ICAM-
1) is one of at least three mechanisms for lymphocyte adhesion to
cultured endothelial cells. J Cell Biol 107:321-331, 1988.
42. Staunton DE, Dustin ML, Springer TA: Functional cloning of
ICAM-2, a cell adhesion ligand for LFA-1 homologous to ICAM-1.
Nature 339:61-64, 1989.
43. Anderson DC, Springer TA: Leukocyte adhesion deficiency: An
inherited defect in the Mac-1, LFA-1, and p150,95 glycoproteins.
Annu Rev Med 38:175-194, 1987.
44. Thomas C, Le Deist F, Cavazzana-Calvo M, et al: Results of alloge-
neic bone marrow transplantation in patients with leukocyte adhe-
sion deficiency. Blood 86:1629-1635, 1995.
45. Kinashi T, Aker M, Sokolovsky-Eisenberg M, et al: LAD-III, a
leukocyte adhesion deficiency syndrome associated with defective
Rap1 activation and impaired stabilization of integrin bonds. Blood
103:1033-1036, 2004.
46. Xiong JP, Stehle T, Diefenbach B, et al: Crystal structure of the
extracellular segment of integrin alphaV beta3. Science 294:339-
345, 2001.
47. Takagi J, Petre BM, Walz T, et al: Global conformational rearrange-
ments in integrin extracellular domains in outside-in and inside-out
signaling. Cell 110:599-611, 2002.
48. Luo BH, Carman CV, Springer TA: Structural basis of integrin regu-
lation and signaling. Annu Rev Immunol 25:619-697, 2007.
49. Kim M, Carman CV, Springer TA: Bidirectional transmembrane
signaling by cytoplasmic domain separation in integrins. Science
301:1720-1725, 2003.
50. Kucik DF, Dustin ML, Miller JM, et al: Adhesion-activating phorbol
ester increases the mobility of leukocyte integrin LFA-1 in cultured
lymphocytes. J Clin Invest 97:2139-2144, 1996.
51. Cairo CW, Mirchev R, Golan DE: Cytoskeletal regulation couples
LFA-1 conformational changes to receptor lateral mobility and clus-
tering. Immunity 25:297-308, 2006.
52. Shimizu Y, van Seventer GA, Horgan KJ, et al: Roles of adhesion
molecules in T-cell recognition: Fundamental similarities between
four integrins on resting human T cells (LFA-1, VLA-4, VLA-5,
VLA-6) in expression, binding, and costimulation. Immunol Rev
114:109-143, 1990.
53. Elices MJ, Osborn L, Takada Y, et al: VCAM-1 on activated endo-
thelium interacts with the leukocyte integrin VLA-4 at a site distinct
from the VLA-4/fibronectin binding site. Cell 60:577-584, 1990.
54. Hu MC, Crowe DT, Weissman IL, et al: Cloning and expression of
mouse integrin beta p(beta 7): A functional role in Peyer’s patch-spe-
cific lymphocyte homing. Proc Natl Acad Sci U S A 89:8254-8258,
1992.
55. Briskin MJ, McEvoy LM, Butcher EC: MAdCAM-1 has homology
to immunoglobulin and mucin-like adhesion receptors and to IgA1.
Nature 363:461-464, 1993.
56. Carman CV, Springer TA: A transmigratory cup in leukocyte diape-
desis both through individual vascular endothelial cells and between
them. J Cell Biol 167:377-388, 2004.
57. Ostermann G, Weber KS, Zernecke A, et al: JAM-1 is a ligand of
the beta(2) integrin LFA-1 involved in transendothelial migration of
leukocytes. Nat Immunol 3:151-158, 2002.
58. Marchesi VT, Gowans JL: The migration of lymphocytes through
the endothelium of venules in lymph nodes: An electron microscope
study. Proc R Soc Lond B Biol Sci 159:283-290, 1964.
59. Springer TA: Traffic signals on endothelium for lymphocyte recir-
culation and leukocyte emigration. Annu Rev Physiol 57:827-872,
1995.
60. Ohl L, Henning G, Krautwald S, et al: Cooperating mechanisms of
CXCR5 and CCR7 in development and organization of secondary
lymphoid organs. J Exp Med 197:1199-1204, 2003.
61. Lindquist RL, Shakhar G, Dudziak D, et al: Visualizing dendritic cell
networks in vivo. Nat Immunol 5:1243-1250, 2004.
62. Kaldjian EP, Gretz JE, Anderson AO, et al: Spatial and molecular
organization of lymph node T cell cortex: A labyrinthine cavity
bounded by an epithelium-like monolayer of fibroblastic reticular
cells anchored to basement membrane-like extracellular matrix. Int
Immunol 13:1243-1253, 2001.
 302 DUSTIN  |  ADAPTIVE IMMUNITY INCLUDING ORGANIZATION OF LYMPHOID TISSUES
  63. Gretz JE, Norbury CC, Anderson AO, et al: Lymph-borne chemo-
kines and other low molecular weight molecules reach high endothe-
lial venules via specialized conduits while a functional barrier limits
access to the lymphocyte microenvironments in lymph node cortex.
J Exp Med 192:1425-1440, 2000.
  64. Sixt M, Kanazawa N, Selg M, et al: The conduit system transports
soluble antigens from the afferent lymph to resident dendritic cells in
the T cell area of the lymph node. Immunity 22:19-29, 2005.
  65. Nolte MA, Belien JA, Schadee-Eestermans I, et al: A conduit system
distributes chemokines and small blood-borne molecules through the
splenic white pulp. J Exp Med 198:505-512, 2003.
  66. Munoz-Fernandez R, Blanco FJ, Frecha C, et al: Follicular dendritic
cells are related to bone marrow stromal cell progenitors and to myo-
fibroblasts. J Immunol 177:280-289, 2006.
  67. Lee IY, Choe J: Human follicular dendritic cells and fibroblasts share
the 3C8 antigen. Biochem Biophys Res Commun 304:701-707,
2003.
68. Miller MJ, Wei SH, Parker I, et al: Two-photon imaging of lym-
phocyte motility and antigen response in intact lymph node. Sci-
ence 296:1869-1873, 2002.
  69. Miller MJ, Wei SH, Cahalan MD, et al: Autonomous T cell traffick-
ing examined in vivo with intravital two-photon microscopy. Proc
Natl Acad Sci U S A 100:2604-2609, 2003.
  70. Wei SH, Parker I, Miller MJ, et al: A stochastic view of lympho-
cyte motility and trafficking within the lymph node. Immunol Rev
195:136-159, 2003.
  71. Miller MJ, Hejazi AS, Wei SH, et al: T cell repertoire scanning
is promoted by dynamic dendritic cell behavior and random T
cell motility in the lymph node. Proc Natl Acad Sci U S A 101:
998-1003, 2004.
  72. Han SB, Moratz C, Huang NN, et al: Rgs1 and Giα2 regulate the
entrance of B lymphocytes into lymph nodes and B cell motility
within lymph node follicles. Immunity 22:343-354, 2005.
  73. Castellino F, Huang AY, Altan-Bonnet G, et al: Chemokines
enhance immunity by guiding naive CD8+ T cells to sites of CD4+
T cell-dendritic cell interaction. Nature 440:890-895, 2006.
74. Grakoui A, Bromley SK, Sumen C, et al: The immunological syn-
apse: A molecular machine controlling T cell activation. Science
285:221-227, 1999.
  75. Dustin ML, Colman DR: Neural and immunological synaptic rela-
tions. Science 298:785-789, 2002.
  76. Benvenuti F, Lagaudriere-Gesbert C, Grandjean I, et al: Dendritic
cell maturation controls adhesion, synapse formation, and the
duration of the interactions with naive T lymphocytes. J Immunol
172:292-301, 2004.
  77. Brossard C, Feuillet V, Schmitt A, et al: Multifocal structure of the T
cell–dendritic cell synapse. Eur J Immunol 35:1741-1753, 2005.
78. Mempel TR, Henrickson SE: Von Andrian UH: T-cell priming
by dendritic cells in lymph nodes occurs in three distinct phases.
Nature 427:154-159, 2004.
  79. Miller MJ, Safrina O, Parker I, et al: Imaging the single cell dynamics
of CD4+ T cell activation by dendritic cells in lymph nodes. J Exp
Med 200:847-856, 2004.
  80. Shakhar G, Lindquist RL, Skokos D, et al: Stable T cell-dendritic
cell interactions precede the development of both tolerance and
immunity in vivo. Nat Immunol 6:707-714, 2005.
  81. Hugues S, Fetler L, Bonifaz L, et al: Distinct T cell dynamics in lymph
nodes during the induction of tolerance and immunity. Nat Immunol
5:1235-1242, 2004.
  82. Zinselmeyer BH, Dempster J, Gurney AM, et al: In situ character-
ization of CD4+ T cell behavior in mucosal and systemic lymphoid
tissues during the induction of oral priming and tolerance. J Exp Med
201:1815-1823, 2005.
  83. Mandala S, Hajdu R, Bergstrom J, et al: Alteration of lymphocyte
trafficking by sphingosine-1-phosphate receptor agonists. Science
296:346-349, 2002.
  84. Brinkmann V, Lynch KR: FTY720: targeting G-protein-coupled
receptors for sphingosine 1-phosphate in transplantation and
­autoimmunity. Curr Opin Immunol 14:569-575, 2002.
  85. Matloubian M, Lo CG, Cinamon G, et al: Lymphocyte egress from
thymus and peripheral lymphoid organs is dependent on S1P recep-
tor 1. Nature 427:355-360, 2004.
  86. Wei SH, Rosen H, Matheu MP, et al: Sphingosine 1-phosphate type
1 receptor agonism inhibits transendothelial migration of medullary
T cells to lymphatic sinuses. Nat Immunol 6:1228-1235, 2005.
  87. DeFranco AL: Structure and function of the B cell antigen receptor.
Annu Rev Cell Biol 9:377-410, 1993.
  88. Cyster JG, Goodnow CC: Antigen-induced exclusion from follicles
and anergy are separate and complementary processes that influence
peripheral B cell fate. Immunity 3:691-701, 1995.
  89. Rathmell JC, Cooke MP, Ho WY, et al: CD95 (Fas)-dependent elim-
ination of self-reactive B cells upon interaction with CD4+ T cells.
Nature 376:181-184, 1995.
  90. Brink R, Goodnow CC, Crosbie J, et al: Immunoglobulin M and D
antigen receptors are both capable of mediating B lymphocyte activa-
tion, deletion, or anergy after interaction with specific antigen. J Exp
Med 176:991-1005, 1992.
  91. Cavanagh LL, Bonasio R, Mazo IB, et al: Activation of bone marrow-
resident memory T cells by circulating, antigen-bearing dendritic
cells. Nat Immunol 6:1029-1037, 2005.
  92. Qi H, Egen JG, Huang AY, et al: Extrafollicular activation of lymph
node B cells by antigen-bearing dendritic cells. Science 312:1672-
1676, 2006.
  93. Bhakta NR, Oh DY, Lewis RS: Calcium oscillations regulate thymo-
cyte motility during positive selection in the three-dimensional thy-
mic environment. Nat Immunol 6:143-151, 2005.
  94. Bousso P, Bhakta NR, Lewis RS, et al: Dynamics of thymocyte-stro-
mal cell interactions visualized by two-photon microscopy. Science
296:1876-1880, 2002.
  95. Witt CM, Raychaudhuri S, Schaefer B, et al: Directed migration of
positively selected thymocytes visualized in real time. PLoS Biol 3:
e160, 2005.
  96. Villasenor J, Benoist C, Mathis D: AIRE and APECED: Molecular
insights into an autoimmune disease. Immunol Rev 204:156-164,
2005.
  97. Hataye J, Moon JJ, Khoruts A, et al: Naive and memory CD4+ T cell
survival controlled by clonal abundance. Science 312:114-116, 2006.
  98. van Stipdonk MJ, Hardenberg G, Bijker MS, et al: Dynamic pro-
gramming of CD8+ T lymphocyte responses. Nat Immunol 4:361-
365, 2003.
  99. van Stipdonk MJ, Lemmens EE, Schoenberger SP: Naive CTLs
require a single brief period of antigenic stimulation for clonal expan-
sion and differentiation. Nat Immunol 2:423-429, 2001.
100. Kalia V, Sarkar S, Gourley TS, et al: Differentiation of memory B and
T cells. Curr Opin Immunol 18:255-264, 2006.
101. Pulendran B, Ahmed R: Translating innate immunity into immuno-
logical memory: Implications for vaccine development. Cell 124:849-
863, 2006.
102. Sallusto F, Lenig D, Forster R, et al: Two subsets of memory
T ­ lymphocytes with distinct homing potentials and effector func-
tions. Nature 401:708-712, 1999.
103. Dustin LB, Rice CM: Flying under the radar: The immunobiology of
hepatitis C. Annu Rev Immunol 25:71-99, 2007.
104. Cyster JG, Goodnow CC: Pertussis toxin inhibits migration of B and
T lymphocytes into splenic white pulp cords. J Exp Med 182:581-
586, 1995.
105. Muraille E, Giannino R, Guirnalda P, et al: Distinct in vivo den-
dritic cell activation by live versus killed Listeria monocytogenes. Eur
J Immunol 35:1463-1471, 2005.
106. Crispe IN: Hepatic T cells and liver tolerance. Nat Rev Immunol
3:51-62, 2003.
107. Geissmann F, Cameron TO, Sidobre S, et al: Intravascular immune
surveillance by CXCR6+ NKT cells patrolling liver sinusoids. PLoS
Biol 3:e113, 2005.
108. Winau F, Hegasy G, Weiskirchen R, et al: Ito cells are liver-resident
antigen-presenting cells for activating T cell responses. Immunity
26:117-129, 2007.
109. Gong Q, Ou Q, Ye S, et al: Importance of cellular microenviron-
ment and circulatory dynamics in B cell immunotherapy. J Immunol
174:817-826, 2005.
110. Bargatze RF, Jutila MA, Butcher EC: Distinct roles of L-selectin and
integrins alpha 4 beta 7 and LFA-1 in lymphocyte homing to Peyer’s
patch-HEV in situ: The multistep model confirmed and refined.
Immunity 3:99-108, 1995.
111. Salazar-Gonzalez RM, Niess JH, Zammit DJ, et al: CCR6-medi-
ated dendritic cell activation of pathogen-specific T cells in Peyer’s
patches. Immunity 24:623-632, 2006.
112. Niess JH, Brand S, Gu X, et al: CX3CR1-mediated dendritic cell
access to the intestinal lumen and bacterial clearance. Science
307:254-258, 2005.
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
113. Chieppa M, Rescigno M, Huang AY, et al: Dynamic imaging of den-
dritic cell extension into the small bowel lumen in response to epi-
thelial cell TLR engagement. J Exp Med 203:2841-2852, 2006.
114. Ohl L, Mohaupt M, Czeloth N, et al: CCR7 governs skin dendritic
cell migration under inflammatory and steady-state conditions.
Immunity 21:279-288, 2004.
115. Redmond WL, Hernandez J, Sherman LA: Deletion of naive CD8 T
cells requires persistent antigen and is not programmed by an initial
signal from the tolerogenic APC. J Immunol 171:6349-6354, 2003.
116. Redmond WL, Marincek BC, Sherman LA: Distinct requirements
for deletion versus anergy during CD8 T cell peripheral tolerance in
vivo. J Immunol 174:2046-2053, 2005.
117. Redmond WL, Sherman LA: Peripheral tolerance of CD8 T lympho-
cytes. Immunity 22:275-284, 2005.
118. Zehn D, Bevan MJ: T cells with low avidity for a tissue-restricted
antigen routinely evade central and peripheral tolerance and cause
autoimmunity. Immunity 25:261-270, 2006.
119. Theobald M, Biggs J, Hernandez J, et al: Tolerance to p53 by A2.
1-restricted cytotoxic T lymphocytes. J Exp Med 185:833-841, 1997.
120. Olivares-Villagomez D, Wang Y, Lafaille JJ: Regulatory CD4(+) T
cells expressing endogenous T cell receptor chains protect myelin
basic protein-specific transgenic mice from spontaneous autoimmune
encephalomyelitis. J Exp Med 188:1883-1894, 1998.
121. Sakaguchi S: Naturally arising Foxp3-expressing CD25+CD4+ regu-
latory T cells in immunological tolerance to self and non-self. Nat
Immunol 6:345-352, 2005.
122. Tadokoro CE, Shakhar G, Shen S, et al: Regulatory T cells inhibit
stable contacts between CD4+ T cells and dendritic cells in vivo. J
Exp Med 203:505-511, 2006.
123. Tang Q, Adams JY, Tooley AJ, et al: Visualizing regulatory T cell
control of autoimmune responses in nonobese diabetic mice. Nat
Immunol 7:83-92, 2006.
124. Shiow LR, Rosen DB, Brdickova N, et al: CD69 acts downstream
of interferon-alpha/beta to inhibit S1P1 and lymphocyte egress from
lymphoid organs. Nature 440:540-544, 2006.
125. Mora JR, Cheng G, Picarella D, et al: Reciprocal and dynamic
control of CD8 T cell homing by dendritic cells from skin- and
gut-associated lymphoid tissues. J Exp Med 201:303-316, 2005.
126. Iwata M, Hirakiyama A, Eshima Y, et al: Retinoic acid imprints gut-
homing specificity on T cells. Immunity 21:527-538, 2004.
127. Sigmundsdottir H, Pan J, Debes GF, et al: DCs metabolize sunlight-
induced vitamin D3 to ‘program’ T cell attraction to the epidermal
chemokine CCL27. Nat Immunol 8:285-293, 2007.
128. Jacob J, Kassir R, Kelsoe G: In situ studies of the primary immune
response to (4-hydroxy-3-nitrophenyl)acetyl, I: The architecture and
dynamics of responding cell populations. J Exp Med 173:1165-1175,
1991.
129. Jacob J, Kelsoe G, Rajewsky K, et al: Intraclonal generation of antibody
mutants in germinal centres. Nature 354:389-392, 1991.
130. Jacob J, Kelsoe G: In situ studies of the primary immune response
to (4-hydroxy-3-nitrophenyl)acetyl, II: A common clonal origin for
periarteriolar lymphoid sheath-associated foci and germinal centers.
J Exp Med 176:679-687, 1992.
131. Han S, Hathcock K, Zheng B, et al: Cellular interaction in germinal
centers: Roles of CD40 ligand and B7-2 in established germinal cen-
ters. J Immunol 155:556-567, 1995.
132. Allen CD, Ansel KM, Low C, et al: Germinal center dark and light
zone organization is mediated by CXCR4 and CXCR5. Nat Immunol
5:943-952, 2004.
303
133. Chtanova T, Tangye SG, Newton R, et al: T follicular helper cells
express a distinctive transcriptional profile, reflecting their role as
non-Th1/Th2 effector cells that provide help for B cells. J Immunol
173:68-78, 2004.
134. Schwickert TA, Lindquist RL, Shakhar G, et al: Germinal center
imaging reveals a dynamic open structure. Nature 446:83-87, 2007.
135. Allen CD, Okada T, Tang HL, et al: Imaging of germinal center
selection events during affinity maturation. Science 315:528-531,
2007.
136. Sun Z, Unutmaz D, Zou YR, et al: Requirement for RORgamma
in thymocyte survival and lymphoid organ development. Science
288:2369-2373, 2000.
137. Mebius RE: Organogenesis of lymphoid tissues. Nat Rev Immunol
3:292-303, 2003.
138. Cupedo T, Jansen W, Kraal G, et al: Induction of secondary and ter-
tiary lymphoid structures in the skin. Immunity 21:655-667, 2004.
139. Luther SA, Bidgol A, Hargreaves DC, et al: Differing activities of
homeostatic chemokines CCL19, CCL21, and CXCL12 in lym-
phocyte and dendritic cell recruitment and lymphoid neogenesis. J
Immunol 169:424-433, 2002.
140. Yu P, Wang Y, Chin RK, et al: B cells control the migration of a sub-
set of dendritic cells into B cell follicles via CXC chemokine ligand
13 in a lymphotoxin-dependent fashion. J Immunol 168:5117-5123,
2002.
141. Weninger W, Carlsen HS, Goodarzi M, et al: Naive T cell recruit-
ment to nonlymphoid tissues: A role for endothelium-expressed CC
chemokine ligand 21 in autoimmune disease and lymphoid neogen-
esis. J Immunol 170:4638-4648, 2003.
142. Takemura S, Klimiuk PA, Braun A, et al: T cell activation in rheu-
matoid synovium is B cell dependent. J Immunol 167:4710-4718,
2001.
143. Masopust D, Ahmed R: Reflections on CD8 T-cell activation and
memory. Immunol Res 29:151-160, 2004.
144. Pearce EL, Mullen AC, Martins GA, et al: Control of effector CD8+
T cell function by the transcription factor Eomesodermin. Science
302:1041-1043, 2003.
145. Szabo SJ, Sullivan BM, Peng SL, et al: Molecular mechanisms regu-
lating Th1 immune responses. Annu Rev Immunol 21:713-758,
2003.
146. Hsieh CS, Macatonia SE, Tripp CS, et al: Development of TH1
CD4+ T cells through IL-12 produced by Listeria-induced macro-
phages. Science 260:547-549, 1993.
147. Ivanov II, McKenzie BS, Zhou L, et al: The orphan nuclear recep-
tor RORgammat directs the differentiation program of proinflam-
matory IL-17+ T helper cells. Cell 126:1121-1133, 2006.
148. Veldhoen M, Hocking RJ, Atkins CJ, et al: TGFbeta in the context
of an inflammatory cytokine milieu supports de novo differentiation
of IL-17-producing T cells. Immunity 24:179-189, 2006.
149. Langowski JL, Zhang X, Wu L, et al: IL-23 promotes tumour inci-
dence and growth. Nature 442:461-465, 2006.
150. Mangialaio S, Ji H, Korganow AS, et al: The arthritogenic T cell
receptor and its ligand in a model of spontaneous arthritis. Arthritis
Rheum 42:2517-2523, 1999.
151. Ji H, Korganow AS, Mangialaio S, et al: Different modes of patho-
genesis in T-cell-dependent autoimmunity: Clues from two TCR
transgenic systems. Immunol Rev 169:139-146, 1999.
152. Mandik-Nayak L, Allen PM: Initiation of an autoimmune response:
Insights from a transgenic model of rheumatoid arthritis. Immunol
Res 32:5-13, 2005.
 18
KEY POINTS
HLA molecules are among the most highly variable
(polymorphic) proteins encoded in the genome.
HLA variability is a major factor in controlling immune
­responses.
HLA variability is likely to be directly responsible for a portion
of the genetic susceptibility to autoimmune diseases.
Genetic associations, including HLA associations, must be
interpreted with caution because of confounding ­factors,
such as linkage disequilibrium and hidden population
­stratification.
Large-scale genetic analysis of the entire genome is revealing
additional risk genes for autoimmunity, independent of HLA.
The current genetic data strongly suggest that multiple genes
are involved in autoimmunity, and that many of these genes
predispose to multiple different autoimmune disorders.
Currently, the genetic analysis of human disease is undergo-
ing a dramatic transformation. Any textbook chapter on the
genetics of human rheumatic diseases becomes outdated in
many details soon after publication. The principles underly-
ing genetics are more stable, however, than the details; a
firm understanding of basic genetic principles should permit
practitioners and researchers to interpret the wealth of new
genetic findings that will be forthcoming in the next few
years. This chapter emphasizes approaches and concepts
at least as much as genetic findings, in the hope that this
provides a foundation for keeping up with advances in the
field. The first section deals with the major histocompatibil-
ity complex (MHC) because the human leukocyte antigens
(HLAs) encoded in this region form a cornerstone for inte-
grating immunology and genetics into the understanding of
rheumatic diseases. The second section of the chapter deals
with rapidly emerging data on relevant genes outside of the
MHC and provides a background on the approaches and
technologies that ultimately will bring genetic knowledge
into the daily practice of rheumatologists.
MAJOR HISTOCOMPATIBILITY COMPLEX
The MHC, which encodes the HLAs, has been associ-
ated with susceptibility to many different diseases since the
late 1970s. The chromosomal region containing the MHC
was originally identified because of the ability of genes in
this region to regulate transplant rejection1 and to control
the immune responses of mice and guinea pigs to simple
­antigens,2 a series of observations that led to the 1980
Genetics of Rheumatic
Diseases
PETER K. GREGERSEN
Nobel prize. The HLA molecules and their counterparts in
rodents subsequently were shown to be directly responsible
for immune response differences between individuals and
for determining the likelihood of graft rejection.1-4 There
are numerous different HLA genes within the MHC, and
they exhibit an enormous degree of structural variability.
In addition to influencing immune response patterns, many
of these alleles are associated with susceptibility to a wide
spectrum of autoimmune diseases, making the MHC an
essential starting point for anyone wishing to understand
the genetics of rheumatic diseases.
HUMAN LEUKOCYTE ANTIGEN MOLECULES
AND ANTIGEN-SPECIFIC T CELL RECOGNITION
The primary function of HLA molecules is the presentation
of antigenic peptides to T cells. In the case of α/β T cells,
most antigen recognition events involve the formation of a
trimolecular complex consisting of the HLA molecule, its
bound peptide, and the α/β T cell receptor (see Chapters 9
and 17). When this recognition occurs in the appropriate
context, it may result in signal transduction and activation
of the T cell. The requirement for MHC molecules to pres-
ent antigenic peptides to T cells is frequently referred to as
MHC-restricted T cell recognition. In each individual, T
cells generally are restricted to recognize antigens presented
by the individual’s own HLA molecules. The allelic varia-
tions among different HLA molecules are a major factor
accounting for differences in the types of antigenic peptides
to which an individual responds or in the types of T cells
that are used in an immune response.
HUMAN LEUKOCYTE ANTIGEN CLASS I
AND CLASS II MOLECULES
The original serologic and biochemical studies of HLA mol-
ecules revealed the presence of two major isotypes: HLA
class I and HLA class II. The basic structural features of
these classic HLA molecules are summarized in Figure 18-1.
HLA class I molecules consist of a 45-kD α chain encoded
within the MHC that is noncovalently associated with the
12-kD β2-microglobulin chain (encoded on chromosome
15). HLA class II molecules consist of noncovalently asso-
ciated α (32 kD) and β (28 kD) chains, both of which are
encoded within the MHC. HLA class I and class II mol-
ecules are cell surface glycoproteins, anchored to the mem-
brane by hydrophobic transmembrane segments.
A major breakthrough in the understanding of HLA
molecules came in 1987, when Bjorkman and colleagues5,6
reported the crystal structure of the HLA class I molecule,
HLA-A2. This work was followed by the solution of other
305
 306 GREGERSEN  |  Genetics of Rheumatic Diseases

MHC MHC
class I class II
α2
α1
α2 S α1 β1 S α1
S S

α3 S S β 2m β2 S S α2
S S S S N

C C C C
Cytoplasm Membrane

Figure 18-1  Schematic comparison of the structural features of major C

histocompatibility complex (MHC) class I and class II molecules. MHC β2m
class I molecules are anchored in the membrane by a single transmem-
brane segment contained in the 45-kD α chain. The MHC class I α chain is α3
noncovalently associated with β2-microglobulin. There are four external
domains, three of which contain intramolecular disulfide bonds, as indi-
cated. In contrast, MHC class II molecules consist of noncovalently associ-
A
ated α (32 kD) and β (28 kD) chains, both of which are anchored within
the membrane. The overall domain organization of the two molecules is
highly similar, however. Glycosylation sites on both molecules are indi-
cated by the black dot (•).

MHC class I and class II structures relevant to rheumatic dis-

ease.7,8 A side view of the class I molecule taken from Bjork-
man’s original article is shown in Figure 18-2A. The base
of the molecule (directly adjacent to the cell membrane)
is formed by β2-microglobulin and the immunoglobulin-
like α3 domain. The α1 and α2 domains form a distinct cleft
or groove at the top of the molecule. The function of this
cleft is to bind antigenic peptides for presentation to T cells.
A top view of this cleft is shown in Figure18-2B. The “floor”
of the peptide-binding cleft consists of β-sheets, whereas
the “walls” of the cleft are bounded by extended regions
of α-helical structure. The size of the HLA class I cleft is
approximately 10 to 20 Å, and it generally can accommo-
date antigenic peptides that are 9 amino acids long.9,10
HLA class II molecules have a structure that is highly
similar to that of class I molecules, with a prominent pep-
tide-binding cleft at the membrane distal portion, sitting on
top of a base formed by the α2 and β2 immunoglobulin-like
domains. The overall size and shape of the cleft in the two
classes of HLA molecules are almost superimposable. Subtle
differences exist, however, particularly at the ends of the
cleft, and this allows for some differences in the sizes of anti-
genic peptides that are presented by HLA class II molecules
compared with class I molecules. Direct analysis of peptides
bound to HLA class II molecules has shown that their size
commonly varies from 12 to 19 amino acids.10 Relatively
longer peptides lie in the cleft of class II molecules and may
extend beyond the ends of the cleft, whereas class I mol-
ecules contain much shorter peptides that are buried within
the cleft at either end.
Finally, x-ray crystallographic analyses have been done of
the entire trimolecular complex consisting of an HLA mol-
ecule, its bound peptide, and the T cell receptor. ­Figure 18-3
shows an example of this structure for an HLA-DRB1*0401
N
B
Figure 18-2  Three-dimensional structure of an HLA class I molecule,
based on the x-ray crystallographic analysis of Bjorkman.5 A, A side view
is shown. A peptide-binding cleft is formed by the α1 and α2 domains at
the top of the molecule. The α3 and β2-microglobulin domains are similar
in structure to immunoglobulin domains; essentially, they act as a plat-
form on which the peptide-binding cleft rests and provide contact sites
for the CD8 molecule during CD8+ T cell recognition. B, A top view of
the empty peptide-binding cleft is shown. This “T cell view” of the major
histocompatibility complex molecule would normally include a peptide
bound within the cleft. The disulfide bond connects the α-helix of the α2
domain with the floor of the cleft.
allele presenting influenza Ha peptide to its cognate α/β T
cell receptor.11 Three-dimensional versions of this HLA
structure (entry code 1J8H) can be viewed on the Protein
Data Bank (PDB) website at http://www.rcsb.org/pdb/home/
home.do.
HUMAN LEUKOCYTE ANTIGEN CLASS I
AND CLASS II ISOTYPES: FUNCTIONAL
CORRELATES
HLA class II molecules have a restricted tissue distribu-
tion, generally limited to antigen-presenting cells of the
immune system, such as B cells, macrophages, dendritic
cells, and some subsets of T cells. This distribution reflects
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
Figure 18-3  Ribbon diagram derived from the three-dimensional ­crystal
structure of the trimolecular complex of a human α/β-T cell ­receptor (top,
green), influenza Ha antigen peptide, and the major histocompatibility
complex class II molecule DRB1*0401.11 The peptide is contained with
the peptide-binding cleft of the HLA-DR molecule. The polymorphisms
associated with the “shared epitope” are located on α helical rim (DRB1
chain) of the peptide-binding cleft where they may interact with either
the bound peptide antigen or the T cell receptor.
the fact that HLA class II molecules are primarily involved
in ­presenting foreign antigens to CD4+ T cells during the
initiation and propagation of the immune response. The
expression of HLA class II molecules also can be induced on
a variety of other cell types by inflammatory cytokines, such
as ­ interferon-γ, enabling these cells to engage in antigen
presentation to CD4+ T cells. In contrast, HLA class I mol-
ecules are widely distributed on all somatic cells except for
red blood cells. This distribution reflects their predominant
role in presenting antigen to CD8+ effector or cytotoxic
T cells.
Another major functional difference between class I and
class II molecules is related to the source of peptide antigens
that are found in the antigen-binding cleft. Generally, class
I molecules present peptide antigens derived from proteins
that are actively synthesized within the endoplasmic reticu-
lum, whereas HLA class II molecules present antigens that
are taken up from outside of the cell by endocytosis. These
differences are reflected in the antigen-processing machin-
ery and the different trafficking patterns of class I and class II
molecules inside the cell. This complex process is discussed
in detail in Chapters 8 and 17.
307
GENETIC ORGANIZATION OF THE HUMAN MAJOR
HISTOCOMPATIBILITY COMPLEX
The human MHC extends over approximately 4 million
base pairs on the short arm of chromosome 6 (6p21.3). The
HLA class I and class II gene clusters are found in distinct
locations, as indicated on the highly abridged genetic map
shown in Figure 18-4. Only genes that are traditionally
associated with immune function are shown in Figure 18-4.
More than 200 genes have been identified in the MHC,
one of the most gene-rich regions in the human genome.12
Detailed genetic maps with the complete DNA sequence of
the MHC and a complete list of genes in the region can be
found online at http://www.sanger.ac.uk/HGP/Chr6/MHC.
shtml.
The HLA class I α chain genes are found on the telomeric
side of the MHC, including the classic class I genes, HLA-
A, HLA-B, and HLA-C; these three genes also are referred
to as the class Ia genes. Several other class I genes have been
defined, including the HLA-G, HLA-E, and HLA-F loci,
also known as class Ib genes. Initially, these class Ib genes
were thought to be nonfunctional because they exhibit lim-
ited polymorphism. Some of these genes do have significant
immune functions, however. HLA-G and HLA-E can act as
ligands for natural killer cell inhibitory receptors.13 In a sur-
prising twist, HLA-E molecules bind and present peptides
that are derived from the leader peptide of the class Ia genes
and HLA-G.14
The HLA class II genes are situated centromeric to the
class I region and have a more complicated organization.
The three major subregions of the class II cluster are desig-
nated DR, DQ, and DP. Each of these subregions contains
a variable number of α and β chain genes. Particularly in
the case of the DR subregion, this variability has led to
­confusion regarding the nomenclature to describe these
genes. An international standard for this nomenclature has
been agreed on.15
The DR subregion contains a single α chain gene, des-
ignated DRA, which does not exhibit significant allelic
variation. In contrast, the genes encoding the DR β chains
(DRB) are highly polymorphic and vary in number among
different individuals in the population. This is shown in
the boxed area of Figure 18-4, in which several examples of
common DR haplotypes are displayed. (A haplotype refers
to a group of alleles at closely linked loci that are commonly
inherited together.) Many of these DRB genes are nonfunc-
tional pseudogenes (indicated by the symbol ψ), although
all haplotypes contain at least one functional DRB1 gene,
and many haplotypes contain a second functional DRB gene
(DRB3, DRB4, or DRB5).
The DQ subregion contains one pair of functional α
and β chain genes, designated DQA1 and DQB1. These
two genes encode all the known HLA-DQ molecules. Pro-
tein products of the DQA2 and DQB2 loci have not been
reported. A similar situation exists in the DP subregion, in
which only the DPA1 and DPB1 genes give rise to known
protein products, the HLA-DP molecules.
In addition to the HLA class II molecules, several other
genes distributed within the class II region are involved in
peptide antigen processing. The TAP1 and TAP2 genes
have been of particular interest because they exhibit a mod-
est degree of polymorphism and are involved in delivering
 308 GREGERSEN  |  Genetics of Rheumatic Diseases

HLA class I region “Central” MHC HLA class II region

HLA-F HLA-A HLA-E HLA-B NFKBIL1 AIF1 Ly-6 family members BTNL2 DR DQ DP

Telomere 6p21

HLA-G HLA-C
LTa-TNF-α-LTB C4A-C4B-factorB-C2

DQA1 DQA2 DPA1 DPA2

DRA DRB1 DQB1 DQB2 DPB1 DPB2

ψDRB DRB1
DR1,10
DRB5 ψDRB DRB1 Loci encoding peptide loading
DR15,16 (DMA/DMB), peptide transport
DRB3 ψDRB DRB1 (TAP1 and TAP2) and proteo-
DR3,11,12,13,14 some subunits (LMP1 and LMP2)
DRB4 ψDRB ψDRB DRB1
DR4,7,9
DRB1
DR8

DRB gene organization varies by haplotype family

Figure 18-4  Map of the human major histocompatibility complex (MHC). The HLA class I and class II molecules are encoded in distinct regions of the
MHC. The HLA class II region contains three subregions—DR, DQ, and DP. Each of these contains a variable number of α and β chain genes. HLA class II
loci with known functional protein products are labeled in the shaded red box. In the case of DR, different numbers of DRB genes are present in different
haplotypes. A summary of the most common of these is shown in the box. The DQ and DP subregions each contain one pair of functional α and β chain
genes. Many genes involved in antigen processing and presentation by class I molecules are situated between the DP and DQ subregions. The HLA class
I region contains the three classic class I genes—HLA-A, HLA-B, and HLA-C—and other related class I molecules (see text). The “central MHC” also con-
tains many genes related to immune function, including the complement components (C4A, C4B, C2, and factor B) and tumor necrosis factor (TNF)-α  
and TNF-β. Other potentially interesting genes in this region (NFKB1L1, AIF1, and BTNL2) are discussed in the text and by Price and colleagues.24

peptides for loading onto HLA class I molecules.16 Other
proteins encoded in this region are involved in peptide
loading onto class II molecules, such as the DM molecule
(encoded by the DMA and DMB genes) and the DO/DN
heterodimer (encoded by DOB and DNA).
The “central” MHC is located in between the MHC class
I and class II regions. It contains numerous genes involved
in immune function, including those for tumor necrosis
factor (TNF)-α and TNF-β and the complement compo-
nents C4A, C4B, C2, and factor B. The central MHC is
gene dense, and it contains many other genes with potential
relevance to immune function and disease susceptibility.17
Several of these, such as NFKBIL1, AIF1, and BTNL2, have
been implicated in various autoimmune and inflammatory
disorders.18-20 The role of many genes in central MHC and
class I regions is still largely unknown, however.
POLYMORPHIC NATURE OF HUMAN LEUKOCYTE
ANTIGEN MOLECULES
One of the most dramatic features of the HLA system is
the extreme degree of polymorphism at most of these loci.
The formal definition of polymorphism (Table 18-1) usu-
ally requires that the most common allele at the locus does
not exceed a frequency of 98%. In contrast, at many HLA
loci, it is uncommon for a single HLA allele to exceed a fre-
quency of 50% in the population. The number of different
alleles present in the population is much larger than in any
other known polymorphic locus encoding functional genes.
At the HLA-A locus, more than 100 different alleles have
been reported; at HLA-B, the number of reported alleles is
greater than 200. A similar degree of allelic diversity is seen
at the DRB1 locus and to a lesser degree at DQA and DQB.
Until more recently, the naming of these various HLA
alleles has been a major source of confusion in the literature.
The difficulty with the nomenclature stems partly from the
different methods that have been used to define HLA poly-
morphisms. Originally, HLA class I alleles were detected
through the use of alloantisera (Table 18-2); the prefix allo-
refers to genetic differences that exist between individuals
of the same species. Alloantisera directed toward HLA mol-
ecules are commonly found in the context of pregnancy, in
which the mother mounts an immune response against the
“foreign” HLA molecules carried by the fetus (derived from
the father). Anti-HLA responses also are seen after blood
transfusion because the HLA molecules on the donor cells
are highly immunogenic. In the case of HLA class II alleles,
differences were originally detected using mixed lympho-
cyte responses. When T cells from a responder are mixed
with lymphocytes from another individual, differences in
HLA class II alleles cause the responder’s T cells to prolifer-
ate. Data on mixed lymphocyte culture typing dominated
the early HLA literature, and it was the method first used
to detect the HLA class II associations with rheumatoid
arthritis (RA).21 Subsequently, serologic methods also were
employed to detect class II polymorphisms.
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION 309

Table 18-1  Glossary of Terms

Allele Alternative form, or variant, of a gene at a particular locus
Alloantisera Antisera that detect antigenic differences between individuals in the population; the term is most often used
to refer to sera that detect antigenic (i.e., structural) differences among human leukocyte antigen molecules
carried by different individuals
Haplotype A group of alleles at adjacent or closely linked loci on the same chromosome that are usually inherited
­together as a unit
Heterozygote An individual who inherits two different alleles at a given locus on two homologous chromosomes
Heterozygosity A measure at a particular locus of the frequency with which heterozygotes occur in the population
Linkage The tendency toward the coinheritance within a family of two genes that lie near each other on the ­ 
genome; complete linkage occurs when parents who are heterozygous at each locus are unable to  
produce ­recombinant gametes
Linkage disequilibrium The preferential association in a population of two alleles or mutations that occurs more frequently than
predicted by chance; linkage disequilibrium is detected statistically, and except in unusual circumstances, it
implies that the two alleles lie near each other on the genome
Polymorphism The degree of allelic variation at a locus within a population; specific criteria differ, but a locus is said to be
polymorphic if the most frequent allele does not occur in >98% of the population; occasionally, polymor-
phism can be used in the same way as allele to refer to a particular genetic variant
Penetrance The conditional probability of disease (or phenotype) given the presence of a risk genotype

Table 18-2  Comparison between Modern, Sequence-based Nomenclature, and Older Naming Conventions  
for Class I and Class II Alleles Belonging to HLA-B27 and HLA-DR4 Serologic Groups*
HLA-B Locus: HLA-B27 Alleles HLA-DRB1 Locus: HLA-DR4 Alleles
Definitive Nomenclature Serologic ­Designation Definitive Nomenclature Serologic ­Designation
Based on DNA (Defined by Based on DNA (Defined by Cellular Typing
Sequence ­Alloantisera) Sequence ­Alloantisera) (Based on MLC)
B*2701 B27 DRB1*0401 DR4 Dw4
B*2702 B27 DRB1*0402 DR4 Dw10
B*2703 B27 DRB1*0403 DR4 Dw13
B*2704 B27 DRB1*0404 DR4 Dw14
B*2705 B27 DRB1*0405 DR4 Dw15
*The list of alleles is incomplete. The B27 allele family contains at least 17 members, and the HLA-DR4 allele family contains at least 35 members.
See http://www.ebi.ac.uk/imgt/hla/for a complete list of all HLA alleles.
MLC, mixed lymphocyte culture.

The current names of the HLA class I and class II alleles
are attached to the specific DNA sequence and locus for each
allele and are definitive.22 Many older publications have
used the serologically derived names for alleles, however.
It is important to have some concept of how these naming
conventions are related. The modern definitive (sequence-
based) allele names are derived from the older serologic names
because the serologic techniques frequently detected whole
groups of related alleles. Two examples of this are shown in
Table 18-2. The designation of HLA-B27 was developed for
individuals carrying an HLA-B allele that was recognized
by the B27-specific alloantisera. Sequencing of the HLA-B
alleles carried by these individuals revealed the existence of
at least 17 different alleles, however, 5 of which are listed in
Table 18-2. A similar situation exists for HLA class II allele
families, such as HLA-DR4 (see Table 18-2). In this case,
the DR4 allospecificity already was known to detect many
different alleles that could be discriminated further on the
basis of mixed lymphocyte culture typing.23 These alleles
have been defined and named by their sequence (see Table
18-2). A full list of all HLA alleles at the major loci can be
found at http://www.ebi.ac.uk/imgt/hla/.
Despite the precision of the molecular definition of HLA
alleles, the old serologic names are often used in oral discus-
sion because they are less cumbersome. The DRB1*03011
allele is common in white populations (10% in some
­populations) and is often referred to as simply DR3, after its
original serologic designation. There are at least 16 distinct
alleles that are detected by such DR3 alloantisera, and the
term DR3 is imprecise. When used in the context of a dis-
cussion about white populations, however, DR3 is assumed
to refer to the predominant DRB1*03011 allele.
LINKAGE DISEQUILIBRIUM OF HUMAN
LEUKOCYTE ANTIGEN ALLELES
In addition to their highly polymorphic nature, a character-
istic feature of HLA genes is the tendency for certain HLA
alleles to be found together on the same haplotype. This phe-
nomenon is termed linkage disequilibrium, and it is central to
understanding the significance of HLA associations (or any
other genetic associations) with disease. Linkage disequi-
librium exists when the frequency of two alleles occurring
together on the same haplotype exceeds that ­predicted by
chance. A common haplotype that exhibits linkage disequi-
librium in whites carries a certain combination of alleles—
A*0101-B*0801-DRB1*03011—commonly referred to as
the A1-B8-DR3 haplotype, and more recently designated
the “8.1” haplotype.24 This haplotype is ­ present in about
9% of the Danish population, a typical white Northern
 310 GREGERSEN  |  Genetics of Rheumatic Diseases
­ uropean group. To understand why this haplotype reflects
E Table 18-3  Contingency Table for Cohort Study*
the presence of linkage disequilibrium, consider the fact
that the A1 allele is present in 17% of Danes and the B8
allele is present in 12.7% of Danes. They could be expected
to be found together only 12.7% × 17% = 2.1% of the time,
much less than what is observed (9%).
There are three likely explanations for linkage disequi-
librium. First, the population may have originated from a
mixture of two populations, one of which had a very high
frequency of a particular haplotype (in this case, A1-B8-
DR3). If this happened recently, there would not have been
time (i.e., a sufficient number of generations) to randomize
alleles at closely linked loci by recombination at meiosis.
Inasmuch as human history is marked by large popula-
tion migrations,25 it is probable that population admixture
explains many examples of linkage disequilibrium.
A second explanation, related to the first, rests on the
observation that certain regions of the genome tend to
exhibit low levels of meiotic recombination. Genetic vari-
ants within these regions tend to stay together on the same
haplotype over many generations, even if haplotypes were
introduced into a population in the distant past. The impor-
tance of this concept for understanding linkage disequilib-
rium is still under active debate in the genetics community
and is discussed further in a later section.
A third explanation for linkage disequilibrium posits
that the alleles in linkage disequilibrium may be maintained
together because of a selective advantage. A1 and B8 (and
other genes closely linked to them) could confer an advan-
tage for immune defense when they are present together in
the same individual. Although plausible, this hypothesis is
difficult to prove for any particular haplotype, and there is
no direct evidence for it.
HUMAN LEUKOCYTE ANTIGEN
ASSOCIATIONS WITH RHEUMATIC
DISEASES
POPULATION ASSOCIATION STUDIES
AND CALCULATION OF THE ODDS RATIO,
AN ESTIMATE OF RELATIVE RISK
The ideal way to establish whether a genetic variant (allele)
confers risk for a disease is by performing a prospective
cohort study. In this kind of study, a group of individuals car-
rying (exposed to) the allele is compared with a matched
control group that does not carry the allele. These two
groups are followed over time (preferably over a lifetime)
to see if disease develops more frequently in the exposed
group. The results can be displayed in contingency tables
(Tables 18-3 and 18-4). On examining Table 18-3, it is
apparent that the fraction of exposed individuals who get
the disease is a/(a + b), whereas the fraction of unexposed
individuals who develop the disease is c/(c + d). The ratio of
these two fractions is known as the relative risk = a/(a + b) ÷  pocket. This fact leads naturally to the question of whether
c/(c + d) = (ac + ad)/(ac + bc). If the disease is rare in the pop-
ulation, ac is very small, and the relative risk is approximated
by (a × d)/(b × c), also referred to as the cross product.
In reality, such prospective cohort studies are usually
impractical, and a retrospective case-control design is used.
In this type of study, subjects are initially identified ­according
to whether they have the disease, and individuals without
Disease No Disease
Exposed a b
Not Exposed c d
*a, b, c, d = number of individuals observed in each category.
Table 18-4  Contingency Table for Case-Control  
Study*
Exposed Not Exposed
Disease a b
No Disease c d
*a, b, c, d = number of individuals observed in each category.
the disease are the controls. The data can be tabulated as in
Table 18-4. In this case, the cross product or (a × d)/(b × c)
is known as the odds ratio. In practice, this quantity is
often reported as the estimated relative risk because the
cross product is close to the relative risk when the disease
is rare. An odds ratio of 1 indicates that the genetic fac-
tor confers no risk for the disease. An odds ratio less than
1 suggests that the genetic factor under study is negatively
associated with the disease. (Odds ratios of <1 are occasion-
ally reported as the negative inverse value; an odds ratio
of >0.5 also may be reported −2.0.) With the exception of
HLA-B27-associated diseases, most HLA associations with
rheumatic diseases have odds ratios of less than 10. Several
examples of typical HLA associations with rheumatic and
autoimmune disorders are shown in Table 18-5.
HUMAN LEUKOCYTE ANTIGEN CLASS I
ASSOCIATIONS: HLA-B27 AND
SPONDYLOARTHROPATHIES
One of the strongest and earliest26 reported HLA associa-
tions with rheumatic diseases is the association of HLA-B27
with ankylosing spondylitis. In white populations, more
than 90% of patients with ankylosing spondylitis carry
HLA-B27, in contrast to approximately 8% of normal indi-
viduals, giving estimated relative risk values of 50 to 100 or
higher. The consistency of this finding across most ethnic
groups lends support to the contention that the HLA-B27
alleles are directly involved in the pathogenesis of anky-
losing spondylitis.27,28 HLA-B27 also is associated with
reactive arthritis, including Reiter’s syndrome, and with
arthritis seen in the context of inflammatory bowel disease.
As shown in Table 18-5, the strength of these associations
is lower in terms of estimated relative risk compared with
ankylosing spondylitis.
The serologic specificity of HLA-B27 encompasses many
distinct HLA class I alleles. These alleles differ from one
another at many amino acid positions, most of which involve
amino acid substitutions in and around the peptide-binding
there are differences among these B27 alleles in terms of
disease association. Most data indicate that this is not the
case, although there may be some exceptions in some popu-
lations.27 These exceptions may provide clues to the role of
the HLA-B27 molecule in pathogenesis. Overall, however,
it seems that most of the structural differences among the
B27 alleles do not affect disease risk.28
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
Table 18-5  Common HLA Associations with Rheumatic and Autoimmune Diseases
HLA Allele
Disease (Serologically Defined)
Ankylosing spondylitis B27
Reiter’s syndrome B27
Spondylitis in inflammatory B27
bowel disease
Rheumatoid arthritis DR4
Systemic lupus ­erythematosus DR3
Multiple sclerosis DR2
Juvenile diabetes mellitus   DR4
(type 1)
Some hypotheses can be derived by combining the
sequence information on the various B27 alleles with struc-
tural information about the HLA-B27 molecule and the pep-
tides bound to it.7,29 The structure of the B*2705 allele (the
most common allele in white populations) has been solved
by x-ray crystallography and reveals the presence of a char-
acteristic “pocket” in the floor of the peptide-binding cleft.
This has been referred to as the “45 pocket” because amino
acid position 45 is situated at the base of this pocket, and in
the case of the B27 alleles, it contains a negatively charged
glutamic acid.7 The peptides that bind to B27 alleles are all 9
amino acids long and invariably contain a positively charged
arginine at the second position from the N terminus.7 This
arginine appears to be situated within the 45 pocket when a
peptide is bound to the HLA-B27 molecule. This sequence
motif is distinct from peptides that are bound to other HLA
class I alleles, such as HLA-A2, which lacks a glutamic acid
in the 45 pocket. Many other sequence motifs of B27-associ-
ated peptides also have been described.
These data suggest that the reason for the HLA-B27 asso-
ciation with spondylitic disease may be related to the spe-
cific peptides that are bound and presented to CD8 T cells
by these molecules. Because a negatively charged 45 pocket
is not uniquely present in B27 alleles, but also is found in
class I molecules that are not associated with spondyloar-
thropathies, this feature cannot be a complete explanation
for the B27 associations with ankylosing ­spondylitis.
HUMAN LEUKOCYTE ANTIGEN CLASS II
ASSOCIATIONS WITH AUTOIMMUNE DISEASES
Numerous HLA class II associations with autoimmune dis-
eases have been described.30 Generally, the disease associa-
tions with HLA class II alleles are weaker and more complex
than those seen with HLA-B27. RA has received particu-
larly intense scrutiny, but the precise reasons for the HLA
associations with this disease are still unknown. In the case
of systemic lupus erythematosus (SLE) and related illnesses,
many of the HLA class II alleles are associated with the pres-
ence of specific autoantibodies or clinical phenotypes. More
recent data in RA indicate that the major HLA-DR associa-
tions are with anticitrullinated protein antibody–positive
disease, suggesting that control of autoantibody responses
may be a primary mechanism underlying these associations
in RA as well.
311
Approximate Allele Approximate Allele
Frequency in White Frequency in White Approximate
Patients (%) Controls (%) Relative Risk
90 8 90
70 8 40
50 8 10
70 30 6
45 20 3
60 20 4
75 30 6
RHEUMATOID ARTHRITIS: HLA-DRB1
ASSOCIATIONS AND THE SHARED EPITOPE
The first associations of RA with HLA class II alleles were
reported in the 1970s by Stastny.21 Stastny used cellular31
and antibody reagents32 that are no longer routinely used for
HLA typing; however, as discussed earlier, the nomenclature
for HLA alleles still derives from these early typing methods.
The DRB1*0401 allele (corresponding to the “Dw4” type in
Stastny’s original report31) was the first HLA polymorphism
to be associated with RA. Numerous studies have generally
confirmed that this allele is the most strongly associated with
RA, at least in white populations.33-35 Several other HLA-
DRB1 alleles also have been associated with RA, although
the strength of these associations varies.34,36,37
In some ethnic groups, RA is not associated with HLA-
DR4 alleles, but rather with HLA-DR138 or HLA-DR10.39
It is now widely accepted that the following alleles are
the major contributors to RA risk at the DRB1 locus:
DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0101, and
DRB1*1001. In addition, minor variants of these alleles and
others (DRB1*140240) may contribute to susceptibility, and
DRB1*0901 is a susceptibility in Asians, where this allele
is common.41 Most of these risk alleles share a common
sequence, as shown in Table 18-6. This consensus amino
acid sequence 70Q or K-R-R-A-A74 has been termed the
shared epitope.42 This structural feature is located on the
α-helical portion of the DR β chain in a position where
it may influence peptide binding and T cell receptor
interactions with the DRB1 molecule. (In the case of the
DRB1*1001 risk allele, one amino acid varies from this con-
sensus by a conservative change, with an R at position 70, as
does DRB1*0901, which is commonly associated with RA
in Asian populations) (Table 18-7).
Many different hypotheses have been advanced to
explain the shared epitope association with RA.43,44 Two of
these follow directly from knowledge about the role of HLA
molecules in antigen presentation and immune regulation.
It has been suggested that a particular peptide antigen, or
set of related antigens, may be involved in the initiation or
propagation of RA, and that shared epitope positive DRB1
alleles possess a unique, or enhanced, ability to bind or pre­
sent these peptides to the immune system.43 It has been diffi-
cult to address this hypothesis directly because the identity of
these putative disease-causing peptide antigens is unknown.
 312 GREGERSEN  |  Genetics of Rheumatic Diseases

Table 18-6  Amino Acid Substitutions That Compose DQB1 DQA1 DRB1 DQB1 DQA1 DRB1
the Shared Epitope at Positions 70 through 74 of DRB1 *0201 *0201 *0301 *0201 *0501 *0701
Alleles ­Associated with Rheumatoid Arthritis
Amino Acid Position
DQβ DQα DQβ DQα
DRB1 chain chain chain chain
Alleles 70 71 72 73 74
0101 Gln Arg Arg Ala Ala
DQ molecule encoded DQ molecule encoded
0401 — Lys — — — on a “DR3-DQ2” on a “DR7-DQ2”
0404 — — — — — haplotype haplotype
0405 — — — — — Figure 18-5  Combinatorial diversity of HLA-DQ molecules. The se-
rologically defined “DQ2” molecule may contain the same DQβ chain
0408 — — — — —
paired with different DQα chain alleles. This is different from HLA-DR
1402 — — — — — molecules, in which the DRα chain does not vary among different major
1001 Arg — — — — histocompatibility complex haplotypes.

(SE) positive alleles carry the same degree of genetic risk,

Table 18-7  Genotype Relative Risks of DRB1   lations. Generally, DRB1*0101 alleles carry lower levels of
Genotypes for Rheumatoid Arthritis
DRB1 Genotype Relative Risk P Value
0101/DRX 2.3 10−3
0401/DRX 4.7   10−12
0404/DRX 5 10−9
0101/0401 6.4 10−4
0401/0404 31.3   10−33
Data from Hall FC, Weeks DE, Camilleri JP, et al: Influence of the HLA-DRB1 locus
on susceptibility and severity in rheumatoid arthritis. QJM 89:821-829, 1996.
In view of the strong association of the shared epitope alleles
with anticitrullinated protein antibodies,45 it is of interest
that citrullinated peptides may have a particular affinity for
DRB1*0401 alleles.46 A second major hypothesis posits that
these risk alleles regulate the formation of the peripheral T
cell repertoire, by acting to select for particular T cell recep-
tors during thymic selection.44 There is elegant experimen-
tal evidence in humans to support a role for DR4 alleles in
shaping the peripheral T cell repertoire.47 It is unclear, how-
ever, whether this effect on the T cell receptor repertoire is
actually related to disease susceptibility. Many other inter-
esting hypotheses have been proposed, involving molecular
mimicry,48,49 allele-specific differences in intracellular traf-
ficking,50 and regulation of nitric oxide production,51 but
these require further experimental confirmation.
More recently, the shared epitope hypothesis itself has
come under renewed scrutiny, with some investigators pro-
posing a direct role for HLA-DQ polymorphisms,52,53 based
partly on studies in transgenic mice.54 As can be seen in
Figure 18-5, the HLA-DQ α and β chains are encoded just
centromeric to DRB1, and alleles at this locus are in strong
linkage disequilibrium with DRB1 alleles. The strong linkage
disequilibrium between the DR and DQ loci makes it difficult
to tease apart the effects of DR versus DQ based solely on
population genetic studies; the arguments for a DQ effect gen-
erally depend on showing the enrichment of rare genotypes
in the RA patient group compared with controls. Overall, a
primary role for DQ alleles is not strongly supported by large
HLA association studies that have examined this issue.55
Regardless of whether HLA-DQ alleles are involved in
RA susceptibility, the shared epitope hypothesis is not a
complete explanation for the HLA associations with RA.
This is evident from the fact that not all shared epitope
and the strength of the association varies in different popu-
relative risk for RA than the DRB1*0401 and DRB1*0404
alleles,35 yet DRB1*0101 is the major risk allele in some eth-
nic groups.56,57 The shared epitope itself does not seem to
associate strongly with RA in African-American and some
Hispanic populations.58,59 Certain combinations of DRB1
alleles carry especially high risk, as originally observed by
Nepom and colleagues.60 The combination of DRB1*0401
with DRB1*0404 carries a relative risk of more than 30 in
white populations35; this compares with relative risk values in
the range of 4 or 5 for either allele alone. Some of these rela-
tionships are summarized in Table 18-6. Attempts have been
made to formalize the gradient of risk conferred by the various
shared epitope alleles.61 It is unclear, however, whether these
effects are mediated by the HLA-DR molecules themselves,
or reflect the action of other genes on these haplotypes.
HLA-DQ ASSOCIATIONS WITH AUTOIMMUNE
DISEASES
Many of the first HLA class II associations with autoim-
mune disorders were detected using alloantisera for HLA-DR
alleles, as indicated in Table 18-5. As knowledge increased
about the genetic organization of the class II region, it became
apparent that for some diseases, the genetic associations are
stronger with HLA-DQ alleles. Although juvenile diabetes
does exhibit HLA associations with HLA-DR4 and HLA-
DR3, it is likely that a group of associated HLA-DQ alleles
are responsible for these observations.62 As discussed subse-
quently, the HLA associations with particular autoantibodies
in SLE also probably reflect the effects of HLA-DQ alleles.
The DQ subregion presents special challenges for the new-
comer to HLA because the old serologic nomenclature does
not usually have a simple correlation with a group of alleles
at a single locus. Because most of the HLA correlations with
autoantibodies in SLE involve the DQ loci, it is important
to understand this at the outset. The problem arises because
the α and β chains are polymorphic in DQ molecules. The
serologic specificity of DQ2 may detect one of three closely
related DQB1 alleles—DQB1*0201, DQB1*0202, or
DQB1*0203. This is similar to the DR serologic specificity
detecting a group of related DRB1 alleles (see Table 18-2).
In the case of DQ, the DQ2 serologic specificity also detects
these alleles on several different haplotypes that may
encode quite different DQ α chains. (This is ­different from
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
HLA-DR molecules, in which the DR α chain structure is
constant and does not vary among haplotypes.) In white
populations, the DQB1*0201 allele is commonly found on
DR3 haplotypes (associated with DQA1*0501) and DR7
haplotypes (associated with DQA1*0201), but both of these
haplotypes would type serologically as DQ2 (see Fig. 18-5).
Especially when reading the older literature and discussing
DQ polymorphisms, it is important to distinguish serologi-
cally defined polymorphisms, which may vary within the
group of alleles on the α and β chains, from polymorphisms
defined by sequence at a specific locus (DQA1 or DQB1).
The HLA associations with the Ro (SS-A) and La (SS-B)
autoantibody systems have been thoroughly studied. The
anti-Ro response is present in 25% to 50% of patients with
SLE63 and even more frequently in patients with primary
Sjögren’s syndrome.64 Although early serologic studies
indicated an association with HLA-DR3 and HLA-DR2,
a detailed molecular analysis of these HLA haplotypes has
provided evidence that HLA-DQ alleles in linkage disequi-
librium with HLA-DR2 and HLA-DR3 are responsible for
controlling this autoantibody response; heterozygous indi-
viduals who inherit a DR2-DQ1 haplotype and a DR3-DQ2
haplotype tend to have very high anti-Ro antibody titers in
the setting of SLE or Sjögren’s syndrome.65 The strongest
associations involve a DQA1*0501DQB1*0201 haplotype
(frequently found in linkage disequilibrium with DR3) and
a DQA1*06-DQB1*06 haplotype (frequently found in link-
age disequilibrium with DR2).
Reveille and colleagues66 attempted to refine further these
associations by looking at DQ alleles in Ro antibody–posi-
tive patients who do not inherit one of these two associated
haplotypes. These data led to the hypothesis that particular
amino acid substitutions in the DQ α chain (glutamine at
position 34) and DQ β chain (leucine at position 26) may
be involved in risk for developing a Ro antibody response.66
Proof of this hypothesis is unlikely to come solely from addi-
tional genetic studies, but rather from experiments that
directly test the influence of these DQ polymorphisms on
the immune response to specific autoantigen.67,68 HLA-DQ
associations also have been reported for other autoantibody
systems, such as antiphospholipid antibodies69 and anti-Sm
responses.70 The overall pattern of HLA-DQ associations
with these antibody responses is similar to that seen for anti-
Ro responses, although the alleles involved are different.
POPULATION ASSOCIATION STUDIES:
WHAT DO THEY MEAN?
Almost all of the studies on HLA and disease involve
population associations that are detected by means of ret-
rospective case-control studies. It is essential to understand
the strengths and weaknesses of this approach to genetic
analysis to judge the significance of these HLA associations.
Generally, there are three possible reasons for detecting an
association between a particular allele and a disease.
First, the allele under investigation may be directly
involved in the pathogenesis of the disease. This assump-
tion underlies most of the foregoing discussion on HLA and
rheumatic disease. The studies described reflect the search
for an ever more precise definition of particular amino acid
substitutions or unifying structural characteristics of disease-
associated alleles. This effort derives from the idea that HLA
313
DR2 DR3
DR4 DR5
DR3
DR4
Figure 18-6  Family structure used for determination of haplotype rela-
tive risk. The affected child carries the DR3 and DR4 alleles. A fictitious
“control” individual can be constructed from the noninherited DR2 and
DR5 alleles and used for a relative risk calculation. Such families also can
be used for transmission disequilibrium testing, as discussed in the text.
alleles directly predispose to disease by virtue of their ability
to control the immune response.4 As discussed previously,
this may involve many mechanisms, including preferential
peptide binding and the influence of the MHC on thymic
selection of the peripheral T cell repertoire.
The second possible reason for observing an HLA asso-
ciation is that a gene in linkage disequilibrium with HLA
may be the disease gene. Linkage disequilibrium over long
distances is a particularly prominent feature of the HLA
region, particularly for certain haploytpes.24,71 Many genes
with immunologic function within the MHC complex may
themselves be directly involved in predisposing to autoim-
munity. As shown in Figure 18-4, genes involved in anti-
gen processing (TAP1 and TAP2), complement activation
(C4, C2, factor B), and cytokines such as TNF-α all are
encoded in this region and could be responsible for dis-
ease risk. As discussed in a later section, it is likely that for
many autoimmune diseases, MHC genes other than, or in
addition to, the classic HLA alleles are involved in disease
­susceptibility.
A third reason to consider with any HLA association
is the possibility that the result is an artifact of population
stratification of patients and controls. The specific concern
is that the control group may not be genetically matched to
the disease group at loci that are unrelated to disease. This
often results from a failure to study a control group that is
ethnically matched to the disease group. This is emerging as
a major issue generally in genetic case-control studies, and
several approaches to control for this have been proposed,17
including the use of single-nucleotide polymorphism (SNP)
markers that can specifically determine ethnic background.72
As yet, these methods for correcting for underlying popula-
tions have not been widely used.
ALTERNATIVES TO THE CASE-CONTROL METHOD
FOR DETECTING DISEASE ASSOCIATION
To avoid the confounding effects of population stratifica-
tion in case-control studies, family-based controls also can
be used for doing association studies. Consider the family
shown in Figure 18-6. The affected child carries DR4 and
DR3, each of which is inherited from one parent. The laws of
mendelian inheritance specify that one DR haplotype from
each parent is not inherited by any given offspring—in this
example, DR2 in the father and DR5 in the mother. These
two noninherited haplotypes can be thought of as forming
 314 GREGERSEN  |  Genetics of Rheumatic Diseases
a genotype for a “control” individual. In this manner, issues
of population stratification are eliminated because patients
and controls are sampled from the identical (parental) gene
pool. This approach to disease association was originally
proposed by Falk and Rubinstein73 and was called the haplo-
type relative risk method. Its validity depends on numerous
assumptions, including that the genetic marker under study
does not influence mating preference or the production of
gametes.
A popular extension of this approach is called the trans-
mission disequilibrium test.74 Using the family in Figure 18-
6, for a given heterozygous parent (e.g., the father carrying
DR2 and DR4), there is a probability of 0.5 that any given
allele, such as DR4, would be transmitted to the child. If
the DR4 allele has no bearing on disease risk, the probabil-
ity of transmission (T) to an affected child is equal to the
probability of nontransmission (NT). This can be stated
simply as P (T|D) = P (NT|D), in which D indicates the
presence of disease in the offspring. If the allele being exam-
ined is associated with disease risk, however, P (T|D) >
P (NT|D). If large numbers of heterozygous parents with
affected offspring are examined, transmission disequilibrium
testing can establish an association between disease and
the test allele compared with the (noninherited) control
alleles. The advantages for getting around the problem of
case-control matching are compelling. The transmission
disequilibrium test does require the availability of parents,
however, and case-control studies still have a major role to
play, particularly for diseases with adult onset where parents
are unlikely to be available in many cases.
COMPLEXITY OF HLA ASSOCIATIONS
WITH RHEUMATIC DISEASES: EVIDENCE
FOR MULTIPLE-RISK GENES WITHIN
THE MAJOR HISTOCOMPATIBILITY COMPLEX
The foregoing discussion largely assumes that for a given
disease or autoimmune phenotype, a set of alleles at a
single HLA locus is likely to explain the association. For
many autoimmune diseases, however, it is likely that mul-
tiple genes within the MHC contribute independently
to disease risk. In the case of RA, numerous studies have
indicated that a separate locus in the central MHC may
be associated with the disease, independently of the HLA-
DRB1 locus.75-77 In addition, there is evidence that genes
in the MHC class I region may influence the risk conferred
by certain HLA-DRB1*0404 haplotypes,77 or may interact
with other non-MHC genes.78 Similar analyses in SLE,79,80
juvenile arthritis,81,82 multiple sclerosis,83 and myasthenia
gravis84 all point to the fact that multiple different genes
within the MHC can contribute to disease susceptibility.
This issue is currently a major focus of research efforts in
autoimmune diseases, and it is highly likely that additional
risk genes in the MHC will be defined in the next few years
using the dense SNP mapping techniques discussed in the
next section.
GENETICS IN THE WHOLE-GENOME ERA
As pointed out in the introduction, there has been an explo-
sion in new genetic knowledge over the past few years, and this
is likely to continue for some time. A major ­foundation was
laid by the completion of the Human Genome ­Project,85,86
which provided a reference sequence for the entire 3.2 billion
base pairs of the human genome. Subsequently, the human
HapMap Project87 has provided a catalog of the common
sequence variations across the human genome, with more
than 10 million SNPs now defined. In addition to SNPs,
there are tens of thousands of variable numbers of tandem
repeats, a common form of which is termed a microsatellite.
Microsatellites have been used extensively for linkage analy-
sis over the past decade.88 It also has become apparent more
recently that large insertions and deletions are common
across the genome,89 some of which arise de novo,90 provid-
ing another source of individual genetic variation that can
be associated with human disease.
Against this background of basic information about DNA
variation, there has been sweeping technologic change, so
that it is now possible to obtain hundreds of thousands or
even millions of SNP genotypes on many individuals in a
matter of days.91 This change has provoked the develop-
ment of sophisticated statistical approaches to large data-
sets, at the same time highlighting the need to integrate
the genetic knowledge with clinical data in ways that are
manageable and meaningful to researchers and ultimately
to practitioners. Although many of the basic principles of
association and linkage disequilibrium discussed in the con-
text of the HLA association are still valid, these have been
extended in new ways, as discussed subsequently in the sec-
tion on haplotype block structure.
Another unsettled issue concerns the overall genetic
“architecture” of human disease.92 Until more recently,
there has been an assumption that common allelic vari-
ants are likely to account for most of the genetic risk for
autoimmunity in the population. The HLA alleles that are
associated with rheumatic diseases are examples, as are sev-
eral of the new associations with RA and other autoimmune
disorders, such as PTPN22.93 By common variants, we gen-
erally mean variants that are present in the population at
frequencies of 5% or more and not less than 1%. There is
no a priori reason, however, to reject the hypothesis that
lots of rare variants account for a significant fraction of the
genetic burden of disease. The main reason that common
variants have been a focus of research is because the current
technologies are particularly well suited to investigate them.
This situation is about to change, with the advent of new
technologies that permit resequencing on a massive scale,
with routine resequencing of individual human genomes
probably less than a decade away. Some examples in the lit-
erature now show that lots of different rare genetic variants
can account for a common phenotype, such as plasma levels
of lipoproteins.94,95 We are still early in the whole-genome
era of genetic mapping.
ESTIMATING THE SIZE OF THE GENETIC
CONTRIBUTION TO RHEUMATIC DISEASES
Since the last edition of this chapter, several genes outside
of the MHC have been definitively associated with auto-
immune and rheumatic diseases, including PTPN22,96,97
IRF5,98,99 and several genes in the interleukin (IL)-12/IL-23
pathway (see later). Until these observations were made,
however, the evidence for additional risk genes outside of
the MHC was largely based on the epidemiology of familial
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
aggregation and twin concordance for autoimmune diseases.
The general approach is to compare disease prevalence in
groups of individuals with different degrees of genetic relat-
edness. The most common groups used for such studies are
genetically identical monozygotic individuals (MZ twins),
individuals who share approximately 50% of their genes in
common (dizygotic twins and siblings), and unrelated indi-
viduals in the population whose overall degree of genetic
similarity (at polymorphic loci) is relatively low, with about
0.1% difference over the entire genome. A 0.1% difference
among unrelated subjects implies approximately 3 million
base pair differences over the entire genome of 3.2 billion
base pairs, assuming only SNPs. On average, siblings and
dizygotic twins should differ by about 1.5 million SNPs.
The use of such family and background population preva-
lence data to calculate risk ratios remains a primary method
of estimating the overall size of the genetic component in
complex diseases.100 One can estimate the relative risk of
disease for siblings of affected individuals compared with the
general population. This leads to a value called λs, or rela-
tive risk to sibs, which is calculated as follows:
Disease prevalence in siblings of affected individuals
λs = SCREENING THE ENTIRE GENOME FOR DISEASE
Diisease prevalence in general population
Obtaining a reliable value of λs depends on having accu-
rate estimates of disease prevalence in the two comparison
groups. This is not a trivial matter. In the case of RA, the
estimation of population prevalence is fraught with poten-
tial sources of error. A firm diagnosis of RA is difficult to
make in large surveys, with errors in both directions possible.
Underestimation may occur because of the lack of reporting
of disease that is no longer active. Overestimation may result
from inadequate distinction between RA and other forms
of polyarthritis. Similar problems may occur in estimates of
population prevalence of SLE, in which mild disease may
be overlooked, or variability in phenotypic expression may
confound the issue. Accurate detection and assessment of
disease phenotype also are involved in the determination of
sibling affection rates.
Despite these difficulties, a range of values for λs has been
established for many of the common autoimmune disor-
ders.101 Representative examples are shown in Table 18-8.
With the exception of ankylosing spondylitis, most autoim-
mune disorders seem to have a λs in the range of 10 to 20.
Table 18-8 also shows the estimates for λMZ. Analogous to
the calculation for λs, λMZ is calculated as follows:
Disease prevalence in identical (MZ)
cotwins of affecteed individuals
λ MZ =
Disease prevalence in general population
The value λMZ can be interpreted as an estimate of the maxi-
mal genetic risk for the disease. This interpretation assumes
that all of the increased risk to an MZ cotwin of an affected
individual results from the fact that they share the same
genetic polymorphisms. This is clearly not the case because
at least some environmental sharing probably contributes to
the risk. It is notable, however, that the estimated value for
λMZ is quite high for many autoimmune disorders, and this
emphasizes the fact that MZ twin concordance rates must
315
Table 18-8  Familial Clustering of Selected  
Autoimmune Diseases, as Measured by the Relative  
Risk to Siblings (λS) and Monozygotic Twins (λMZ)
Disease λS λMZ
Type 1 diabetes mellitus 15 60
Rheumatoid arthritis 3-10 20-60
Multiple sclerosis 20 250
Systemic lupus   20 250
erythematosus
Ankylosing ­spondylitis 54 500
From Lander ES, Linton LM, Birren B, et al: Initial sequencing and analysis of
the human genome. Nature 409:860-921, 2001.
be interpreted in light of the background population preva-
lence of the disease. A low MZ twin concordance rate does
not imply a low genetic component to the disorder. The
absence of complete concordance for disease among MZ
twins indicates the need to include environmental, devel-
opmental, and stochastic factors to understand the role of
genetics fully.
GENES: APPROACHES BASED ON LINKAGE
Case-control association studies have been the primary
means of detecting the involvement of HLA or other
genetic loci in susceptibility to the rheumatic diseases.
These types of studies generally are limited to a few can-
didate markers and have the major disadvantage that the
candidate genes to be tested are selected on the basis of
incomplete knowledge about the disease. This is the
“hypothesis-driven” approach to human genetics, and
although still valuable if applied properly, it often has
resulted in the publication of false-positive results, partly
because of publication bias.102 The alternative is to take a
“discovery-driven” approach, in which the entire genome
is interrogated without any a priori assumptions about
what genes are involved.
There are two basic methods of whole-genome analysis—
one based on linkage, and the other based on association.103
Linkage methods depend on the ability to track polymorphic
genetic markers in families and to show that these genetic
markers cosegregate with the disease phenotype in families
in which there are multiple members affected. Multiplex
families are required for linkage analysis. The details of the
statistical methods are complex, but are generally based on
examining the likelihood of a particular pattern of coinheri-
tance of marker and disease (linkage) compared with the
likelihood that there is no linkage (the null hypothesis). A
measure of this likelihood is referred to as the LOD (log
of the odds) score, with a LOD score greater than 3 gener-
ally interpreted to indicate significant evidence of linkage
when markers across the entire genome are examined. Fur-
ther details of linkage methods can be found in Ott.104 One
advantage of linkage analysis is that the entire genome can
be effectively interrogated using less than 500 informative
microsatellite markers.88
Linkage analysis has been applied with great success to
the analysis of rheumatic diseases that exhibit a clear men-
delian pattern of inheritance (e.g., dominant or ­recessive).
 316 GREGERSEN  |  Genetics of Rheumatic Diseases
Markers at 1,2 3,4
locus X:
#1 #2
Possible
1,3 1,3 combinations
1,4 of alleles
2,3 inherited from
2,4 parents
Haplotypes shared by siblings: 0 1 2 diseases in the 1990s and has resulted in a few successes, most
Expected frequency: 25% 50% 25%
Figure 18-7  A nuclear family with two affected children (affected
­sibling pair). The possible distribution of alleles at an autosomal ­ locus,
X, is shown for sib 2, along with the predicted frequency of shared
­haplotypes among the sibs. Such families can be used to detect linkage
using affected sibling pair analysis (see text).
In 1992, familial Mediterranean fever was mapped to chro-
mosome 16,105 and this led to the identification of the gene
for this disease in 1997.106 In addition, an entirely new class
of familial periodic fever syndromes has been localized to
mutations in the TNF receptor 1 gene on chromosome
12.107,108 For highly penetrant mendelian disorders, clas-
sic linkage analysis is a powerful means of identifying the
underlying molecular basis of disease. One of the drawbacks
to classic linkage analysis is, however, that, to be most use-
ful, it should be applied to disorders with a high penetrance
and a known genetic model (e.g., dominant versus reces-
sive). An alternative approach based on linkage, broadly
termed allele sharing, is preferred for the study of autoim-
mune diseases that have a complex genetic basis.103
The most common approach to allele sharing is the
affected sibling pair method. This method is based on a
simple question: When two sibs are affected with a disease,
do they share alleles at particular genetic markers more
frequently than would be expected by chance? The basic
approach is illustrated in Figure 18-7. In this family, two
siblings are affected, and the first-born sibling (sib 1) has
inherited alleles 1 and 3 at a marker locus, X. By the laws of
mendelian inheritance, sibling 2 has a 25% chance of inher-
iting these same two alleles and has a 25% chance of inher-
iting neither of these alleles (i.e., sib 2 inherits 2 and 4 and
shares nothing with sib 1 at locus X). By a similar reasoning,
there is a 50% chance that these two siblings would share
one allele in common. This 25:50:25 distribution of shar-
ing 0, 1, or 2 haplotypes is expected if there is no linkage
between the disease and the marker locus. If a gene that lies
very near the marker locus is involved in disease risk, how-
ever, a significant deviation toward increased sharing among
affected siblings would be observed. The closer the marker
is to the disease locus, the greater the deviation would be
from a 25:50:25 distribution. By examining large numbers
of affected sibling pairs in this manner, the investigator can
develop statistical evidence that this is the case using a stan-
dard χ2 analysis, with the null hypothesis being that there is
no increased sharing at the marker locus.
Affected sibling pair analysis has many distinct advantages
and disadvantages. Only affected individuals are used, and
the problem of falsely assigning a family member as “unaf-
fected” is eliminated. This is a major issue for diseases such
as RA because the disease may not express itself until later
in life. Affected sibling pair analysis can be done without
committing to a specific model of inheritance (i.e., recessive
or dominant). As with linkage in general, the affected sib-
ling pair methods have relatively low power to detect genes
that confer only modest risk.103 This means that quite large
numbers (hundreds or thousands) of multiplex families are
required to obtain statistically significant results.
Linkage analysis based on allele sharing began to be
applied to genetically complex rheumatic and autoimmune
notably the identification of the NOD2 gene as a major risk
factor for Crohn’s disease.109,110 Linkage analysis has pro-
vided evidence for numerous risk genes involved in SLE,111
although precise gene identification has not yet been done
for most of these loci. More recently, the presence of a link-
age peak (LOD score >3.5) on chromosome 2q in RA sib-
ling pairs112 has led to the identification of STAT4 as a risk
gene for RA and SLE.113 Although challenging to carry out,
linkage can be applied successfully to complex diseases that
do not have a clear mendelian pattern of segregation.
WHOLE-GENOME ASSOCIATION STUDIES
Compared with linkage analysis, association methods have
much greater statistical power to detect genetic effects.103
In contrast to the hundreds of markers used for linkage,
however, large numbers of genetic markers are required to
carry out a comprehensive genome-wide study using asso-
ciation methods. Because of dramatic advances in technol-
ogy and the rapid decline in costs of genotyping, we are now
in an era where whole-genome association with hundreds of
thousands (or even 1 million) SNPs is becoming a standard
approach to gene discovery in complex disease. For scan-
ning the whole genome, this has generally replaced linkage
methods except in special situations.
The success of whole-genome association studies
depends on the underlying haplotype structure of the
genome. Although SNPs have been most commonly used
in the context of candidate gene association studies, it is
now apparent that combinations of SNPs can be employed
to define common haplotype “blocks” that exist by virtue
of strong linkage disequilibrium between alleles at adjacent
SNP markers.114,115 This concept emerged from early studies
of a region on chromosome 5q114 that contains numerous
cytokine genes and is associated with inflammatory bowel
disease,116 as shown in Figure 18-8. Consider the first block
on the right of the figure (labeled “block 1”). There are
eight SNPs defined within an 84-kb segment of DNA in this
region of chromosome 5q31. In principle, any combination
of these eight SNPs could occur in this region, giving rise
to 28256 possible combinations. Only two of these (GGA-
CAACC and AATTCGGG) occur with high frequency in
the population (76% and 18%), however, and together they
account for 96% of all the possible 256 haplotypes in the
population (line b in Figure 18-8). Figure 18-8 illustrates
this principle of limited haplotype variation for 10 addi-
tional haplotype blocks in this region. The HapMap project
 SEPT8 IL5 IRFI P4HA2 FACL6
Genes

IL4 IL13 RAD50 OCTN2 OCTN1 PDLIM3 CSF2 IL3

Genes

SNPs
PART 3 
| 

GGACAACC TTACG CGGAGACGA CGCGCCCGGAT CCAGC CCGAT CCCTGCTTACGGTGCAGTGGCACGTATT*CA CGTTTAG ACAACA GTTCTGA TATAG
a GACTGGTCG TTGCCCCGGCT CAACC CTGAC CATCACTCCCCAGACTGTGATGTTAGTATCT TAATTGG GTGACG TGTGCGG TATCA
AATTCGTG CCCAA CGCAGACGA CTGCTATAACC GCGCT CTGAC TCCCATCCATCATGGTCGAATGCGTACATTA TGTT*GA GCGGTG TG*GTAA CGGCG
CTGCCCCAACC CCACC ATACT CCCCGCTTACGGTGCAGTGGCACGTATATCA TGATTAG ACGGTG

BLOCK 1 BLOCK 2 BLOCK 3 BLOCK 4 BLOCK 5 BLOCK 6 BLOCK 7 BLOCK 8 BLOCK 9 BLOCK 10 BLOCK 11
84 kb 3 kb 14 kb 30 kb 25 kb 11 kb 92 kb 21 kb 27 kb 55 kb 19 kb

b 96% 97% 92% 94% 93% 97% 93% 91% 92% 90% 98%

c 76% 77% 36% 37% 35% 41% 40% 38% 36% 42% 29%
26% 14% 9% 9% 14% 8% 10% 8% 16%
18% 19% 28% 19% 13% 29% 27% 31% 33% 36% 51%
21% 35% 18% 12% 7% 9%
Figure 18-8  A schematic map of a 1-megabase region on chromosome 5q31 with a summary of the haplotype block pattern of single-nucleotide polymorphism (SNP) variation in the region. Each tick mark
on the top bar marks 50 kb. The genes in the region are shown above the bar, with direction of transcription as indicated at the left. Section a summarizes the major haplotype blocks, with colors highlight-
ing the most common combinations of SNP alleles for each haplotype block. Section b shows the percent of the total haplotypes in each block that are captured by the major haplotypes. Section c gives the
percentages for each specific haplotype. Two to four haplotypes account for greater than 90% of the haplotypes in each block. This haplotype diversity is dramatically reduced from what would be expected
if each SNP were randomly associated with other SNPs in the same block. A fuller description of these patterns of haplotype diversity can be found in Daly and colleagues.114 (From Daly MJ, Rioux JD, Schaffner
SF, et al: High-resolution haplotype structure in the human genome. Nat Genet 29:229-232, 2001.)
EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
317
 318 GREGERSEN  |  Genetics of Rheumatic Diseases
has shown that this limited haplotype diversity is typical of
the entire human genome.87 The reasons for this are related
partly to the discontinuous nature of meiotic recombina-
tion, so that regions between blocks tend to exhibit higher
rates of recombination than within blocks.115,117 Population
history and migration also contribute to these patterns of
genetic variation.
Effectively, these patterns of haplotype variation lead to
the view that a large portion of the common human varia-
tion can be described as a “bar code” of common haplo-
type blocks. By picking SNPs that “tag” these haplotypes,
it is possible to screen the whole human genome with a
much reduced set of SNPs, much fewer than the 10 million
or more common SNP variants that have been defined in
human populations. Generally, most of the common varia-
tion can be captured with 300,000 to 500,000 SNPs, and
standard platforms are now widely available to perform
these analyses on large populations of cases and controls.
In addition, the International HapMap Project has estab-
lished online resources (www.hapmap.org) that allow users
to explore easily the haplotype structure of any region of
the genome.118 The HapMap website also contains infor-
mative tutorials on genetic diversity and the use of this
resource.
Whole-genome association studies for human disease are
already bearing fruit. In 2005, the complement regulatory
protein factor H was identified as a significant risk factor
for age-related macular degeneration.119,120 In addition, the
IL-23 receptor was identified as a risk gene for Crohn’s dis-
ease in late 2006.121 Many other whole-genome association
studies are in progress for a variety of complex disorders and
phenotypes,122 and a deluge of such data is expected in the
coming year.
OVERLAPPING SUSCEPTIBILITY GENES AND
PATHWAYS FOR AUTOIMMUNE DISEASES
It should be clear from the previous discussion that a full
catalog of common susceptibility genes for autoimmunity is
forthcoming. Nevertheless, some themes are already emerg-
ing from the current data. In particular, common genes and
pathways seem to be involved in multiple different autoim-
mune disorders.
In 2004, an association between the intracellular phos-
phatase, PTPN22, was reported for many autoimmune
diseases, including type 1 diabetes,97 RA,96 SLE,123 and
autoimmune thyroid disease.124 With odds ratios consis-
tently in the range of 1.5 to 2, this was a compelling dem-
onstration that a specific common allelic variant can confer
risk for multiple different autoimmune phenotypes. In this
case, a nonsynonymous change in one of several SH3 bind-
ing sites in PTPN22 (a tryptophan substitution for arginine
at codon 620) was shown to disrupt the normal association
of PTPN22 with a Csk, an intracellular tyrosine kinase.96,97
The exact functional consequence of this finding is uncer-
tain, although an increase in phosphatase activity has
been reported for PTPN22 as a result of this change.125
The role of PTPN22 in immune regulation is still a mat-
ter of debate, but seems partly related to setting thresholds
for T cell receptor signaling through Lck.93 PTPN22 also
is found in many other hematopoietic cells, and its func-
tion in these cells is largely unknown. Nevertheless, since
these first reports, the association of the PTPN22 R620W
allele with multiple autoimmune diseases has been widely
­replicated.93 All of the autoimmune diseases associated
with PTPN22 have a prominent autoantibody component,
and a specific role in regulating humoral immunity seems to
be a unifying feature. Despite a lack of understanding of the
precise mechanisms, PTPN22 is a prime example of how
the discovery of disease associations with relatively mod-
est effect can redirect hypothesis-driven research onto new
pathways.
In 2005, interferon regulatory factor 5 (IRF5) was
shown to be associated with susceptibility to SLE98 and
replicated shortly thereafter.99 This was a satisfying obser-
vation because upregulation of interferon pathways is cen-
tral to the pathogenesis of SLE and related disorders.126
Interferon dysregulation is not a prominent feature of
RA, and IRF5 is not associated with RA. In contrast to
PTPN22, IRF5 seems to be specific for a more narrow
subgroup of SLE-related autoimmune disorders in which
interferon pathways play a significant role. The details
of the genetic data are particularly instructive because
there are complex interactions between several alleles
on the risk haplotype.127 Some risk alleles give rise to
splice variation, others regulate levels of expression, and
a third risk allele is related to an insertion deletion. The
presence of all of these variants on the same haplotype
confers maximal risk. There are three levels of risk, how-
ever, including a protective effect, depending on which
specific alleles are present.127 The IRF5 association with
SLE illustrates that even when a risk gene is identified,
the molecular details and allelic patterns of association
can be quite complex.
Finally, several more recent discoveries in the IL-12/
IL-23/STAT4 pathways show that multiple genes within
related pathways can have contrasting effects on risk for
different autoimmune diseases. As noted earlier, the IL-23
receptor was shown to be associated with Crohn’s disease
in late 2006.121 IL-12B and IL-23R have been associated
with susceptibility to psoriasis.128 Significant associations
between IL-23R and ankylosing spondylitis also have been
observed.128a IL-12 and IL-23 can signal through STAT4,129
and RA and SLE have been associated with polymorphisms
in STAT4.113 The influence of STAT4 gene knockout is
different in animal models—it is protective for arthritis
models,130 but exacerbates nephritis in SLE-prone mouse
strains.131 Inhibition of STAT4 with RNAi can ameliorate
ongoing arthritis in animals,130 suggesting utility as a thera-
peutic target. The STAT4 association also raises the possi-
bility that the balance of T helper type 1 and T helper type
17 may be important for understanding disease susceptibil-
ity because IL-12 and IL-23 have contrasting effects in this
regard.132
These and other genetic discoveries are emerging as
a compelling rationale for focusing attention on par-
ticular disease pathways for hypothesis-driven research.
Even if particular genetic variants play a role in only a
small subset of patients or only in certain environmental
settings, their elucidation nevertheless provides impor-
tant insights into disease pathogenesis and potentially
­provides for diagnostic and therapeutic specificity. This
is the most important reason for pursuing the genetics of
complex diseases.
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
 
Future Directions
This chapter has discussed some of the fundamental
 
concepts and techniques that have been applied to
the study of rheumatic diseases, highlighting the more
­recent emerging genetic data. Although the underlying  
concepts will not change, further advances are around
the corner. As the genetic underpinnings of disease  
are identified, a major challenge will be to define and
­integrate the multiple genetic and environmental factors  
into an overall understanding of disease pathogenesis.
It is likely that a renewed appreciation will develop  
for the commonality between, and heterogeneity of,
­rheumatic diseases. This appreciation will provide  
­opportunities for new and more specific approaches to
diagnosis and ­treatment.  
 
REFERENCES
 
   1. Snell G, Dausset J, Nathanson S: Histocompatibility. New York,
­Academic Press, 1976.  
   2. Benacerraf B: Role of MHC gene products in immune regulation.
Science 212:1229-1238, 1981.
   3. Schwartz RH: T-lymphocyte recognition of antigen in association  
with gene products of the major histocompatibility complex. Annu
Rev Immunol 3:237-261, 1985.  
   4. McDevitt HO, Benacerraf B: Genetic control of specific immune
responses. Adv Immunol 11:31-74, 1969.  
   5. Bjorkman PJ, Saper MA, Samraoui B, et al: Structure of the
human class I histocompatibility antigen, HLA-A2. Nature  
329:506-512, 1987.
   6. Bjorkman PJ, Saper MA, Samraoui B, et al: The foreign antigen
binding site and T cell recognition regions of class I histocompat-  
ibility antigens. Nature 329:512-518, 1987.
   7. Madden DR, Gorga JC, Strominger JL, et al: The structure of HLA-  
B27 reveals nonamer self-peptides bound in an extended conforma-
tion. Nature 353:321-325, 1991.
   8. Dessen A, Lawrence CM, Cupo S, et al: X-ray crystal structure of  
HLA-DR4 (DRA*0101, DRB1*0401) complexed with a peptide
from human collagen II. Immunity 7:473-481, 1997.  
   9. Rammensee HG, Falk K, Rotzschke O: Peptides naturally pre-
sented by MHC class I molecules. Annu Rev Immunol 11:213-244,
1993.  
  10. Engelhard VH: Structure of peptides associated with class I and class
II MHC molecules. Annu Rev Immunol 12:181-207, 1994.  
  11. Hennecke J, Carfi A, Wiley DC: Structure of a covalently stabilized
complex of a human alphabeta T-cell receptor, influenza HA pep-
tide and MHC class II molecule, HLA-DR1. EMBO J 19:5611-5624,  
2000.
  12. The MHC Sequencing Consortium: Complete sequence and gene
map of a human major histocompatibility complex. Nature 401:921-  
923, 1999.
  13. O’Callaghan CA, Bell JI: Structure and function of the human
MHC class Ib molecules HLA-E, HLA-F and HLA-G. Immunol Rev  
163:129-138, 1998.
  14. Llano M, Lee N, Navarro F, et al: HLA-E-bound peptides influence
recognition by inhibitory and triggering CD94/NKG2 receptors:
Preferential response to an HLA-G-derived nonamer. Eur J Immu-  
nol 28:2854-2863, 1998.
  15. Marchini J, Cardon LR, Phillips MS, et al: The effects of human
population structure on large genetic association studies. Nat Genet  
36:512-517, 2004.
  16. van Endert PM: Genes regulating MHC class I processing of antigen.
Curr Opin Immunol 11:82-88, 1999.  
  17. Price AL, Patterson NJ, Plenge RM, et al: Principal components
analysis corrects for stratification in genome-wide association stud-
ies. Nat Genet 38:904-909, 2006.
  18. Okamoto K, Makino S, Yoshikawa Y, et al: Identification of Ikap-  
paBL as the second major histocompatibility complex-linked suscep-
tibility locus for rheumatoid arthritis. Am J Hum Genet 72:303-312,
2003.
319
19. Kimura M, Kawahito Y, Obayashi H, et al: A critical role for allograft
inflammatory factor-1 in the pathogenesis of rheumatoid arthritis.
J Immunol 178:3316-3322, 2007.
20. Rybicki BA, Walewski JL, Maliarik MJ, et al: The BTNL2 gene and
sarcoidosis susceptibility in African Americans and whites. Am J
Hum Genet 77:491-499, 2005.
21. Stastny P: Mixed lymphocyte cultures in rheumatoid arthritis. J Clin
Invest 57:1148-1157, 1976.
22. Marsh SG: Nomenclature for factors of the HLA system, update
January 2007. Tissue Antigens 69:623-625, 2007.
23. Reinsmoen NL, Bach FH: Five HLA-D clusters associated with
HLA-DR4. Hum Immunol 4:249-258, 1982.
24. Price P, Witt C, Allcock R, et al: The genetic basis for the asso-
ciation of the 8.1 ancestral haplotype (A1, B8, DR3) with multiple
immunopathological diseases. Immunol Rev 167:257-274, 1999.
25. Cavalli-Sforza LL, Cavalli-Sforza F: The Great Human Diasporas.
Reading, Mass, Addison-Wesley Publishing, 1995.
26. Brewerton DA, Hart FD, Nicholls A, et al: Ankylosing spondylitis
and HLA-B27. Lancet 1:904-907, 1973.
27. Reveille JD, Ball EJ, Khan MA: HLA-B27 and genetic predisposing
factors in spondyloarthropathies. Curr Opin Rheumatol 13:265-272,
2001.
28. Khan MA, Mathieu A, Sorrentino R, et al: The pathogenetic role of
HLA-B27 and its subtypes. Autoimmun Rev 6:183-189, 2007.
29. Madden DR, Gorga JC, Strominger JL, et al: The three-dimensional
structure of HLA-B27 at 2.1: A resolution suggests a general mecha-
nism for tight peptide binding to MHC. Cell 70:1035-1048, 1992.
30. Tiwari J, Terasaki P: HLA and Disease Associations. Berlin, Springer-
Verlag, 1985.
31. Stastny P: HLA-D and Ia antigens in rheumatoid arthritis and sys-
temic lupus erythematosus. Arthritis Rheum 21:S139-S143, 1978.
32. Stastny P: Association of the B-cell alloantigen DRw4 with rheu-
matoid arthritis. N Engl J Med 298:869-871, 1978.
33. Weyand CM, Hicok KC, Conn DL, et al: The influence of HLA-
DRB1 genes on disease severity in rheumatoid arthritis. Ann Intern
Med 117:801-806, 1992.
34. Nepom GT: Major histocompatibility complex-directed susceptibil-
ity to rheumatoid arthritis. Adv Immunol 68:315-332, 1998.
35. Hall FC, Weeks DE, Camilleri JP, et al: Influence of the HLA-DRB1
locus on susceptibility and severity in rheumatoid arthritis. QJM
89:821-829, 1996.
36. Ollier W, Winchester R: The germline and somatic genetic basis for
rheumatoid arthritis. Curr Dir Autoimmun 1:166-193, 1999.
37. Gregersen PK: T-cell receptor-major histocompatibility complex
genetic interactions in rheumatoid arthritis. Rheum Dis Clin N Am
18:793-807, 1992.
38. Nichol FE, Woodrow JC: HLA DR antigens in Indian patients with
rheumatoid arthritis. Lancet 1:220-221, 1981.
39. Sanchez B, Moreno I, Magarino R, et al: HLA-DRw10 confers the
highest susceptibility to rheumatoid arthritis in a Spanish popula-
tion. Tissue Antigens 36:174-176, 1990.
40. Willkens RF, Nepom GT, Marks CR, et al: Association of HLA-
Dw16 with rheumatoid arthritis in Yakima Indians: Further evidence
for the “shared epitope” hypothesis. Arthritis Rheum 34:43-47, 1991.
41. Lee HS, Lee KW, Song GG, et al: Increased susceptibility to rheu-
matoid arthritis in Koreans heterozygous for HLA-DRB1*0405 and
*0901. Arthritis Rheum 50:3468-3475, 2004.
42. Gregersen PK, Silver J, Winchester RJ: The shared epitope
hypothesis: An approach to understanding the molecular genet-
ics of susceptibility to rheumatoid arthritis. Arthritis Rheum
30:1205-1213, 1987.
43. Buckner JH, Nepom GT: Genetics of rheumatoid arthritis: Is there a
scientific explanation for the human leukocyte antigen association?
Curr Opin Rheumatol 14:254-259, 2002.
44. Roudier J: Association of MHC and rheumatoid arthritis: Associa-
tion of RA with HLA-DR4: The role of repertoire selection. Arthri-
tis Res 2:217-220, 2000.
45. Irigoyen P, Lee AT, Wener MH, et al: Regulation of anti-cyclic
citrullinated peptide antibodies in rheumatoid arthritis: Contrasting
effects of HLA-DR3 and the shared epitope alleles. Arthritis Rheum
52:3813-3818, 2005.
46. Hill JA, Southwood S, Sette A, et al: Cutting edge: The conversion
of arginine to citrulline allows for a high-affinity peptide interaction
with the rheumatoid arthritis-associated HLA-DRB1*0401 MHC
class II molecule. J Immunol 171:538-541, 2003.
 320 GREGERSEN  |  Genetics of Rheumatic Diseases
  47. Walser-Kuntz DR, Weyand CM, Weaver AJ, et al: Mechanisms  
underlying the formation of the T cell receptor repertoire in rheu-
matoid arthritis. Immunity 2:597-605, 1995.
  48. Roudier J, Petersen J, Rhodes GH, et al: Susceptibility to rheuma-  
toid arthritis maps to a T-cell epitope shared by the HLA-Dw4 DR
beta-1 chain and the Epstein-Barr virus glycoprotein gp110. Proc
Natl Acad Sci U S A 86:5104-5108, 1989.
  49. Albani S, Keystone EC, Nelson JL, et al: Positive selection in auto-  
immunity: Abnormal immune responses to a bacterial dnaJ anti-
genic determinant in patients with early rheumatoid arthritis. Nat
Med 1:448-452, 1995.  
  50. Auger I, Toussirot E, Roudier J: HLA-DRB1 motifs and heat shock
proteins in rheumatoid arthritis. Int Rev Immunol 17:263-271,
1998.  
  51. Ling S, Li Z, Borschukova O, et al: The rheumatoid arthritis shared
epitope increases cellular susceptibility to oxidative stress by antago-
nizing an adenosine-mediated anti-oxidative pathway. Arthritis Res  
Ther 9:R5, 2007.
  52. Zanelli E, Huizinga TW, Guerne PA, et al: An extended HLA-DQ-
DR haplotype rather than DRB1 alone contributes to RA predispo-  
sition. Immunogenetics 48:394-401, 1998.
  53. Zanelli E, Gonzalez-Gay MA, David CS: Could HLA-DRB1 be
the protective locus in rheumatoid arthritis? Immunol Today 16:
274-278, 1995.  
  54. Taneja V, Griffiths MM, Luthra H, et al: Modulation of HLA-DQ-
restricted collagen-induced arthritis by HLA-DRB1 polymorphism.
Int Immunol 10:1449-1457, 1998.
  55. Milicic A, Lee D, Brown MA, et al: HLA-DR/DQ haplotype in  
rheumatoid arthritis: Novel allelic associations in UK Caucasians.
J Rheumatol 29:1821-1826, 2002.
  56. Gao XJ, Brautbar C, Gazit E, et al: A variant of HLA-DR4 deter-  
mines susceptibility to rheumatoid arthritis in a subset of Israeli Jews.
Arthritis Rheum 34:547-551, 1991.
  57. Gao X, Gazit E, Livneh A, et al: Rheumatoid arthritis in Israeli Jews:  
Shared sequences in the third hypervariable region of DRB1 alleles
are associated with susceptibility. J Rheumatol 18:801-803, 1991.
  58. Teller K, Budhai L, Zhang M, et al: HLA-DRB1 and DQB typing of
Hispanic American patients with rheumatoid arthritis: The “shared  
epitope” hypothesis may not apply. J Rheumatol 23:1363-1368,
1996.
  59. McDaniel DO, Alarcon GS, Pratt PW, et al: Most African-Ameri-  
can patients with rheumatoid arthritis do not have the rheumatoid
antigenic determinant (epitope). Ann Intern Med 123:181-187,
1995.  
  60. Nepom BS, Nepom GT, Mickelson E, et al: Specific HLA-DR4-
associated histocompatibility molecules characterize patients with
seropositive juvenile rheumatoid arthritis. J Clin Invest 74:287-291,
1984.  
  61. Michou L, Croiseau P, Petit-Teixeira E, et al: Validation of the
reshaped shared epitope HLA-DRB1 classification in rheumatoid
arthritis. Arthritis Res Ther 8:R79, 2006.  
  62. Todd JA, Bell JI: McDevitt HO: HLA-DQ beta gene contributes to
susceptibility and resistance to insulin-dependent diabetes mellitus.
Nature 329:599-604, 1987.
  63. Hamilton RG, Harley JB, Bias WB, et al: Two Ro (SS-A) autoan-  
tibody responses in systemic lupus erythematosus: Correlation of
HLA-DR/DQ specificities with quantitative expression of Ro (SS-  
A) autoantibody. Arthritis Rheum 31:496-505, 1988.
  64. Harley JB, Alexander EL, Bias WB, et al: Anti-Ro (SS-A) and anti-  
La (SS-B) in patients with Sjogren’s syndrome. Arthritis Rheum
29:196-206, 1986.  
  65. Harley JB, Reichlin M, Arnett FC, et al: Gene interaction at
HLA-DQ enhances autoantibody production in primary Sjogren’s  
syndrome. Science 232:1145-1147, 1986.
  66. Reveille JD, Macleod MJ, Whittington K, et al: Specific amino acid
residues in the second hypervariable region of HLA-DQA1 and  
DQB1 chain genes promote the Ro (SS-A)/La (SS-B) autoantibody
responses. J Immunol 146:3871-3876, 1991.
  67. Mamula MJ, Fatenejad S, Craft J: B cells process and present lupus  
autoantigens that initiate autoimmune T cell responses. J Immunol
152:1453-1461, 1994.  
  68. Talken BL, Schafermeyer KR, Bailey CW, et al: T cell epitope map-
ping of the Smith antigen reveals that highly conserved Smith anti-
gen motifs are the dominant target of T cell immunity in systemic  
lupus erythematosus. J Immunol 167:562-568, 2001.
69. Arnett FC, Thiagarajan P, Ahn C, et al: Associations of anti-beta2-
glycoprotein I autoantibodies with HLA class II alleles in three eth-
nic groups. Arthritis Rheum 42:268-274, 1999.
70. Olsen ML, Arnett FC, Reveille JD: Contrasting molecular patterns
of MHC class II alleles associated with the anti-Sm and anti-RNP
precipitin autoantibodies in systemic lupus erythematosus. Arthritis
Rheum 36:94-104, 1993.
71. de Bakker PI, McVean G, Sabeti PC, et al: A high-resolution
HLA and SNP haplotype map for disease association studies in the
extended human MHC. Nat Genet 38:1166-1172, 2006.
72. Seldin MF, Shigeta R, Villoslada P, et al: European population sub-
structure: Clustering of northern and southern populations. PLoS
Genet 2:e143, 2006.
73. Falk CT, Rubinstein P: Haplotype relative risks: An easy, reliable
way to construct a proper control sample for risk calculations. Ann
Hum Genet 51:227-233, 1987.
74. Spielman RS, McGinnis RE, Ewens WJ: Transmission test for link-
age disequilibrium: The insulin gene region and insulin-dependent
diabetes mellitus (IDDM). Am J Hum Genet 52:506-516, 1993.
75. Mulcahy B, Waldron-Lynch F, McDermott MF, et al: Genetic vari-
ability in the tumor necrosis factor-lymphotoxin region influences
susceptibility to rheumatoid arthritis. Am J Hum Genet 59:676-683,
1996.
76. Ota M, Katsuyama Y, Kimura A, et al: A second susceptibility gene
for developing rheumatoid arthritis in the human MHC is localized
within a 70-kb interval telomeric of the TNF genes in the HLA class
III region. Genomics 71:263-270, 2001.
77. Jawaheer D, Li W, Graham RR, et al: Dissecting the genetic com-
plexity of the association between human leukocyte antigens and
rheumatoid arthritis. Am J Hum Genet 71:585-594, 2002.
78. Yen JH, Moore BE, Nakajima T, et al: Major histocompatibility
complex class I-recognizing receptors are disease risk genes in rheu-
matoid arthritis. J Exp Med 193:1159-1167, 2001.
79. Graham RR, Ortmann W, Rodine P, et al: Specific combinations
of HLA-DR2 and DR3 class II haplotypes contribute graded risk for
disease susceptibility and autoantibodies in human SLE. Eur J Hum
Genet, 15:823-830, 2007.
80. Graham RR, Ortmann WA, Langefeld CD, et al: Visualizing human
leukocyte antigen class II risk haplotypes in human systemic lupus
erythematosus. Am J Hum Genet 71:543-553, 2002.
81. Zeggini E, Donn RP, Ollier WE, et al: Evidence for linkage of HLA
loci in juvenile idiopathic oligoarthritis: Independent effects of
HLA-A and HLA-DRB1. Arthritis Rheum 46:2716-2720, 2002.
82. Runstadler JA, Saila H, Savolainen A, et al: HLA-DRB1, TAP2/
TAP1, and HLA-DPB1 haplotypes in Finnish juvenile idiopathic
arthritis: More complexity within the MHC. Genes Immun 5:562-
571, 2004.
83. Rubio JP, Bahlo M, Butzkueven H, et al: Genetic dissection of the
human leukocyte antigen region by use of haplotypes of Tasmanians
with multiple sclerosis. Am J Hum Genet 70:1125-1137, 2002.
84. Vandiedonck C, Beaurain G, Giraud M, et al: Pleiotropic effects of
the 8.1 HLA haplotype in patients with autoimmune myasthenia
gravis and thymus hyperplasia. Proc Natl Acad Sci U S A 101:15464-
15469, 2004.
85. Lander ES, Linton LM, Birren B, et al: Initial sequencing and analy-
sis of the human genome. Nature 409:860-921, 2001.
86. Venter JC, Adams MD, Myers EW, et al: The sequence of the human
genome. Science 291:1304-1351, 2001.
87. Consortium TIH: A haplotype map of the human genome. Nature
437:1299-1320, 2005.
88. Todd JA: La carte des microsatellites est arrivee! [The map of micro-
satellites has arrived!]. Hum Mol Genet 1:663-666, 1992.
89. Wong KK, deLeeuw RJ, Dosanjh NS, et al: A comprehensive analy-
sis of common copy-number variations in the human genome. Am J
Hum Genet 80:91-104, 2007.
90. Sebat J, Lakshmi B, Malhotra D, et al: Strong association of de
novo copy number mutations with autism. Science 316:445-449,
2007.
91. Steemers FJ, Gunderson KL: Whole genome genotyping technolo-
gies on the BeadArray platform. Biotechnol J 2:41-49, 2007.
92. Pritchard JK, Cox NJ: The allelic architecture of human disease
genes: Common disease-common variant … or not? Hum Mol
Genet 11:2417-2423, 2002.
93. Gregersen PK, Lee HS, Batliwalla F, et al: PTPN22: Setting thresh-
olds for autoimmunity. Semin Immunol 18:214-223, 2006.
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
  94. Cohen JC, Pertsemlidis A, Fahmi S, et al: Multiple rare variants
in NPC1L1 associated with reduced sterol absorption and plasma
low-density lipoprotein levels. Proc Natl Acad Sci U S A 103:
1810-1815, 2006.
  95. Cohen JC, Kiss RS, Pertsemlidis A, et al: Multiple rare alleles
contribute to low plasma levels of HDL cholesterol. Science 305:
869-872, 2004.
  96. Begovich AB, Carlton VE, Honigberg LA, et al: A missense single-
nucleotide polymorphism in a gene encoding a protein tyrosine
phosphatase (PTPN22) is associated with rheumatoid arthritis. Am
J Hum Genet 75:330-337, 2004.
  97. Bottini N, Musumeci L, Alonso A, et al: A functional variant of
lymphoid tyrosine phosphatase is associated with type I diabetes.
Nat Genet 36:337-338, 2004.
  98. Sigurdsson S, Nordmark G, Goring HH, et al: Polymorphisms in
the tyrosine kinase 2 and interferon regulatory factor 5 genes are
associated with systemic lupus erythematosus. Am J Hum Genet 76:
528-537, 2005.
  99. Graham RR, Kozyrev SV, Baechler EC, et al: A common haplotype
of interferon regulatory factor 5 (IRF5) regulates splicing and expres-
sion and is associated with increased risk of systemic lupus erythema-
tosus. Nat Genet 38:550-555, 2006.
100. Risch N: Linkage strategies for genetically complex traits, II: The
power of affected relative pairs. Am J Hum Genet 46:229-241, 1990.
101. Vyse TJ, Todd JA: Genetic analysis of autoimmune disease. Cell
85:311-318, 1996.
102. Hirschhorn JN, Lohmueller K, Byrne E, et al: A comprehensive
review of genetic association studies. Genet Med 4:45-61, 2002.
103. Risch NJ: Searching for genetic determinants in the new millen-
nium. Nature 405:847-856, 2000.
104. Ott J: Analysis of Human Genetic Linkage. Baltimore, Johns Hop-
kins University Press, 1999.
105. Pras E, Aksentijevich I, Gruberg L, et al: Mapping of a gene causing
familial Mediterranean fever to the short arm of chromosome 16.
N Engl J Med 326:1509-1513, 1992.
106. Consortium TIF: Ancient missense mutations in a new member of
the RoRet gene family are likely to cause familial Mediterranean
fever. The International FMF Consortium. Cell 90:797-807, 1997.
107. McDermott MF, Aksentijevich I, Galon J, et al: Germline muta-
tions in the extracellular domains of the 55 kDa TNF receptor,
TNFR1, define a family of dominantly inherited autoinflammatory
syndromes. Cell 97:133-144, 1999.
108. Hull KM, Drewe E, Aksentijevich I, et al: The TNF receptor-associ-
ated periodic syndrome (TRAPS): Emerging concepts of an autoin-
flammatory disorder. Medicine (Baltimore) 81:349-368, 2002.
109. Hugot JP, Chamaillard M, Zouali H, et al: Association of NOD2
leucine-rich repeat variants with susceptibility to Crohn’s disease.
Nature 411:599-603, 2001.
110. Ogura Y, Bonen DK, Inohara N, et al: A frameshift mutation in
NOD2 associated with susceptibility to Crohn’s disease. Nature
411:603-606, 2001.
111. Forabosco P, Gorman JD, Cleveland C, et al: Meta-analysis of
genome-wide linkage studies of systemic lupus erythematosus. Genes
Immun 7:609-614, 2006.
112. Amos CI, Chen WV, Lee A, et al: High-density SNP analysis of 642
Caucasian families with rheumatoid arthritis identifies two new link-
age regions on 11p12 and 2q33. Genes Immun 7:277-286, 2006.
113. Remmers EF, Plenge RM, Lee AT, et al: STAT4 and the risk of
rheumatoid arthritis and systemic lupus erythematosus. N Engl J
Med, 357:977-986, 2007.
114. Daly MJ, Rioux JD, Schaffner SF, et al: High-resolution haplotype
structure in the human genome. Nat Genet 29:229-232, 2001.
321
115. Jeffreys AJ, Kauppi L, Neumann R: Intensely punctate meiotic
recombination in the class II region of the major histocompatibility
complex. Nat Genet 29:217-222, 2001.
116. Rioux JD, Daly MJ, Silverberg MS, et al: Genetic variation in the
5q31 cytokine gene cluster confers susceptibility to Crohn disease.
Nat Genet 29:223-228, 2001.
117. Myers S, Bottolo L, Freeman C, et al: A fine-scale map of recom-
bination rates and hotspots across the human genome. Science
310:321-324, 2005.
118. Thorisson GA, Stein LD: The SNP Consortium website: Past, pres-
ent and future. Nucleic Acids Res 31:124-127, 2003.
119. Klein RJ, Zeiss C, Chew EY, et al: Complement factor H poly-
morphism in age-related macular degeneration. Science 308:385-
389, 2005.
120. Zareparsi S, Branham KE, Li M, et al: Strong association of the
Y402H variant in complement factor H at 1q32 with susceptibility
to age-related macular degeneration. Am J Hum Genet 77:149-153,
2005.
121. Duerr RH, Taylor KD, Brant SR, et al: A genome-wide associa-
tion study identifies IL23R as an inflammatory bowel disease gene.
Science 314:1461-1463, 2006.
122. Saxena R, Voight BF, Lyssenko V, et al: Genome-wide association
analysis identifies loci for type 2 diabetes and triglyceride levels.
Science, 316:1331-1336, 2007.
123. Kyogoku C, Langefeld CD, Ortmann WA, et al: Genetic associa-
tion of the R620W polymorphism of protein tyrosine phosphatase
PTPN22 with human SLE. Am J Hum Genet 75:504-507, 2004.
124. Criswell LA, Pfeiffer KA, Lum RF, et al: Analysis of families in the
multiple autoimmune disease genetics consortium (MADGC) col-
lection: The PTPN22 620W allele associates with multiple autoim-
mune phenotypes. Am J Hum Genet 76:561-571, 2005.
125. Vang T, Congia M, Macis MD, et al: Autoimmune-associated lym-
phoid tyrosine phosphatase is a gain-of-function variant. Nat Genet
37:1317-1319, 2005.
126. Baechler EC, Batliwalla FM, Reed AM, et al: Gene expression pro-
filing in human autoimmunity. Immunol Rev 210:120-137, 2006.
127. Graham RR, Kyogoku C, Sigurdsson S, et al: Three functional vari-
ants of IFN regulatory factor 5 (IRF5) define risk and protective hap-
lotypes for human lupus. Proc Natl Acad Sci U S A 104:6758-6763,
2007.
128. Cargill M, Schrodi SJ, Chang M, et al: A large-scale genetic associa-
tion study confirms IL12B and leads to the identification of IL23R as
psoriasis-risk genes. Am J Hum Genet 80:273-390, 2007.
128a. Wellcome Trust Case Control Consortium. Association scan of
14,500 nonsynonymous SNPs in four diseases identifies autoimmu-
nity variants. Nat Genet 39(11):1329-1337, 2007.
129. Watford WT, Hissong BD, Bream JH, et al: Signaling by IL-12 and
IL-23 and the immunoregulatory roles of STAT4. Immunol Rev
202:139-156, 2004.
130. Hildner KM, Schirmacher P, Atreya I, et al: Targeting of the transcrip-
tion factor STAT4 by antisense phosphorothioateoligonucleotides
suppresses collagen-induced arthritis. J Immunol 178:3427-3436,
2007.
131. Jacob CO, Zang S, Li L, et al: Pivotal role of Stat4 and Stat6 in
the pathogenesis of the lupus-like disease in the New Zealand mixed
2328 mice. J Immunol 171:1564-1571, 2003.
132. Mathur AN, Chang HC, Zisoulis DG, et al: Stat3 and Stat4 direct
development of IL-17-secreting Th cells. J Immunol 178:4901-4907,
2007.
 19
KEY POINTS: RHEUMATIC DISEASES—
GENERAL CONCEPTS
Autoantibodies may activate the complement system,
which then contributes to damage at the site of autoantigen
­expression.
Urate crystals, like other types of self-debris, activate innate
immunity, including the complement system.
The complement system plays a key role in the handling
of immune complexes, especially those that arise in the
­circulation. Complement activation is an efficient means to
coat antigens so that they adhere to peripheral blood cells
and are then phagocytosed or carried to the spleen and
liver for disposal. In these organs, the immune complexes
are transferred to tissue macrophages for destruction or an
­immune response.
Complement measurements are helpful in diagnosing and
following patients with systemic lupus erythematosus. A low
C4 and C3 in the presence of antinuclear antibodies has nearly
a 100% specificity for lupus.
In rheumatic diseases featuring complement activation, we
lack sensitive day-to-day markers to monitor disease activity.
Therefore, we do not know how much complement ­inhibition
is required to favorably modulate a disease process. We
also lack knowledge as to whether C3a/C5a, C4b/C3b, or the
membrane attack complex is the key mediator of pathology,
as well as which pathway and at which step to block.
The complement system is activated in the joints in
­rheumatoid arthritis and in vessel walls in vasculitis.
In this chapter, the workings of the complement system are
reviewed, with a focus on how the system functions in rheu-
matic diseases. The involvement of the complement system
has long been recognized in two of the most common inflam-
matory diseases treated by rheumatologists: systemic lupus
erythematosus (SLE) and rheumatoid arthritis (RA). What
has been learned about the complement system’s role in these
two diseases likely applies to related syndromes. The goal of
this chapter is to help the rheumatologist appreciate the bio-
logic and pathologic relevance of the complement system in
rheumatic diseases. This information can also assist in the
clinical interpretation of laboratory tests of complement pro-
teins and in the determination of the significance of comple-
ment activation fragments in pathologic specimens.
HISTORICAL ASPECTS
Today, complement is a collective term designating a group
of plasma and membrane proteins that play a key role in
innate and adaptive immunity. The complement system is
Complement System
John p. atkinson
ancient, predating chordates such as the lamprey and hag-
fish.1,2 Complement was discovered in the late 19th century
by experimental pathologists attempting to understand the
protective basis of vaccination and the cause of transfusion
reactions.3 In these studies, it became apparent that the cell-
free portion of blood contained a lytic substance for bacteria
and for transfused red blood cells (RBCs). The factor was
heat labile (destroyed by heating at 56°C for 30 minutes
or by leaving serum on the bench top overnight) and was
innate or nonspecific (all individuals had this lytic sub-
stance). It was contrasted to antibody, which was heat sta-
ble and specific because immunized animals contained this
substance. Through mixing experiments, two factors were
found to be required for lysis—a specific recognition piece
(antibody) and a nonspecific lytic piece (complement). In
retrospect, this was the discovery of the classic complement
pathway and led to its characterization as a heat-labile bac-
tericidal factor that “complemented” the acquired factor
raised by immunization. Similarly, in the blood of patients
who recovered from transfusion reactions, a heat-stable sub-
stance (antibody) was found that recognized the transfused
erythrocytes. However, it did not lyse the transfused cells;
rather, it cooperated with a second lytic substance (comple-
ment) present in fresh serum.
From this discussion, it is apparent why lysis became
linked to the complement system and why the study of anti-
body and complement dominated the field of immunology
for the next half century. Of interest, the first autoimmune
disease to be recognized as such was a hemolytic anemia
(paroxysmal cold hemoglobinuria) in which the same pair
of reactants, antibody and complement, was shown to medi-
ate RBC lysis.4 Consequently, this double-edged sword
aspect of the complement system was recognized early, but
not until the 1960s was there such a clear-cut demonstra-
tion of its protective versus injurious role. Compared with
normal mice, C5-deficient mice were 100-fold more sensi-
tive to death from pneumococcal infection but 100-fold less
sensitive to tissue destruction by autoantibodies. Even more
to the point, we now know that inherited deficiencies of C1,
C4, and C2 strongly predispose to SLE, yet immune com-
plex (IC) deposition and complement activation contribute
to tissue damage in SLE.
FUNCTION
A major activity of the complement system is to modify
membranes and soluble antigens through strong (cova-
lent) attachment of its activation fragments (Fig. 19-1).
For example, several million C3b molecules can bind to a
bacterial surface in less than 2 minutes. One goal of these
deposited complement proteins is to opsonize the target.
323
 324 Atkinson  |  Complement System
Complement
Membrane modification Cellular activation
Pathogen
Direct lysis Opsonization Degranulation Chemotaxis
Outcomes
1. Microbial destruction
2. Inflammation
3 Instructions to adaptive immunity
Figure 19-1  Function of the complement system. The most impor-
tant function of the complement system is to alter the membrane of a
pathogen by coating its surface with clusters of activation fragments. In
one case, they facilitate the key process of opsonization, as C4b and C3b
interact with complement receptors. In the other case, as with certain
gram-negative bacteria and viruses, the membrane attack complex lyses
the organism. The second critical function of complement is to activate
cells and thus promote inflammatory and immune responses. The com-
plement fragments C3a and C5a (known as anaphylatoxins) stimulate
many cell types, such as mast cells, to release their contents and stimu-
late phagocytic cells to migrate to sites of inflammation (chemotaxis).
Through these phenomena of opsonization and cell activation, com-
plement serves as nature’s adjuvant to prepare, facilitate, and instruct
the host’s adaptive immune response. Because complement activation  NOMENCLATURE
occurs in a few seconds, this innate immune system initially engages
most pathogens, especially those that try to enter the vascular space.
C3b (and its degradation fragments) and C4b are ligands
for complement receptors on peripheral blood cells, tissue
macrophages, and antigen presenting cells such as follicular
dendritic cells. Complement ligands and their receptors are
particularly adept at this coupling process, known as immune
adherence. On phagocytic cells, this often leads to ingestion
of the infectious particle, IC, or debris. Additionally, the
membranes of some microorganisms (e.g., gram-negative
organisms) and some host cells can be lysed by the termi-
nal complement components (C5b-C9). In many cases,
however, microbes possess capsules, making them resistant
to lysis (e.g., most gram-positive organisms). In human dis-
eases featuring autoantibodies, cells and tissues are similarly
opsonized, and membrane integrity can be compromised. A
recent extension of this opsonic function relates to com-
plement’s role in the clearance of cellular debris following
necrosis or apoptosis. In particular, one hypothesis to explain
autoantibody formation in SLE is the complement system’s
failure to properly clear or handle apoptotic cells.5
A second function of the complement system is cellular
activation. It prepares the local environment for defense
against infection (see Fig. 19-1). In the proteolytic steps
of the early complement activation process, mediators are
released that activate nearby cells. These low-molecular-
weight fragments (C4a, C3a, C5a) are termed anaphylatox-
ins because, if released in excessive amounts, they induce
shock. C3a and C5a, the two most powerful mediators,
bind to their respective receptors at sites of complement
activation. This causes histamine release by mast cells and
chemotaxis to the area by phagocytic cells. With improved
reagents and newer technology, receptors for C3a and C5a
have now been shown to be much more widely expressed
Lectin
Classic Alternative
C3b
Inflammation Opsonization
(C3a/C5a) (C3b/C4b)
Membrane attack (lysis)
(C5b-C9)
Figure 19-2  Simplified diagram of the three pathways of comple-
ment activation. Deposition of clusters of C3b on a target is the primary
goal. As shown by the broken line, the alternative pathway also serves as
a feedback loop to amplify C3b deposition (regardless of which cascade
deposited the C3b).
than initially thought; for example, they are expressed by
epithelial cells, T cells, hepatocytes, endothelial cells, neu-
rons, and other cell types. C3a and C5a may engage their
receptors on endothelial and neuronal cells to alter blood
flow and modulate cellular function.
There are three activation cascades: the classic pathway
(CP), the alternative pathway (AP), and the lectin pathway
(LP) (Fig. 19-2). These converge to activate C3 and then
the membrane attack complex (MAC).
The nine proteins of the CP are designated by the upper-
case letter C, followed by a number (Table 19-1). These
numbered components act in numerical order, except for
C4, which is cleaved in parallel with C2 and before C3.
Components of the AP are designated by capital letters
(e.g., factor B). Regulatory proteins are designated by a
descriptive title (e.g., C4 binding protein) or a letter (e.g.,
factor H) (Table 19-2). Single components or multimeric
complexes that have enzymatic activity are designated by
a bar (e.g., C1 ). The loss of hemolytic potential by a com-
ponent is usually designated by a lowercase prefix (e.g.,
iC3b). Fragments generated during complement activa-
tion are designated by a lowercase letter suffix (e.g., C3a,
C3b). Except for the fragments of C2 (which are opposite),
the a fragment is liberated into the surrounding milieu,
whereas the b fragment becomes cell bound and continues
the cascade.
Receptors were named in order of their discovery but are
also commonly identified relative to their ligand specific-
ity and CD number (Tables 19-3 to 19-5). For example,
complement receptor type 1 may be referred to in the litera-
ture as CR1, the C3b/C4b receptor, the immune adherence
receptor, or CD35.
ACTIVATION CASCADES
Because many rheumatic diseases feature autoantibodies
and therefore ICs, an understanding of the general princi-
ples of the activation cascades is essential to characterizing
complement’s role in a disease such as SLE3,6-9 (Fig. 19-3; see
also Fig 19-2).
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION 325

Table 19-1  Components of the Complement   enzymatic or catalytic domain of this complex is C2a, which
Activation Cascades cleaves C3 to form C3a and activated C3b (Fig. 19-7). Acti-
Serum
vated C3b attaches to the target by binding covalently to a
Concentration hydroxyl group (analogous to the interaction of C4b with
Component (µg/mL) Function a hydroxyl or amino group), where it serves as the major
Classic Pathway (CP) opsonin of the complement system. In addition, some of the
newly activated C3b binds covalently to a specific region on
C1 50 Binds IC to trigger CP
  C1q subcomponent Binds Fc portion of IgG,
the C4b component of C3 convertase to form the CP C5
IgM convertase C4bC2aC3b (see Fig. 19-6).
  C1r subcomponent Protease: cleaves C1s CP activation by polyclonal antibodies is efficient on
  C1s subcomponent Protease: cleaves C4 membranes of cells, bacteria, and viruses. In one illustrative
and C2 study, complement activation by IgG binding to a mem-
C4 200-500 Opsonin* (C4b), C4a†
C2 20 Protease: cleaves   brane antigen of nucleated cell produced about 2.5 million
C3 and C5 as part of bound C4b molecules, and about 0.5 million C2a molecules
the convertases subsequently attached to these C4bs to form convertases.11
Lectin Pathway (LP) In less than 5 minutes, 21 million (representing a 10×
MBL 150 (wide Binds sugars to trigger LP amplification over the number of attached C4b molecules)
range) C3b molecules were deposited. Further, only about 10% to
MASP-1 5 Protease 20% of the C4b and C3b molecules generated bound to the
MASP-2 5 Protease: cleaves C4 target. The rest were inactivated by hydrolysis in the fluid
and C2
phase and then degraded by fluid-phase inhibitors. Con-
Alternative Pathway (AP) comitantly, C4a, C3a, and C5a anaphylatoxins were gener-
Factor D 1-2 Protease: cleaves factor B ated equal to the number of C4b, C3b, and C5b fragments
Factor B 150-250 Protease: cleaves C3 produced. Approximately 1 million MACs were formed.
and C5 as part of the Fragment attachment to and amplification on soluble anti-
convertases
Properdin 25 Stabilizes AP convertases gens is a less efficient process, particularly the formation of
Central protein  C3 550-1200 Opsonin† (C3b), C3a† the C3 and C5 convertases.
A single IgM can activate the CP. In contrast, two IgGs
Terminal Pathway (TP)
in close proximity (side by side) are required before C1 can
C5 70 MAC component (C5b), be bound. On erythrocytes, this means that several thou-
C5a†
C6 60 MAC component
sand IgGs must bind to an antigen before activation can
C7 60 MAC component occur. Consequently, antigenic density and display play a
C8 60 MAC component (pore role in determining whether complement is activated. For
formation) example, in warm antibody (IgG)-mediated autoimmune
C9 60 MAC component (pore hemolytic anemia, complement is usually not fixed (despite
formation)
the autoantibody’s being of an appropriate IgG subclass)
*C4b forms part of the CP C3 and C5 convertases. It anchors the enzyme because of low antigenic density.
complex to the target. Likewise, for the AP C3 and C5 convertases, C3b
anchors these enzyme complexes to the target. C3b is also part of the CP
C5 convertase. LECTIN PATHWAY
†
C4a, C3a, and C5a are liberated upon cleavage of C4, C3, and C5.
IC, immune complex; Ig, immunoglobulin; MAC, membrane attack com- Lectins are carbohydrate-binding proteins,5,12,13 (see Fig.
plex; MASP, mannan-associated serine protease; MBL, mannan-binding 19-4). Initially, they were described as proteins capable
­lectin. of agglutinating RBCs. They are important players in
innate immunity and in rheumatic diseases. In particular,
mannan­-binding lectin (MBL) is a hepatocyte-­synthesized
CLASSIC PATHWAY plasma protein that preferentially binds to repeating man-
noses and certain other oligosaccharides of pathogens.
The CP is activated primarily by ICs and a few other sub- MBL resembles C1q (it belongs to the same family of
stances, including C-reactive protein.10 Human immuno- proteins, known as collectins), consisting of an oligomer
globulin (Ig) M and IgG subclasses 1 and 3 efficiently activate with a terminal collagenous domain on one end and a
the CP. Once C1 is bound to an activator—by means of the globular domain on the other (see Fig. 19-4). The main
Fc portion of IgG or IgM interacting with the C1q subunit difference is that the carboxyl-terminus of MBL possesses
of the C1 complex (Fig. 19-4)—the C1r enzymatic subcom- a ­ carbohydrate-recognition domain, whereas C1q has an
ponent autoactivates by proteolysis and then cleaves C1s to Ig-­binding domain. Like C1q binding to the Fc portion of
form C1 . C4 is a substrate for C1 and is cleaved by C1 to IgG, MBL engagement with its sugar ligand leads to acti-
C4b, releasing the C4a fragment (Fig. 19-5). The C4b frag- vation of serine proteases similar to C1r and C1s, called
ment, having had its thioester bond disrupted, now has the mannan-­associated serine proteases (MASPs). MASP-2
transient ability (a few microseconds) to covalently bind to cleaves C4 and C2. MBL was discovered during an inves-
a hydroxyl or amino group on a nearby target. C2 is also a tigation into the cause of bacteremia in young children.
substrate for C1, and C2 is cleaved by C1 to yield C2a and The role of MBL deficiency as a predisposing factor for SLE
C2b. C2a interacts with 10% to 20% of the target-bound and RA, as well as its contribution to disease pathology, is
C4b to form the CP C3 convertase C4bC2a (Fig. 19-6). The under intensive study.
 326 Atkinson  |  Complement System

Table 19-2  Complement Regulatory Proteins

Protein Tissue Distribution Function Disease Association
Initiation
C1 inhibitor Plasma Inactivates C1r, C1s and MASPs; a SERPIN Hereditary angioedema
Convertases
Factor I Plasma Cleaves C3b and C4b; requires a cofactor Infections (secondary to low C3), HUS*
protein
Membrane cofactor protein Most cells Cofactor for cleavage of C4b and C3b HUS*
Decay-accelerating factor Most cells Decays C3 and C5 convertases PNH
C4-binding protein Plasma Cofactor for cleavage of C4b; decays CP C3   None†
and C5 convertases
Factor H Plasma Cofactor for cleavage of C3b; decays AP C3   Infections (secondary to low C3), HUS*,
and C5 convertases type II MPGN, AMD
Complement receptor type 1 Blood cells Receptor for C3b and C4b; cofactor activity   None‡
(CR1) for C3b and C4b; decays C3 and C5   Reduced levels in diseases featuring  
convertases immune complexes
Membrane Attack Complex
S protein (vitronectin) Plasma Blocks fluid-phase MAC None‡
CD59 Most cells Blocks MAC on host cells PNH
Anaphylatoxins
Anaphylatoxin inactivator Plasma Inactivates C3a, C4a, and C5a Urticaria, angioedema
*Most patients with HUS are heterozygous for function-altering mutations. 18

†
Insufficient number of cases of complete deficiency described to establish an association.
‡
Complete deficiency has not been reported.
AMD, age-related macular degeneration; AP, alternative pathway; CP, classic pathway; HUS, hemolytic uremic syndrome; MAC, membrane attack complex;
MASP, mannan-associated serine protease; MPGN, membranoproliferative glomerulonephritis; PNH, paroxysmal nocturnal hemoglobinuria; SERPIN, serine
protease inhibitor.

Table 19-3  Receptors for C3 and C4

Receptor Primary Ligand Location Function
CR1 C3b/C4b Peripheral blood cells (except for most T cells   Immune adherence > phagocytosis; antigen localization and
and platelets), FDCs, B cells, podocytes retention; complement regulation
CR2 C3dg/C3d B lymphocytes, FDCs Coreceptor for signaling through B cell receptor; antigen  
localization and processing
CR3/CR4 iC3b Myeloid lineage Phagocytosis > adherence
The CD numbers are as follows: CR1, CD35; CR2, CD21; CR3, CD11b/CD18; CR4, CD11c/CD18.
FDC, follicular dendritic cell.

Table 19-4  Receptors for Anaphylatoxins Table 19-5  Receptors for C1q*
Receptor Ligand Location Function Receptor Ligand Location Function
C3aR C3a Myeloid lineage, Cell activation, including CD91 Collagen-like   Phagocytic cells Enhance phagocy-
including mast granule exocytosis,   region tosis
cells; smooth upregulation of
CD93 Collagen-like   Cell surface; Enhance phagocy-
muscle, epithelial, adhesins, chemotaxis,
region monocytes, tosis
endothelial, and cytoskeletal effects
platelets, en-
neuronal cells
dothelial cells,
C5aR C5a Similar to C3aR Similar to C3aR, but with neutrophils
more chemotactic
Calreticulin Globular head Intracellular Bridge between
effects
C1q and CD93
and CD91
The LP shares many features with the CP.7,8,12,13 The dif- CR1 Globular head Hematopoietic Adherence
ferences occur during the first few steps when MBL binds cells
to an activating surface, such as bacteria with the appropri- Others Globular head CR1 Probably facilitates
ate repeating oligosaccharides. Each element of the MBL- adherence
MASP complex is structurally and functionally homologous *See reference 25 for a progress report in this evolving area.
to the corresponding elements in the CP. C1q and MBL have CR1, complement receptor 1 (CD35).
globular ends through which they attach to their respective
substrates. The MASP-2 in the complex cleaves C4 to form MBL and related lectins such as surfactants and ficolins bind
C4a and C4b (see Fig. 19-5) and C2 to form C2a and C2b— to their cognate residues on microbes to trigger complement
identical to C1 cleaving C4 and C2. The C4b2a complex activation. At one time, MASPs (and possibly C1) directly
formed is the same C3 convertase generated in the CP (see activated C3, but through the evolution of C4 and C2, a
Fig. 19-6). The LP is a key player in innate immunity, where more efficient C3 activating process was engineered.13
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION 327

Activation
Lectin -C1s or MASP-2
C4 C4b + C4a
Classic MASP Alternative
C1
C4 C3 Degradation
B, D, P
C2 C3b Factor I plus
Positive C4b C4d + C4c
cofactor protein
feedback
loop Figure 19-5  C4 activation and degradation. The boxed activation
Inflammation Opsonization fragment is the one that remains covalently attached to the target. The
(C3a/C5a) (C3b/C4b) other fragments are released into the surrounding milieu. C4a is a weak
Membrane attack (lysis) anaphylatoxin but has direct killing activity for bacteria. Factor I is a ser-
(C5b-C9) ine protease and cannot cleave C4b at a specific site unless accompanied
by a cofactor protein. In the plasma, C4bp serves this role; on cells, it is a
function of MCP (CD46) or CR1 (CD35).
Figure 19-3  The complement activation pathways. Note that C4 and
C2 serve identical purposes in the classic pathway (CP) and the lectin
pathway (LP). They are cleaved by mannan-associated serine protease-2
(MASP-2) in the LP and by C1 in the CP. The convertases that cleave C3 Convertase Classic/Lectin Alternative
are shown in Figure 19-6. C1 connects immune complexes to the CP as
the C1q subcomponent binds to the Fc of antibody and the C1s subcom-
ponent cleaves C4 and C2. In the LP, lectin carries a MASP that cleaves C3 C4bC2a C3bBbP
C4 and C2.
C3b C3b
C5
IgG and IgM Carbohydrate C4bC1a C3bBbP
binding site binding site

Figure 19-6  Proteins of the C3 and C5 convertases. C4b or C3b an-

chors the enzyme complex to the target. The catalytic domains are the
serine proteases C2a and Bb. The C3 convertase becomes a C5 conver-
tase when a C3b attaches to a preferred acceptor site on C4b or C3b.
Properdin (P) stabilizes the alternative pathway convertases.

Activation
C3 convertases
C1q MBL C3 C3b + C3a
Figure 19-4  Similarity of C1q and mannan-binding lectin (MBL)
structure. Ficolins, surfactins, and other members of the collectin fam-
ily of proteins also share this general structure. Many activate the lectin Degradation
pathway upon interaction with ligand. The C1r and C1s in the case of Factor I plus
the classic pathway and mannan-associated serine protease-2 (MASP-2) C3b iC3b + C3f
in the case of the lectin pathway are wrapped around the stalk of these cofactor protein
tulip-like structures. It is the nonantigen or Fc portion of immunoglobulin
(Ig) G and IgM that engage the C1q subcomponent of C1. Factor I plus
iC3b C3dg + C3c
CRI
ALTERNATIVE PATHWAY
Proteases
The AP was the second complement activation pathway C3dg C3d + C3g
discovered (hence the name), but it is phylogenetically the
most ancient.1,2 In contrast to the CP and LP, the AP does Figure 19-7  C3 activation and degradation. The boxed activation
fragment is the one that remains covalently attached to the target. The
not require antibody or a lectin to be triggered. It likely plays other fragments are released into the surrounding milieu. C3a is a potent
a larger role in rheumatic diseases than previously appreci- anaphylatoxin and kills bacteria directly. C3f may be a bone marrow– 
ated because of its so-called feedback or amplification loop.14 mobilizing factor for neutrophils. C3c and C3g have no known function.
Thus, if C3b is deposited on a target by the CP or LP, the
AP can enhance C3b deposition many-fold. The AP is also It is stabilized by properdin (P). The catalytic serine protease
continuously active at a low level (like an idling car). The Bb of the C3bBbP enzyme complex then cleaves C3 to C3a
plasma inhibitors factor H and factor I and host cell surface and C3b; thus, a feedback loop is set up. Some of the newly
regulators keep the system in check. In the absence of these generated C3b molecules bind covalently to C3b already
regulators, as exemplified by inherited deficiencies, the sys- deposited on the target to form a C5 convertase (C3b2Bb).
tem fires to exhaustion. The AP routinely “kicks into gear” In vivo, the C3 and C5 AP convertases are short-lived owing
in the setting of microbes. If C3b deposits on an activating to spontaneous decay, unless stabilized by properdin.
surface (i.e., one that lacks regulators), the system’s feedback
loop allows several million C3b molecules to be deposited
MEMBRANE ATTACK COMPLEX
in a few minutes. In this pathway, factor B, structurally and
functionally homologous to C2, binds to the deposited C3b The terminal pathway begins with the cleavage of C5 by
and is then cleaved by the constitutively active factor D to either the CP-LP or the AP C5 convertase. The liberated
Bb and Ba. C3bBb is the AP C3 convertase (see Fig. 19-6). C5a fragment is a potent cell activator (anaphylatoxin) and
 328 Atkinson  |  Complement System

chemotactic factor. The C5b binds C6, which in turn engages DAF: Decay accelerating activity
C7 to yield the fluid phase C5b67. This lipophilic complex C4b
can now interact with cell membranes. Following membrane C2a
insertion, it next binds C8. The C5b678 complex forms an Binding
initial pore in the plasma membrane and then recruits mul-
Decay
tiple (5 to 10) C9 molecules to yield C5b6789. This complex DAF
DAF
is an efficient membrane channel former and is responsible
for cell lysis. Many organisms and cell types are not easily
C4c
lysed, however. The MAC has multiple nonlytic effects on
MCP: Cofactor activity
cells as well. The majority of these lead to activation of sig-
naling pathways, as has been seen in the case of neurons and
kidney cells. Some of the organ dysfunction seen in SLE,
especially if transient or correctable with treatment, may be Binding Factor I C4d
secondary to these nonlytic effects of the MAC.
MCP MCP MCP

REGULATORS
PHYSIOLOGIC REGULATION A

Unregulated, the complement system would fire to exhaus- DAF: Decay accelerating activity
tion, as illustrated by inborn errors of plasma regulatory C3b
proteins.15-17 The complement system has evolved to allow Bb
unimpeded activation on a microbe but to limit the proc­ Binding
ess in time and space. The reaction must be finite in time Decay
to avoid excessive consumption of components in any one DAF DAF Membrane
reaction and finite in space to minimize damage to surround-
ing host tissue. Thus, a typical complement reaction on a
microbial target occurs within a few minutes, and during MCP: Cofactor activity
this time, self-tissue is protected by plasma and membrane C3f
regulators. Many of these inhibitors act at the critical step
of convertase formation. The convertase enzyme complexes
Binding Factor I
intrinsically have short half-lives, and many of the inhibi-
tors act to prevent their formation or to dissociated (decay)
MCP MCP MCP
already formed complexes.

FLUID-PHASE AND MEMBRANE INHIBITORS B

These inhibitors, distributed both in plasma and on cells,
regulate primarily at the initiating events (see Fig. 19-3),
the formation of the C3 and C5 convertases (see Fig. 19-6),
and the insertion of the MAC. A major function of the
plasma inhibitors is to prevent fluid-phase activation (no
target), while the membrane inhibitors prevent activa-
tion on self-tissue (wrong target). In some situations,
the plasma inhibitors bind to damaged cells and exposed
extracellular matrix, where they now function as a mem-
brane regulator.7,8,18
C1 inhibitor is a serine protease inhibitor (SERPIN)
and binds to C1r and C1s and to MASPs. The C1 com-
plex is thereby disrupted, leaving C1q bound to antibody
in the IC. C1q may then interact with C1q receptors to
facilitate disposal of the IC. C1 inhibitor also prevents
excessive and chronic fluid-phase C1 activation.19 The C3
and C5 convertases are regulated by a family of proteins
that include the membrane proteins decay-­accelerating
factor (DAF, or CD55) and membrane cofactor protein
(MCP, or CD46) and the serum inhibitors C4-binding
protein and factor H.7-9,15-17 These proteins act in three
ways: they prevent convertase assembly, disassemble
convertases (decay-accelerating activity; Fig. 19-8A),
and serve as cofactors for the proteolytic inactivation of
C4b and C3b (cofactor activity; Fig. 19-8B). The latter
Figure 19-8  Regulation of convertases. A, Classic pathway C3 conver-
tase. Decay-accelerating factor (DAF) irreversibly displaces the protease
C2a from the anchoring piece C4b. C4b is cleaved by factor I. This reac-
tion requires a cofactor protein, membrane cofactor protein (MCP); C4bp
in plasma and CR1 can serve this role. The residual bound C4d has no
known biologic activity. Classic pathway C5 convertase is similarly inac-
tivated. B, Alternative pathway C3 convertase. The alternative pathway
C3 and C5 convertases are disassembled in a similar fashion by DAF and
MCP. In the case of C3b, it is cleaved to iC3b and then to C3dg. C3dg and
C3d serve as adjuvants to promote an immune response. They accom-
plish this by reacting with the complement receptor 2 (CR2; CD21) on
follicular dendritic cells and B lymphocytes. These two activities of DAF
and MCP are synergistic in their control of the convertases. They prevent
complement activation in blood and on normal host cells and tissues.
­process is mediated by the plasma serine protease factor I.
The MAC is also regulated by plasma and cell-anchored
protein. CD59 is a widely expressed glycolipid-anchored
membrane protein that binds C8 and C9 to prevent their
membrane insertion, while the plasma protein vitronec-
tin (S protein) inactivates fluid-phase MAC.
As a result of this regulatory activity, complement attack
is focused on foreign surfaces (which usually lack comple-
ment regulators) and is held in check on host cells and
in body fluids. Interestingly, a number of microorganisms
have “captured” complement regulators (e.g., pox viruses)
or have evolved proteins (herpesviruses) that inhibit
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
­complement activation (virulence factors).20 However, the
activation of the CP by antibody is extremely rapid and
efficient, such that the inhibitors generally have modest
effects on limiting damage by complement-fixing antibod-
ies. The host inhibitors, however, are efficient at prevent-
ing fluid-phase activation and especially amplification via
the feedback loop.
COMPLEMENT RECEPTORS
The complement system exerts many of its effects through
receptors (see Tables 19-3 to 19-5). During the activation
process, target-bound and locally released fragments serve
as ligands for complement receptors. For example, the vaso-
modulatory and chemotactic effects of C3a and C5a are due
to interaction with their respective receptors. The opsonic
fragments C4b and C3b mediate clearance of ICs and bac-
teria through adherence and phagocytosis to CR1. Degrada-
tion of C3b by regulators leads to the formation of iC3b and
then C3dg, which in turn interact with CR3/CR4 and CR2,
respectively.
CR1 plays an important role in IC clearance. On eryth-
rocytes, CR1 binds C3b/C4b-coated ICs (the immune
adherence phenomenon) for processing and transport to
the liver and spleen.21 In these organs, the ICs are trans-
ferred from the erythrocyte to tissue macrophages, allow-
ing the erythrocyte to return to the circulation for another
round of clearance. CR1 on granulocytes and monocytes
binds and ingests ICs, whereas CR1 on B lymphocytes, tis-
sue macrophages, and follicular dendritic cells facilitates
trapping and processing of IC in lymphoid organs. CR2 is
expressed by B lymphocytes and follicular dendritic cells,
where it facilitates antigen trapping and is a coreceptor
for activation of the B cell antigen receptor.22 Several new
receptors for C3-bearing ICs have been described.23,24 The
identification and role of C1q receptors remain problem-
atic, but they are likely important in the proper handling
of antigens that have undergone complement activation
on their surface.25
Table 19-6  Complement System in Immunity
First line of defense as part of innate immune response (takes place
within seconds)
  Mediates inflammatory response
  Modifies membranes of microbes
Instructive role to adaptive immunity
  Facilitates antigen identification, processing, transportation, and
retention
  Activates cells to synthesize costimulatory molecules and to
secrete cytokines and other mediators of an immune response;
lowers threshold for B lymphocyte activation
Effector arm of humoral immunity (“complements” antibody)
Recognition of injured, apoptotic, and necrotic cells to enhance
cleanup, proper disposal (without an adaptive immune response),
and wound healing
Recognition of extracellular debris (crystals, pigments, lipids,   Target
proteins)*
*Examples are urate crystals in gout, lipofusion pigments in drusen in
age-related macular degeneration; oxidized lipids in atherosclerosis, and
amyloid in Alzheimer’s disease.
329
COMPLEMENT IN THE INNATE
AND ADAPTIVE IMMUNE RESPONSE
INNATE IMMUNITY
The complement system is activated by at least three mech-
anisms that are independent of an adaptive immune response
(Table 19-6): natural antibodies, lectins, and the AP itself
(Fig. 19-9). Complement activation is therefore one of
the earliest reactions to microbes at sites of infection. The
complement response opsonizes organisms for adherence,
phagocytosis, and antigen processing while releasing frag-
ments to activate immunocompetent cells and trigger an
inflammatory milieu.
ADAPTIVE IMMUNITY
As pointed out earlier, complement was discovered because
of its role as an effector arm of humoral immunity. IgM and
IgG subclasses 1 and 3 efficiently activate the CP. More
recently, an important role for complement on the afferent
side has been “rediscovered.”3,22
Accumulating evidence indicates that complement is
an instructor of the adaptive immune response. Nearly
30 years ago, the injection of cobra venom factor was
used to destroy an animal’s complement activity. In such
experimental systems, a requirement for complement in
an animal’s immune response was clearly demonstrated.
An attenuated IgM response, a lack of class switching from
IgM to IgG, and a failure to generate memory B cells were
features of a complement-deficient animal. Similarly, in
multiple subsequent studies, C4- or C2-deficient guinea
pigs and humans were shown to have a defective response
to the intravenous administration of a phage antigen.
Although they produced IgM antibody, these two species
did not class-switch from IgM to IgG antibody or dem-
onstrate immunologic memory. In other studies, block-
ing CR2 reduced the antibody response to T-dependent
antigens and prevented isotype switching. Further, ani-
mals with a targeted gene deletion of CR1/CR2 also had a
lower IgM response and failed to isotype-switch. If larger
doses of antigen or antigen plus adjuvants were adminis-
tered, the complement-deficient animals responded nor-
mally. Finally, coating of antigen with C3d increases its
immunogenicity up to 10,000-fold.22,26,27 Consequently,
vaccines containing complement-coated antigens are
likely to be more immunogenic. Taken together, these
data indicate a contribution of the CP and complement
receptors to an adaptive immune response.
Natural Ab
Alternative
Lectins
pathway
Figure 19-9  Activation of the complement system in innate immu-
nity. Shown are three means of complement activation in a nonimmune
host.
 330 Atkinson  |  Complement System

cells, especially erythrocytes (immune adherence). Erythro-

CLEARANCE OF DEAD, DAMAGED,
AND APOPTOTIC CELLS
The complement system likely plays an important role in
facilitating the removal of injured cells and cellular debris.5-8,28
Natural antibodies, lectins, and the AP recognize certain altered
surface characteristics of damaged cells and, through opsoniza-
tion, promote their proper removal. Such a system probably
evolved to efficiently remove injured tissue, especially trauma-
tized skin and apoptotic cells. In this process, a second goal is to
avoid an immune response. If this system fails, as might happen
in C1q or C4 deficiency, the individual is predisposed to devel-
oping autoantibodies (the so-called garbage- or waste-disposal
hypothesis to explain autoimmunity in SLE). Ischemia-reper-
fusion injury is a related situation in which complement acti-
vation has clearly been shown to mediate damage to viable but
at-risk tissues.29 Altered self-debris engages the innate immune
system. Examples are urate crystals in gout, amyloid proteins
in Alzheimer’s disease, oxidized lipids in atherosclerosis, and
lipofusion (pigments) in age-related macular degeneration. In
these chronic processes, relatively minor changes in comple-
ment regulatory activity may accelerate tissue damage. This
has been shown most clearly in age-related macular degenera-
tion, where about 50% of the genetic risk appears to be due to a
subtle loss of function in a polymorphic variant of factor H.30
IMMUNE COMPLEX CLEARANCE
The complement system is important for the processing and
clearance of ICs.6-9,21,31 As ICs form, activation of the CP
leads to C4b and C3b deposition, which in turn prevents the
ICs from precipitating in a vessel wall or tissue site (called
maintenance of IC solubility). The deposition of C3b on the
antibody and antigen reduces the antibody’s ability to cross-
link and thereby precipitate ICs. Even preformed ICs can
be solubilized by exposure to fresh serum. The soluble IC-
­bearing clusters of C3b become bound to peripheral blood
cytes possess more than 80% of the CR1 in blood. They
serve as a “taxi” or “shuttle” to transport the ICs to the liver
and spleen, where they are dissociated from the RBCs. Most
such ICs are then destroyed by macrophages in the liver
or spleen. This transfer of ICs from the red cells to tissue
macrophages may be mediated by simple affinity differences,
because tissue monocytes or macrophage possess multiple
types of C3 and Fc receptors. Further, there is evidence for a
proteolytic cleavage event at tissue sites that occurs near the
stalk of CR1 to release ICs. The RBCs return to the circula-
tion, possibly minus a few complement receptors, but ready
for another round of immune adherence. This processing
system for ICs evolved to prevent ICs from depositing in
undesirable locations, such as the kidney glomerulus. This
clearance process could fail due to (1) a CP component defi-
ciency, up to and including C3; (2) a complement receptor
deficiency; (3) synthesis of a non-CP fixing antibody, such
as IgG4 or IgA; (4) severe hepatic dysfunction; or (5) sple-
nectomy.31 This concept of IC clearance plays out in many
infectious diseases and is especially pertinent to syndromes
featuring ICs, such as SLE, mixed cryoglobulinemia, serum
sickness, and other vasculitic syndromes.
COMPLEMENT MEASUREMENT
Complement can be assessed by antigenic and functional
assays (Tables 19-7 and 19-8). In clinical practice, the most
common functional measurement is the total hemolytic or
whole complement assay (THC or CH50). The assay is based
on the ability of the patient’s serum sample to lyse sheep
erythrocytes optimally sensitized with a rabbit antisheep cell
antibody. All nine components of the CP (i.e., C1 through
C9) are required for a normal THC. A result of 200 units
indicates that at a dilution of 1:200, the test serum lysed
50% of the antibody-coated sheep ­ erythrocytes. THC is a
useful screening tool for detecting a homozygous deficiency

Table 19-7  Assays for Complement Activation in Human Disease

Method Use Comments
CH50 or THC Screen for component deficiency or activation of Functional assay—requires appropriate sample
classic pathway handling
AP50 Screen for component deficiency or activation of Functional assay—requires appropriate sample
alternative pathway handling
Antigenic (ELISA, immunodiffusion, Standard method for C3, C4, factor B, C1 inhibitor, Widely available, easy to perform, reliable, inexpensive
nephelometry) MBL, and factor H
Antigenic or hemolytic assay of an Further define a suspected deficiency Samples usually sent to labs specializing in comple-
individual component ment assays
Activation fragments C3a, C5a, Bb, May be elevated in the setting of normal comple­ Expensive; sample collection important; commercial
C1r/C1s, C5b-9 (neoantigen) ­ ment levels labs often perform these assays; more sensitive than  
assays of static levels
C1 inhibitor function Clinical picture consistent with HAE, but C1 inhibitor 15% of HAE kindreds have normal or elevated levels of
levels by antigenic assay are normal or elevated a nonfunctional protein
Immunofluorescence Demonstration of complement activation fragments C1q and cleavage fragments of C4 and C3 are most
in tissue commonly analyzed, especially in kidney and skin
biopsies
Antiglobulin testing (non-gamma Demonstration of C3 fragments on erythrocytes in Usual fragment detected is C3d
Coombs) hemolytic anemias
AP50, alternative pathway equivalent; CH50 or THC, total hemolytic assay for classic pathway; ELISA, enzyme-linked immunosorbent assay; HAE, hereditary
angioedema; MBL, mannan-binding lectin.
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION 331

Table 19-8  Interpretation of Results of Complement Determinations

THC (units/mL) C4 (mg/dL) C3 (mg/dL) Interpretation
150-250 16-40 100-180 Normal range
250 40 200 Acute-phase response (all are high)
100 10 80 CP activation (low C4 and C3)
100 30 50 AP activation (normal C4 and low C3)
<10 or 0 30 140 Inherited deficiency or in vitro activation*
50 <8 100 Partial C4 deficiency or fluid-phase activation†
*In vitro activation is more common than an inherited deficiency state. The lack of activity (THC <10) in the setting of normal C4 and C3 antigenic levels
­suggests (1) an improperly handled sample, (2) cold activation (e.g., by cryoglobulins) following collection of the sample, or (3) homozygous component
deficiency (most commonly C2 with a lupus presentation or, with a Neisseria infection, an AP or membrane attack complex component).
†
Because THC is detectable, this cannot be a complete deficiency of C4. A partial C4 deficiency, such as of C4A, could give this result. Some types of immune
complexes, especially cryoglobulins, and a deficiency of the C1 inhibitor (hereditary angioedema) also give this pattern. In these cases, measurement of C2 is
often helpful: a low value suggests activation, whereas a normal value suggests an inherited, partial C4 deficiency. Also, C4A and C4B alleles can be assessed
by commercial laboratories.
AP, alternative pathway; CP, classic pathway; THC, total hemolytic complement or CH50.
Modified from Primer on the Rheumatic Diseases, 12th ed. 2001, p 71.

Table 19-9  Clinical Manifestations of Complement Gaining in use are tests for the detection of activation
Component Deficiency fragments (e.g., C3a, C5a, C4d, C3d, Bb). Their clinical
Deficient Component Clinical Syndrome*
utility relates to the fact that they are dynamic parameters
and thus reflect ongoing turnover of the system. Also, they
Classic Pathway
are not affected by partial inherited deficiencies or altera-
C1q SLE, infections tions in synthetic rates. However, they are more costly,
C1r/C1s SLE, infections
C4 SLE, infections
not as widely available, and unnecessary in most clinical
C2 SLE, infections situations. A potential advance in this area of biomark-
ers is the measurement of C4 and C3 fragments bound to
Lectin Pathway
RBCs, platelets, and lymphocytes.32 Analogous to monitor-
MBL Infections ing blood glucose by measuring HbA1c in diabetes mellitus,
Central component
  C3 Severe infections, GN, SLE the magnitude of complement activation is proportional to
Membrane attack component the quantity on the cell surface. Thus, RBCs reflect disease
  C5, C6, C7, C8 or C9 Neisseria infections activity over the past several months while platelets reflect
Alternative Pathway disease activity over the past week. Longitudinal studies are
Properdin, factor D Neisseria infections
in progress to assess the utility of this approach in rheumatic
diseases featuring complement activation.
*With early component deficiencies of the classic pathway (C1, C4, or C2),
infections are caused by the commonly encountered pyogenic organisms.
With a late component (C5–C9) or an alternative pathway component COMPLEMENT DEFICIENCY
­deficiency, Neisseria infections predominate, especially meningococcal
­infections. Inherited deficiencies of complement components predis-
GN, glomerulonephritis; MBL, mannan-binding lectin; SLE, systemic lupus pose to bacterial infections (this was expected) or to autoim-
erythematosus.
munity (this was unexpected), especially SLE7-9,28,33 (Tables
19-9 to 19-11). Most deficiencies are inherited as autosomal
of a single component (C1 through C8), because total defi- codominant (recessive) traits, except for the C1 inhibitor,
ciency of a component produces a result of less than 10 units which is autosomal dominant, and properdin and factor D,
or undetectable THC. Deficiency of C9 results in a low but which are X-linked. A thorough analysis of this subject has
detectable THC. THC can screen for total deficiency as well been published, including tables listing every reported case
as provide an overall assessment of complement activation. of early complement component deficiency in humans.28
It is particularly recommended in the initial evaluation of Several other pertinent reviews are also available.33-36
most SLE patients.
Other commonly used tests, especially to follow a patient’s CLASSIC PATHWAY
clinical course, are antigenic assays for C4 and C3. They
are widely available, relatively inexpensive, and simply and The prevalence of lupus in homozygous C1q, C4, or C2
accurately measured by nephelometric-based immunoassays. deficiency is 90%, 75%, and 15%, respectively.28,33-36 The
They are useful in the initial diagnosis of lupus and related female-male ratio is approximately 1:1 in C1q or C4 defi-
syndromes and for following the course of patients undergo- ciency but 7:1 in C2 deficiency. Concordance rates for SLE
ing treatment, particularly if the concentrations were reduced are 2% among dizygotic twins and 24% among monozy-
when the disease process was active. Upon treatment, a gotic twins; concordance of SLE between siblings with
return to normal values correlates with clinical improvement C1q, C4, or C2 deficiency is 90%, 80%, and 58%, respec-
and bodes a better outcome. Table 19-8 provides examples tively. These complement-deficient individuals with SLE
of serum complement test results and their interpretation in usually present before age 20 years and often have promi-
rheumatic diseases. nent cutaneous (90%) and renal (50%) manifestations.
 332 Atkinson  |  Complement System
Table 19-10  Autoimmune Disorders in Complete Complement Component Deficiencies
Component Number of Cases
C1q 35
C1r/s 11
C4 24
C2 110
C3 21
*Infections may be the presenting manifestation and dominate the clinical course, particularly in C1q, C4, or C2 deficiency.
†
A few cases of Sjögren’s syndrome, systemic sclerosis, Henoch-Schönlein purpura, and other immune-mediated syndromes have been reported with C2
deficiency.
GN, glomerulonephritis; SLE, systemic lupus erythematosus.
Table 19-11  Classic Pathway Complement   system ­manifestations but more cutaneous (prominent pho-
Component Deficiency Leading to Autoimmunity
There are two leading hypothesis; they are not mutually exclusive.
1.  Defective immune complex handling
  For foreign antigens
  For self-antigens (apoptotic and necrotic debris)
2.  Defective immune responses
  Reduced humoral immune responsiveness
  Failure to regulate autoreactive B cells
Antinuclear antibody tests are positive in about 75% of
patients. Most patients are DNA negative, and antibodies
to extractable nuclear antigens (especially antibodies to
Ro) are detected in about 70%. The disease process tends
to be more severe in C1q and C4 than in C2 deficiency.
Less than 1% of lupus patients have a complete comple-
ment component deficiency, with C2 deficiency being the
most common.
About one third of SLE patients develop C1q deficiency
secondary to anti-C1q antibody. These antibodies probably
develop after the onset of SLE and are therefore a secondary
phenomenon.28,37 These patients tend to have renal disease
and low complement levels (C4 and C3).
The C4 genes are duplicated (all four must be deleted
or defective to result in total C4 deficiency), giving rise
to C4A (acidic) and C4B (basic) types.38 C4A deficiency
occurs in about 15% of Caucasian SLE patients (com-
pared with 1% to 3% of controls). Because C4A prefer-
entially binds amino groups, it reacts more efficiently with
some types of ICs than does C4B. This may explain why
homozygous C4A deficiency is an independent risk factor
for SLE. The association generally occurs across multiple
ethnic backgrounds as well as in the absence of the Euro-
pean ancestral autoimmune haplotype (HLA-A1, C7, B8,
C4AQ*0, C4B1, DR3, DQ2). The clinical course is simi-
lar to that of idiopathic lupus, but there may be less renal
disease.39-41 In fact, one group has argued for caution in the
use of immunosuppressive therapy because of the generally
favorable course of renal disease in most patients.39 Assess-
ment of the number of C4A and C4B genes is available
through commercial laboratories.
The most common homozygous complement deficiency
is C2 deficiency (occurring in 1 in 10,000 to 20,000 indi-
viduals).28 Heterozygotes are present in 1% to 2% of the
Caucasian population. SLE occurs in about 15% of homo-
zygous C2 null individuals. Notable differences in the set-
ting of C2 deficiency are fewer renal and central nervous
Dominant Phenotype (%) Other Phenotype*
SLE (94) Infections
SLE (55) Infections
SLE (75) Infections
SLE (32) Infections†
Severe infections (75) GN, SLE, vasculitis
tosensitivity) involvement, earlier age of onset, and higher
frequency of anti-Ro antibodies. Heterozygosity for C2 defi-
ciency does not appear to predispose to SLE.
ALTERNATIVE PATHWAY
C3 deficiency is associated with recurrent pyogenic infections
and, less commonly, glomerulonephritis. For the latter, anti-
nuclear antibodies are usually not detected, and other systemic
features of SLE are lacking. Deficiency of properdin, factor
B (one case), and factor D (a few cases) is strongly associated
with meningococcal infections but not autoimmunity.42
LECTIN PATHWAY
Polymorphisms producing low serum MBL are associ-
ated with SLE across several ethnic backgrounds. In
one series, the incidence of SLE was increased twofold
in patients with MBL deficiency,43 but this association
does not appear to be holding up. Impressively, infectious
complications, particularly severe pneumonias in associa-
tion with immunosuppressive therapy, were much more
frequent in MBL-deficient patients. This combination of
MBL deficiency and C4 gene deficiency may be a particu-
larly strong predisposing factor for SLE. In RA, MBL defi-
ciency may be a modest risk factor.44 Of perhaps greater
interest, MBL deficiency was associated with earlier age
of onset, more severe erosive disease, and increased fre-
quency of infectious complications during treatment.
Again, as more studies have been reported, these associa-
tions have become controversial.
ACQUIRED COMPLEMENT DEFICIENCY STATES
Acquired complement deficiency usually results from accel-
erated consumption. CP activation is observed in more
than 50% of lupus patients. Generally, the more active the
disease process, the more likely that complement levels will
be low as consumption progressively outstrips the liver’s
synthetic capacity. Low complement values (C4, C3, or
THC) are a poor prognostic factor and correlate with anti-
bodies to native DNA, nephritis, and, overall, more severe
disease. Complement deposition in tissue, especially in the
glomerulus (as detected by immunofluorescence), assists in
the diagnosis and classification of lupus nephritis. Anti-
bodies to C1q occur in about 30% of SLE patients and are
associated with low complement concentrations and renal
disease.28,37
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
C3 nephritic factor is an autoantibody against the AP
C3 convertase. The autoantibody stabilizes the convertase
to the point that excessive C3 cleavage and a secondary
deficiency of C3 result. Patients with C3 nephritic factor
are predominantly children, who may present with glomer-
ulonephritis, partial lipodystrophy, or frequent infections
with encapsulated bacteria. In many cases, an underlying
deficiency of factor I or factor H predisposes to excessive
AP turnover, leading to the secondary formation of an auto-
antibody to the C3 convertase.
KEY POINTS: SYSTEMIC LUPUS
ERYTHEMATOSUS AND RELATED SYNDROMES
Deficiency of C1q, C4, or C2 is the only single-gene defect that
causes SLE in humans.
Partial deficiency of C4 also predisposes to SLE.
Once autoantibodies are present and ICs are deposited in
undesirable tissue sites, such as the kidney, complement acti-
vation contributes to the inflammation and tissue damage.
Complement measurements are helpful in following ­lupus
­patients, particularly if C4 and C3 were low at the time of
­diagnosis. If so, the levels usually increase in response to
treatment and may become a useful marker of disease
­activity. However, this point has to be established for each
patient.
The complement system is activated in most SLE patients,
even though serum levels are not always reduced. Evidence
for this statement comes from the assessment of complement
turnover studies, the measurement of activation fragments,
and, most convincingly, the demonstration of complement
fragments in more than 70% of patients on skin biopsy (the
old lupus band test). Even in normal skin, there is a remark-
able deposition of C3 fragments at the dermal-epidermal
junction.
Anti-C1q antibodies are present in about 30% of SLE patients
and in about 70% of lupus patients with nephritis. They prob-
ably arise in response to the high quantities of C1-fixing IC.
They likely contribute to the hypocomplementemia that is
common in such patients, as well as to glomerular damage.
In mouse models of SLE, decreased activity of the early
components of the CP accelerate disease development, while
deficiencies of the late components (C5 to C9) ameliorate
disease severity. The literature in this area is not easy to inter­
pret because the models may or may not accurately reflect
­human disease, and the mouse complement system has many
­differences from its human counterpart. For instance, mouse
complement is a less potent activating system overall, and Fc
receptors seem to be responsible for more of the proinflam-
matory reaction in mice than in humans.
MODELS OF INFLAMMATORY DISEASE
FEATURING COMPLEMENT ACTIVATION
In general, in vivo models have explored complement acti-
vation as a mediator of tissue damage and inflammation in
diseases featuring complement as an effector arm of humoral
immunity.7-9,14 More recently, complement’s role in promot-
ing inflammation and cellular migration, enhancing the
afferent limb of the immune response, and handling debris
(including apoptotic cells) have received attention.
333
DEFICIENT ANIMALS
Before knockout mice were available, guinea pigs deficient
in C4, C2, or C3; rats and rabbits deficient in C6; dogs defi-
cient in C3; and pigs deficient in factor H had been char-
acterized.3 Further, in the 1960s, many inbred mouse strains
were shown to be deficient in C5. Experiments with these
species clearly indicated the double-edged sword aspect of
the complement system. The deficient animals were more
sensitive to challenges with bacteria but were more resistant
to antibody-mediated tissue damage. They were also widely
used to determine the role of complement in inflammatory-
immune models—the Arthus reaction, serum sickness, and
Forssman shock. These models are discussed briefly because
they demonstrate the range of reactions requiring comple-
ment activation.
In the Arthus model, antibodies to a foreign antigen are
raised, and then the antigen is reinjected into the immune
host. If the antigen is injected in a joint, ICs form in the
joint space, complement is activated, Fcγ receptors are
engaged, and an inflammatory reaction ensues. It is a tran-
sient and nondestructive process unless antigen is injected
repeatedly.
Emphasis has recently been placed on separating the
effects of IgG antibody working through Fcγ receptors
(FcγRs) on phagocytes and B cells from those induced by
antibody-activating complement.3,14,45-48 For example, it had
generally been believed that the Arthus reaction was medi-
ated by complement activation. The immigration of neu-
trophils in this process was thought to be caused primarily
by the local formation of C5a. This concept was challenged
when it became possible to generate, by homologous recom-
bination, mice with C3 deficiency or FcγR deficiency. In the
mouse, FcγRIII but not complement was required for the
Arthus reaction in skin. However, complement was critical
in IC-mediated peritonitis in mice and IC-mediated lung
disease in rats. Interestingly, complement is also of major
importance in IC-mediated skin disease in guinea pigs and
rats. Thus, the species, the tissue site of activation, the
genetic background, and undoubtedly additional factors are
critical in determining the importance of complement ver-
sus FcγR in disease pathogenesis. In most cases, these two
effector systems operate conjointly, both in the response to
pathogens and in autoimmunity.
In the usual serum sickness model in the rabbit employ-
ing bovine serum albumin(BSA)–anti-BSA, a polyarthritis
develops that is mediated by the deposition of IC in joints. In
the preantibiotic era, a serum sickness reaction was observed
in patients receiving horse serum containing antibodies to
treat infectious diseases. The human or rabbit clinical ill-
ness lasted 1 to 2 weeks, resolving as the host went into
antibody excess and cleared the foreign protein. Of course,
if more antigen was injected, an accelerated serum sickness
reaction developed. These conditions are likely to be met in
SLE and in mixed cryoglobulinemia secondary to chronic
hepatitis B or hepatitis C infection. In these conditions,
nuclear antigens from cellular turnover and viral antigens
synthesized in the liver are continuously present in a host
with preformed antibodies.
Forssman antigen is a widely distributed lipopolysaccha-
ride.3 Species that are Forssman negative, such as the rab-
bit, develop an immune response to the RBCs of sheep, a
 334 Atkinson  |  Complement System
Forssman-positive species. The rabbit responds to the injec-
tion of sheep RBCs with high-titer anti-Forssman antibody.
Upon intravenous injection of this rabbit antibody into
guinea pigs, a Forssman-positive species, the antibody trav-
els to the lung, where it causes alveolar capillary fluid leak-
age and hemorrhage that is CP dependent. Phagocytes are
not important in this cataclysmic reaction that causes death
in a few minutes secondary to pulmonary edema. The lytic
activities of complement are essential, however. This model
and the Arthus reaction have been used to test the effect of
anticomplement reagents in preventing tissue damage.
GENE TARGETED DEFICIENCIES
Most components, regulators, and receptors have now been
deleted by gene targeted insertional mutagenesis.6-8,14,33,49,50
C1q, C4, C2, C3, and CR1/CR2 mice demonstrated sub-
stantial defects in T cell–dependent immune responses
and have the expected defects in IC clearance. Thus, for
most strains with a defect in the CP or AP, defective clear-
ance of bacteria and viruses was shown. The C1q- and
C4-deficient mice (depending on the genetic background)
have enhanced humoral immunity, glomerulonephritis
(lupus-like picture), and problems in the clearance of apop-
totic bodies. The C5aR-deficient mice have a decrease in
inflammatory cell infiltrates. Although there are substantial
differences between the human and mouse complement
systems, these mice have been useful in clarifying comple-
ment’s role in host defense against infection, the immune
response, and autoimmunity.
ANTIPHOSPHOLIPID ANTIBODY SYNDROME
The antiphospholipid syndrome is often associated with
hypocomplementemia in the absence or presence of a posi-
tive test for antinuclear antibodies. In a recent review of
a large number of such patients, hypocomplementemia was
just as frequent as in patients with SLE.51 Complement acti-
vation is also a common event in recurrent pregnancy loss.52
In a mouse model of antiphospholipid antibody–induced
pregnancy loss, complement-deficient mice were protected
from disease development.53 Complement activation by the
antiphospholipid antibody is a required intermediary event
in the pathogenesis of fetal injury. In particular, complement
activation by the autoantibodies causes a dysregulation of
angiogenic factors such as vascular endothelial growth fac-
tor.54 Clinical trials are under way to assess the role of com-
plement activation and angiogenesis in patients with the
antiphospholipid syndrome.
COMPLEMENT ACTIVATION IN POLYARTHRITIS
Human Rheumatoid Arthritis
A long-standing observation in RA and juvenile RA is that
complement is activated locally, in the synovial fluid and
surrounding joint tissue.14,55,56 Thus, if the functional activity
of joint fluid C4, C2, or C3 is determined, it is low compared
with that obtained for a concomitant antigenic level. Such
data have established that complement components in RA
joint fluid are antigenically intact but functionally inactive.
In other words, the components have already been engaged
in an immune reaction, and activation fragments are largely
being measured. Further, essentially every complement acti-
vation fragment is elevated in RA joint fluid. The activa-
tion profile is primarily that of the CP (low C4 and C2),
although there is also substantial evidence for contribution
of the AP. Clinical correlations with measures of comple-
ment activation in RA include a more severe, erosive, and
seropositive disease process. From about 1950 to 1980, B
cells, rheumatoid factors, and complement were postulated
to play a predominant role in mediating the synovial inflam-
mation in RA. Then, for the next 2 decades, T cell–medi-
ated immunity was thought to be the predominant system
responsible for the synovial reaction in RA. Today, a more
prominent role for B cells, autoantibodies, and complement
is again thought to exist; their critical role in animal models
of RA has been recognized, and antibodies to citrullinated
peptides have been identified. Both humoral and cellular
immunity are almost certainly required to produce a persis-
tent synovitis in the RA syndrome.
KEY POINTS: RHEUMATOID ARTHRITIS
AND RELATED SYNOVITIDES
Complement activation fragments are present in joint fluid
and deposited in synovial tissue.
Autoantibodies (e.g., to citrullinated peptides) may activate
the complement system to trigger inflammation.
Rheumatoid factor augments the effects of IgG bound to
an antigen by promoting both agglutination of the IC and
complement activation.
Low C4 and C3 are common in rheumatoid vasculitis, indicat-
ing CP activation by ICs; similarly, cryoglobulins and other
types of ICs activate the CP.
In mouse models of rheumatoid arthritis featuring autoanti-
bodies, complement inhibitors and complement deficiency
protect against disease development and reduce its severity.
Collagen-Induced Arthritis
Complement activation products are prominent in the joint
fluid and synovial tissue of rat and mouse models of col-
lagen-induced arthritis.14,56-58 The disease is mild or absent
in C5-deficient mice, mice treated with a monoclonal
antibody to C5, mice treated with the potent complement
inhibitor soluble CR1, and factor B–deficient mice.49,59 The
C5 monoclonal antibody treatment also ameliorated estab-
lished disease. These results implicate the AP in generating
C5a, C5b to C9 (MAC), or a combination thereof as being
responsible for synovitis. One might have predicted that C3
activation products would be major contributors to disease
pathogenesis, but that is not the case.
Mouse Model
A spontaneous mouse model was generated by crossing the
T cell receptor (TCR) transgenic mouse line (known as
KRNxC56BL/6) with the nonobese diabetic (NOD) mouse
strain.60,61 Autoantibodies that arise in the K/BxN mouse
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
Table 19-12  Complement Activation Inhibitors Undergoing Clinical Trials
Product Description
TP10 Soluble recombinant CR1 (sCR1)
H5G1.1; h5G1.1-scFv Humanized, high-affinity anti-C5   Blocks cleavage of C5 by C5   Alexion Pharmaceuticals
mAb; single-chain version
CA, cofactor activity; DAA, decay-accelerating activity; mAb, monoclonal antibody.
recognize the ubiquitously expressed enzyme glucose-6-
phosphate isomerase. Important features of this model are as
follows: (1) a destructive small-joint arthritis develops that
resembles RA; (2) the autoantibodies are pathogenic, as the
disease can be transferred to other strains by antibody alone;
and (3) complement is required for the arthritis to develop.
Surprisingly, the CP is not necessary; mice deficient in C1q or
C4 are as susceptible as wild-type mice. Mice deficient in C3
or AP component factor B, however, did not develop arthritis.
Moreover, C5-deficient mice or mice treated with a monoclo-
nal antibody to C5 were resistant to the disease. C5a recep-
tor–deficient mice were protected from developing arthritis.
Thus, AP-derived C5a interacts with its receptor to mediate
the complement effect required for disease development.
Another insight from this model is a putative role for
the complement regulatory proteins in “allowing” a local
inflammatory process.60 Thus, the pathogenic antibody in
this model also binds to the kidney glomerulus, but there
is no excessive C3b deposition and no development of glo-
merulonephritis. A likely explanation is that both antigen
accessibility and a permissive local environment for com-
plement activation are required. Joint cartilage is relatively
devoid of complement regulatory proteins. In this setting,
the AP is engaged. The CP is not activated because the
autoantibody is predominantly of the IgG1 subclass, which,
in the mouse, has poor complement-fixing ability. Neverthe-
less, antibodies bound to an antigen can protect the cascade
from regulation by inhibitors and thereby trigger the AP.62
Though it remains unclear why the autoantibody arises, this
model has provided provocative insights into how an auto-
antibody- and complement-mediated inflammatory arthri-
tis could occur in humans. Further, the recent discovery of
an autoantibody system in about one third of human RA
patients (anticitrullinated peptide antibodies) provides a
parallel with the mouse model system.63
THERAPEUTIC IMPLICATIONS
The study of the complement system has undergone a
renaissance. Much of this revival relates to (1) the devel-
opment of inhibitors for experimental and clinical use;
(2) the association of deficiency states with autoimmunity
(especially the C1q, C4, C2–lupus association); (3) greater
appreciation of the role of the innate immune system and
its role in instructing the adaptive immune system; (4) the
availability of gene targeted mice to more precisely assess
the role of complement in the normal immune response and
in mouse models of autoimmunity; and (5) whole genome
screens with single nucleotide polymorphism analyses that
have identified partial deficiencies and polymorphisms in
complement regulatory proteins as causing a predisposition
to atypical hemolytic uremic syndrome64 and age-related
macular degeneration.30
335
Actions Company
Degrades C3b/C4b (CA) and decays   Avant Immunotherapeutics  
C3/C5 convertases (DAA) ­(Needham, Mass)
 
conver­­­­tases (New Haven, Conn)
The development of complement inhibitors for clinical
use is a long-sought goal. One such compound, a humanized
monoclonal antibody to C5, has been used successfully to treat
a complement-mediated hemolytic anemia65-68 (Table 19-12).
This agent and others ameliorate inflammatory responses in
animal models, where considerable evidence has already
been accumulated relative to complement’s participation
(e.g., Arthus reaction, serum ­ sickness, SLE). Complement
inhibition, however, also reduced infarct size in experimental
models of myocardial infarction and tissue damage in many
other types of ischemia-reperfusion injury.29 Further, a role for
complement in clearing apoptotic cells is likely.28 A comple-
ment inhibitor for clinical use is much anticipated.
REFERENCES
  1. Zarkadis IK, Mastellos D, Lambris JD: Phylogenetic aspects of the
complement system. Dev Comp Immunol 25:745-762, 2001.
  2. Nonaka M, Kimura A: Genomic view of the evolution of the comple-
ment system. Immunogenetics 58:701-713, 2006.
  3. Frank MM, Atkinson JP: Complement system. In Austen KF,
Frank MM, Atkinson JP, et al (eds): Samter’s Immunologic Dis-
eases, 6th ed. Philadelphia, 2001, Lippincott Williams & Wilkins,
pp 281-299.
  4. Silverstein AM: A History of Immunology. San Diego, Academic
Press, 1989.
  5. Nauta AJ, Daha MR, van Kooten C, et al: Recognition and clear-
ance of apoptitic cells: A role for complement and pentraxins.
Trends Immunol 24:148-154, 2003.
  6. Volanakis JE: Overview of the complement system. In Volanakis JE,
Frank MM (eds): The Human Complement System in Health and
Disease, 10th ed. New York, Marcel Dekker, 1998, pp 9-32.
  7. Walport MJ: Complement: First of two parts. N Engl J Med
344:1058-1066, 2001.
  8. Walport MJ: Complement: Second of two parts. N Engl J Med
344:1140-1144, 2001.
  9. Morgan BP: Complement: Clinical Aspects and Relevance to Disease.
London, Harcourt Brace Jovanovich, 1990.
10. DuClos TW: Function of C-reactive protein. Ann Med 32:274-278,
2000.
11. Ollert MW, Kadlec JV, David K, et al: Antibody-mediated comple-
ment activation on nucleated cells: A quantitative analysis of the
individual reaction steps. J Immunol 153:2213-2221, 1994.
12. Turner MW: Mannose-binding lectin: The pluripotent molecule of
the innate immune system. Immunol Today 17:532-540, 1996.
13. Fujita T: Evolution of the lectin-complement pathway and its role in
innate immunity. Nat Rev Immunol 2:346-353, 2002.
14. Thurman JM, Holers VM: The central role of the alternative comple-
ment pathway in human disease. J Immunol 176:1305-1310, 2006.
15. Liszewski MK, Farries TC, Lublin DM, et al: Control of the comple-
ment system. Adv Immunol 61:201-283, 1996.
16. Morgan BP, Harris CL: Complement regulators in therapy. In ­Morgan
BP, Harris CL (eds): Complement Regulatory Proteins. San Diego,
Academic Press, 1999, p 250.
17. Parker CM: Membrane Defenses against Attack by Complement and
Perforins. Berlin, Springer-Verlag, 1992.
18. Zipfel PF, Skerka C, Caprioli J, et al: Complement factor H and hemo-
lytic uremic syndrome. Int Immunopharmacol 1:461-468, 2001.
19. Davis AE III: C1 inhibitor gene and hereditary angioedema. In
­Volanakis JE, Frank MM (eds): The Human Complement System in
Health and Disease. New York, Marcel Dekker, 1998, pp 455-480.
 336 Atkinson  |  Complement System
20. Kotwal GJ: Poxviral mimicry of complement and chemokine system
components: What’s the end game? Immunol Today 21:242-248,
2000.
21. Hebert LA: The clearance of immune complexes from the circulation
of man and other primates. Am J Kidney Dis 17:353-361, 1991.
22. Nielsen CH, Leslie RG: Complement’s participation in acquired
immunity. J Leukoc Biol 72:249-261, 2002.
23. Kang YS, Do Y, Lee HK, et al: A dominant complement fixation path-
way for pneumococcal polysaccharides initiated by SIGN-R1 interact-
ing with C1q. Cell 125:47-58, 2006.
24. Helmy KY, Katschke KJ Jr, Gorgani NN, et al: CRIg: A macrophage
complement receptor required for phagocytosis of circulating patho-
gens. Cell 124:915-927, 2006.
25. Tarr J, Eggleton P: Immune function of C1q and its modulators CD91
and CD93. Crit Rev Immunol 25:305-330, 2005.
26. Fearon DT, Locksley RM: The instructive role of innate immunity in
the acquired immune response. Science 272:50-54, 1996.
27. Dempsey PW, Allison ME, Akkaraju S, et al: C3d of complement as a
molecular adjuvant: Bridging innate and acquired immunity. Science
271:348-350, 1996.
28. Pickering MC, Botto M, Taylor PR, et al: Systemic lupus erythe-
matosus, complement deficiency, and apoptosis. Adv Immunol 76:
227-324, 2000.
29. Moore FD Jr: Therapeutic regulation of the complement system in
acute injury states. Adv Immunol 56:267-299, 1994.
30. Hageman GS, Anderson DH, Johnson LV, et al: A common haplotype
in the complement regulatory gene factor H (HF1/CFH) predisposes
individuals to age-related macular degeneration. Proc Natl Acad Sci
U S A 102:7227-7232, 2005.
31. Atkinson JP, Schifferli JA: Complement system and systemic lupus
erythematosus. In Kammer GM, Tsokos GC (eds): Lupus: Molecu-
lar and Cellular Pathogenesis. Totowa, NJ, Humana Press, 1999, pp
529-540.
32. Navratil JS, Manzi S, Kao AH, et al: Platelet C4d is highly specific for
systemic lupus erythematosus. Arthritis Rheum 54:670-674, 2006.
33. Barilla-LaBarca ML, Atkinson JP: Rheumatic syndromes associated
with complement deficiency. Curr Opin Rheumatol 15:55-60, 2003.
34. Welch TR: The complement system in renal diseases. Nephron
88:199-204, 2001.
35. Sullivan KE: Complement deficiency and autoimmunity. Curr Opin
Pediatr 10:600-606, 1998.
36. O’Neil KM: Complement deficiency. Clin Rev Allergy Immunol
19:83-108, 2000.
37. Trendelenburg M: Antibodies against C1q in patients with systemic
lupus erythematosus. Springer Semin Immunopathol 27:276-285,
2005.
38. Atkinson JP, Schneider PM: Genetic susceptibility and class III com-
plement genes. In Lahita RG (ed): Systemic Lupus Erythematosus, 3rd
ed. San Diego, Academic Press, 1999, pp 91-104.
39. Welch TR, Frenzke M: Glomerulonephritis associated with deficien-
cies and polymorphisms of complement components encoded in the
class III region of the MHC. Front Biosci 6:D898-903, 2001.
40. Welch TR, Brickman C, Bishof N, et al: The phenotype of SLE associ-
ated with complete deficiency of complement isotype C4A. J Clini
Immunol 18:48-51, 1998.
41. Petri M, Watson R, Winkelstein JA, et al: Clinical expression of sys-
temic lupus erythematosus in patients with C4A deficiency. Medicine
(Baltimore) 72:236-244, 1993.
42. Figueroa JE, Densen P: Infectious diseases associated with complement
deficiencies. Clin Microbiol Rev 4:359-395, 1991.
43. Garred P, Madsen HO, Halberg P, et al: Mannose-binding lectin poly-
morphisms and susceptibility to infection in systemic lupus erythema-
tosus. Arthritis Rheum 42:2145-2152, 1999.
44. Garred P, Madsen HO, Halberg P, et al: The association of variant
mannose-binding lectin genotypes with radiographic outcome in
rheumatoid arthritis. Arthritis Rheum 43:515-521, 2000.
45. Takai T: Roles of Fc receptors in autoimmunity. Nat Rev Immunol
2:580-592, 2002.
46. Ravetch JV, Bolland S: IgG Fc receptors. Annu Rev Immunol 19:
275-290, 2001.
47. Baumann U, Chouchakova N, Gewecke B, et al: Distinct tissue site-
specific requirements of mast cells and complement components C3/
C5a receptor in IgG immune complex-induced injury of skin and lung.
J Immunol 167:1022-1027, 2001.
48. Shushakova N, Skokowa J, Schulman J, et al: C5a anaphylatoxin is a
major regulator of activating versus inhibitory Fc gamma receptors in
immune complex-induced lung disease. J Clin Invest 110:1823-1830,
2002.
49. Holers VM: Phenotypes of complement knockouts. Immunopharma-
cology 49:125-131, 2000.
50. Einav S, Pozdnyakova OO, Ma M, Carroll MC: Complement C4
is protective for lupus disease independent of C3. J Immunol 168:
1036-1041, 2002.
51. Ramos-Casals M, Campoamor MT, Chamorro A, et al: Hypo-
complementemia in systemic lupus erythematosus and primary
antiphospholipid syndrome: Prevalence and clinical significance in
667 patients. Lupus 13:777-783, 2004.
52. Caucheteux SM, Kanellopoulos-Langevin C, Ojcius DM: At the
innate frontiers between mother and fetus: Linking abortion with
complement activation. Immunity 18:169-172, 2003.
53. Holers VM, Girardi G, Mo L, et al: Complement C3 activation is
required for antiphospholipid antibody-induced fetal loss. J Exp Med
195:211-220, 2002.
54. Girardi G, Yarilin D, Thurman JM, et al: Complement activation
induces dysregulation of angiogenic factors and causes fetal rejection
and growth restriction. J Exp Med 203:2165-2175, 2006.
55. Neumann E, Barnum SR, Tarner IH, et al: Local production of com-
plement proteins in rheumatoid arthritis synovium. Arthritis Rheum
46:934-945, 2002.
56. Linton SM, Morgan BP: Complement activation and inhibition in
experimental models of arthritis. Mol Immunol 36:905-914, 1999.
57. Wang Y, Kristan J, Hao L, et al: A role for complement in antibody-
mediated inflammation: C5-deficient DBA/1 mice are resistant to
collagen-induced arthritis. J Immunol 164:4340-4347, 2000.
58. Wang Y, Rollins SA, Madri JA, et al: Anti-C5 monoclonal antibody
therapy prevents collagen-induced arthritis and ameliorates estab-
lished disease. Proc Natl Acad Sci U S A 92:8955-8959, 1995.
59. Banda NK, Thurman JM, Kraus D, et al: Alternative complement
pathway activation is essential for inflammation and joint destruction
in the passive transfer model of collagen-induced arthritis. J Immunol
177:1904-1912, 2006.
60. Ji H, Ohmura K, Mahmood U, et al: Arthritis critically dependent on
innate immune system players. Immunity 16:157-168, 2002.
61. Matsumoto I, Maccioni M, Lee DM, et al: How antibodies to a ubiq-
uitous cytoplasmic enzyme may provoke joint-specific autoimmune
disease. Nat Immunol 3:360-365, 2002.
62. Fearon DT, Austen KF: Current concepts of immunology: The alter-
native pathway of complement—a system for host defense to micro-
bial infection. N Engl J Med 303:259-263, 1980.
63. Cantaert T, De Rycke L, Bongartz T, et al: Citrullinated proteins in
rheumatoid arthritis: Crucial . . . but not sufficient! Arthritis Rheum
54:3381-3389, 2006.
64. Dragon-Durey M-A, Fremeaux-Bacchi V: Atypical haemolytic urae-
mic syndrome and mutations in complement regulator genes. Springer
Semin Immunopathol 27:359-374, 2005.
65. Liszewski MK, Atkinson JP: Complement inhibition. In Smolen J,
Lipsky P (eds): Biological Therapy in Rheumatology. London, Martin
Dunitz, 2002, pp 453-461.
66. Klickstein LB, Moore FD Jr, Atkinson JP: Therapeutic inhibition
of complement activation with emphasis on drugs in clinical trials.
In Austen KF, Burakoff SJ, Strom TB, et al (eds): Therapeutic
Immunology Malden, Mass, Blackwell Science, 2000, pp 287-301.
67. Kirshfink M: Targeting complement in therapy. Immunol Rev 180:
177-189, 2001.
68. Bhole D, Stahl GL: Therapeutic potential of targeting the comple-
ment cascade in critical care medicine. Crit Care Med 31(1 Suppl):
S97-S104, 2003.
 20
KEY POINTS
Mitogen-activated protein kinase (MAPK) pathways are a
cascade of kinases engaged by environmental stress, leading
to the activation of multiple transcription factors.
Inhibitor of κB (IκB) sequesters nuclear factor κB (NFκB) in the
cytoplasm and after stimulation by the IKK signal complex, IκB
is marked for degradation, allowing for NFκB to translocate to
the nucleus and activate transcription.
Ligand binding to Toll-like receptors initiates a signal cascade
that involves MyD88, IRAK1, and IRAK4 and results in the
activation of MAPK pathways and the NFκB pathways.
The extrinsic pathway of apoptosis is initiated by the binding
of death ligands to receptors (TNFR1, Fas, TRAIL).
The intrinsic pathway is regulated by the Bcl-2 protein family,
which is divided into proapoptotic and antiapoptotic
members.
p53 stimulates cell cycle arrest and DNA repair by inducing
p21 and GADD45 and can induce the apoptotic cascade
through activation of proapoptotic Bcl-2 family members.
Signal transduction is a biochemical event by which cells
transmit a signal from the cell exterior (e.g., by a hormone,
growth factor, cytokine, or chemokine) through the cell
membrane and into the cytoplasm. This event involves
many molecules, including receptors, kinases, and secon­
dary messengers. The result of multiple signal transduction
pathways is the binding of transcription factors to DNA,
affecting gene transcription. Signal transduction pathways
are highly diverse, yet show an extraordinary degree of spec­
ificity for transcription factors. The binding of extracellular
signaling molecules to their receptors may directly induce
transcription factors such as nuclear factor κB (NFκB) or
NFAT or activate signal transduction pathways, including
mitogen-activated protein kinases (MAPKs), resulting in
the subsequent activation of transcription factors such as
AP-1 and ATF-2. Many transcription factors and their sig­
naling pathways have been implicated in the expression of
genes that mediate the inflammatory response and are con­
sidered novel therapeutic targets for treating inflammatory
diseases.
MITOGEN-ACTIVATED PROTEIN
KINASE FAMILY
MAPKs control many cellular functions, including cell
proliferation and hormonal responses.1,2 MAPK cascades
contain at least three upstream kinases and are activated by
multiple environmental stress. Extracellular signal-regulated
Signal Transduction
John C. Scatizzi  • Harris Perlman
kinases (ERKs) are generally activated by growth factors
and phorbol esters, whereas c-Jun aminoterminal kinases
(JNKs) and p38 isoforms are activated by cellular stress
and cytokine stimulation. MAPKs are activated by mito­
gen-activated protein kinase kinases (MAPKKs), which
are phosphorylated by upstream mitogen-activated protein
kinase kinase kinases (MAPKKKs). MAPKKKs are engaged
by small guanosine triphosphate (GTP) proteins such as Ras
or other upstream kinases (Fig. 20-1). MAPKs are serine/
threonine kinases, which are activated by dual phosphoryla­
tion of threonine and tyrosine residues located within their
activation loops. MAPKs form tight complexes with their
substrates and phosphorylate serine/threonine residues.
Phosphorylation of MAPKs either directly or indirectly
regulates at least three transcription factors—activated pro­
tein-1 (AP-1), NFκB, or signal transducers and activators of
transcription (STAT), each of which is linked to inflamma­
tory diseases.3
There are five ERK proteins (ERK1 through ERK5)
that are activated in response to growth factors and phor­
bol esters. ERK1 and ERK2 are expressed in all tissues and
are activated by dual sequential phosphorylation, first on
a tyrosine then on a threonine residue. Two major events
that initiate the signal cascade are the binding of GTP
ligand to Ras and phosphorylation of active Raf-1. In rest­
ing cells, ERK1 and ERK2 are found throughout the cell;
however, after activation, ERK1 and ERK2 accumulate in
the nucleus. Phosphorylated ERK1 and ERK2 bind to the
transcription factor Pax6, steroid receptor coactivator-1
(SRC-1), NFATc, or STAT3,4 whereas ERK5 influences
c-Jun-mediated transcription. Taken together, these data
suggest that ERK1 and ERK2 are the major isoforms that
are activated during an inflammatory response.
JNKs (also called stress-activated protein kinases) are
activated by cytokines and cellular stress, including ultra­
violet irradiation, growth factor deprivation, or agents that
interfere with DNA or protein synthesis. There are three
different JNK isoforms: JNK1 and JNK2 are ubiquitously
expressed, whereas JNK3 is primarily expressed in neural
tissue.5 JNKs are activated by the dual phosphorylation of
tyrosine and threonine residues separated by a proline resi­
due in their activation loops. Activated JNK1 and JNK2
phosphorylate two serine residues on the amino terminus
of the transcription factor c-Jun, which results in marked
increase in transcriptional activity. JNK phosphorylates
p53, the transcription factors Elk-1 and NFAT4, the glu­
cocorticoid receptor, and members of the Bcl-2 family.5
p38 is a MAPK that is activated by environmental
stresses (oxidative stress, hypoxia, and ultraviolet irradia­
tion) and inflammatory cytokines. There are four isoforms
of p38, each of which is encoded by a separate gene. p38 α
337
 338 SCATIZZI  |  Signal Transduction
A B Cellular stress Phorbalesters or
Signal or cytokines growth factors
MAPKKK MAPKKK MAPKKK
MAPKKK
MAPKK MAPKK3/6 MAPKK4/7 MAPK1/2
MAPK p38α/β/γ/δ JNK1/2 ERK1/2
Transcription SAP-1 c-Jun EIK-1
factor
Figure 20-1  Overview of mitogen-activated protein kinase (MAPK) sig-
naling pathways. A, Following an extracellular signal, a MAP kinase kinase
kinase (MAPKKK) is activated, which phosphorylates a MAP kinase kinase
(MAPKK). MAPKK phosphorylates a MAPK, which activates various tran-
scription factors. B, Cellular stress or proinflammatory cytokines activate
either p38 or JNK, resulting in the activation of SAP-1 and c-Jun transcrip-
tion factors. Phorbol esters, growth factors, and some cytokines activate
ERK, which results in the activation of the Elk-1 transcription factor.
and β are widely expressed; γ is predominantly expressed
in skeletal muscle; and δ is expressed in testes, pancreas,
synovium, and small intestine.6 p38 is localized in the cyto­
plasm and nucleus of resting cells, but the significance of its
distribution within the cell is not well understood. Activa­
tion of p38 results from the dual phosphorylation of tyrosine
and threonine residues in the TGY motif. p38 then phos­
phorylates and enhances the activity of many transcription
factors, including ATF-1 and ATF-2, SAP-1, NFκB, and
p53.6 p38 also regulates the expression of genes by stabiliz­
ing the mRNA through the binding to AU-rich elements.6
These data suggest that p38 mediates the transcription or
message stability of various genes involved in the immune
response.
AP-1 is a transcription factor that controls the regula­
tion of the inflammatory response, including the induced
transcription of proinflammatory cytokines and matrix
metalloproteinase (MMP) genes. AP-1 transcription fac­
tors are divided into two groups, Jun (c-jun, jun B, and
jun D) and Fos (c-fos, fos B, Fra-1, and Fra-2), which form
Jun-Jun homodimers, Jun-Jun heterodimers, or Jun-Fos
heterodimers. AP-1 is classified in the basic region leucine-
zipper (bZip) family. The basic domain allows transactiva­
tion, and the leucine-zipper domain mediates dimerization
and DNA binding. All three MAPKs are involved in tran­
scriptional regulation of Jun and Fos family genes through
the phosphorylation and subsequent activation of the
Ets family transcription factors ELK-1 or SAP-1. MAPKs
also are responsible for post-translational modifications of
AP-1. Phosphorylated AP-1 associates with CBP/p300
and RNA polymerase III and has enhanced transcriptional
activity.7 JNK1 and JNK2 phosphorylate two aminotermi­
nal serine residues found in the transactivation domain of
c-jun, which results in a substantial increase in the tran­
scriptional activity of AP-1. ATF-2 is another transcription
factor in the bZip family, which is activated by p38 or JNK.
ATF-2 binds to DNA either as a homodimer or as a het­
erodimer with c-jun or other members of the bZip family.
ATF-2 transcriptional activity is induced by phosphoryla­
tion of its transactivation domain.
NUCLEAR FACTOR κB TRANSCRIPTION
FACTOR
NFκB was first described as a B cell–specific protein that
binds to a short DNA sequence motif located in the immu­
noglobulin κ light-chain enhancer. It is expressed in virtu­
ally all cell types and has been shown to play a broad role in
gene transcription, including interleukin (IL)-2, IL-6, IL-8,
granulocyte-macrophage colony-stimulating factor (GM-
CSF), intercellular adhesion molecule (ICAM)-1, vascular
adhesion molecule (VCAM)1, and major histocompatibility
class I.8 The NFκB family has been implicated in the tran­
scriptional activation of numerous targets in rheumatoid
arthritis, including tumor necrosis factor (TNF)-α, IL-1,
IL-6, monocyte chemotactic protein-1, GM-CSF, MMP-1,
MMP-3, ICAM, VCAM, inducible nitric oxide synthase,
cyclooxygenase-2, Bcl-2, and Bcl-xL.9 The family consists of
five members (Rel A [p65], c-Rel, Rel B, p50, and p52) that
share an N-terminal Rel homology domain, which is nec­
essary for DNA binding, dimerization, and nuclear trans­
location. p65, c-Rel, and RelB, but not p50 or p52, have
C-terminal transcriptional activation domains. p50 and
p52 are synthesized as p105 and p100 precursors, which are
processed into the smaller isoforms and may dimerize with
other NFκB members to become transcriptionally active.
NFκB members are retained in the cytoplasm as a
result of their association with their inhibitor, IκB (α, β,
ε, γ).10 IκB blocks the nuclear localization signal on NFκB
either by masking it or by covering it. IκBα has a nuclear
export signal that prevents nuclear entry by NFκB. IκBα
and β preferentially associate with p65, but not with
c-Rel, whereas IκBε binds to p65 and c-Rel.11,12 For NFκB
to become transcriptionally active, it must be released
from IκB. This release is mediated by phosphorylation
and ubiquitination of IκB followed by its degradation
(Fig. 20-2). IκB proteins are phosphorylated (Ser32 and
Ser36 for IκBα and Ser 19 and Ser21 for IκBβ) by a protein
complex (IKK signalosome) that consists of two catalytic
subunits IKKα and IKKβ and a regulatory subunit, IKKγ
NFκB essential modulator (NEMO). IKK signalosome is
activated by a multitude of cytokines and Toll-like recep­
tor (TLR) agonists, including TNF-α, IL-1β, and lipopoly­
saccharide (LPS).
Phosphorylated IκB is targeted for ubiquitination and
subsequent degradation by the 26S proteasome. IKKγ is
crucial for transporting IKKβ toward its activating kinase.
TNF-α stimulation leads to recruitment of TAK1, TAB2,
TAB3, MEKK3, TRAF2, and RIP1 to the cytoplasmic tail
of TNF-α receptor. IKKγ is required for the attachment of
lysine 63-linked polyubiquitin chain to the coiled region
of RIP1, which is necessary for IKK signaling and NFκB
activity.13 Selective disruption of IKKβ results in a failure
to phosphorylate IκB in response to stimulation with TNF,
IL-1, or TLR agonists. Deletion of p65, IKKβ, or IKKγ results
in the identical phenotype—embryonic lethality associated
with massive hepatic apoptosis.14 These data indicate that
IKKβ plays a central role in phosphorylation of IκB, and
that IKKγ is required for IκB phosphorylation even though
it lacks a catalytic subunit. Although IKKα may have a
redundant role in the phosphorylation of IκB,15 studies have
shown that IKKα mediates an alternative pathway. This
pathway involves the lymphotoxin-β or BAFF receptor
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
Stress, cytokines, Extracellular space
and pathogens
IKK signal
complex Cytoplasm
P P C.
B.
P P
uuuuu
A.
D.
I-κB
NF-κB dimer E. NF-κB directed
gene transcription
Nucleus
Figure 20-2  Schematic of nuclear factor κB (NFκB) activation. A, NFκB
dimers normally form a complex with inhibitor κB (IκB). The binding of
IκB covers the nuclear localization signal on the NFκB dimers, sequester-
ing the complex in the cytoplasm. B, Activation of NFκB begins with an
extracellular signal activating the IKK signal complex to phosphorylate
IκB. C, Phosphorylation of IκB results in the addition of a ubiquitin tag.
D, The ubiquitin tag marks IκB for proteolytic degradation and subse-
quently releases the nuclear localization signal on the NFκB dimer. 
E, NFκB dimers translocate to the nucleus and bind promoter regions of
NFκB-responsive genes, directing their transcription.
mediated activation of NFκB-inducing kinase (NIK), which
leads to the processing of p100 protein into p52 and forma­
tion of RelB dimers.16
JAK/STAT/SOCS FAMILY
IL-6, interferon (IFN)-α, IFN-β, and IFN-γ in addition to
various other cytokines activate the JAK (Janus kinases)
family, which associate with their cognate receptors and
lead to the phosphorylation of the cytoplasmic domains
to create docking sites for src homology 2 (SH2)–contain­
ing proteins. The STAT (signal transducers and activators
of transcription) family of proteins are one of the most
important substrates for JAKs.17 The SOCS (suppressor
of cytokine signaling) proteins have been shown to func­
tion as inhibitors of JAK/STAT proteins and are induced
through a negative feedback loop.17 The importance of
STAT is shown in mice lacking the various components
of the JAK/STAT/SOCS family. Mice expressing a knock-
in mutant of the gp130 receptor, which has all the STAT
binding sites deleted, have gastrointestinal ulceration and
severe arthritis.18 SOCS1−/− mice are normally lethal, but
are viable on an IFN-γ−/− background. These data show that
the JAK/STAT/SOCS family is crucial for the proper signal
transduction pathway that occurs by the docking of cyto­
kines to their receptors.
TOLL-LIKE RECEPTOR SIGNALING
TLRs are a family of evolutionarily conserved ­ receptors,
which are crucial for initiating the innate immune
response.19,20 TLRs and IL-1 receptors (IL-1Rs) have con­
served regions located in their cytoplasmic tails known as
339
Toll/IL-1 receptor (TIR) domains, which are required for
downstream signaling. The extracellular domains of TLRs
contain leucine-rich sequences (LRR domains) and hydro­
phobic sequences, whereas IL-1Rs have three immuno­
globulin domains in their extracellular portion. Despite the
conservation among the extracellular domains of TLRs,
different TLRs can recognize structurally unrelated ligands.
Location of TLRs also dictates ligand specificity: TLR3
(double-stranded RNA), TLR7 (single-stranded RNA),
and TLR9 (CpG-containing DNA) are not expressed on
the cell surface, whereas TLR1 (triacyl lipopeptides), TLR2
(peptidoglycans from gram-positive bacteria, lipoproteins, or
yeast), and TLR4 (LPS) are located on the cell surface and are
recruited to phagosomes after their activation.
After ligand binding, TLRs/IL-1Rs dimerize and undergo
a conformational change resulting in the recruitment of
downstream signaling molecules. Myeloid differentiation
primary-response protein 88 (MyD88) is one of the initial
signaling molecules recruited to the activated TLR. MyD88
contains an aminoterminal death domain (DD) and a car­
boxyterminal TIR domain, which dimerizes with the TIR
domain located on the cytoplasmic tail of the TLR. MyD88 is
an adapter molecule that recruits and associates with IRAK,
a serine/threonine kinase with an N-terminal death domain.
There are four IRAK proteins (IRAK1 through IRAK4);
however, only IRAK1 and IRAK4 have detectable kinase
activity, which increases after TLR stimulation and is nec­
essary for the activation of NFκB. TLR ligand engagement
results in the activation of IRAK4, which associates and
phosphorylates IRAK1. TNF ­ receptor–­associated factor 6
(TRAF6) is recruited to IRAK1, which results in the dis­
sociation of IRAK1 and TRAF6 from IRAK4. TRAF6 is
a member of a family of evolutionary ­ conserved adapter
proteins, which also play a major role in TNF receptor
superfamily signaling. TRAF6 contains a RING finger-zinc
finger region required for downstream signaling events.
IRAK1 and TRAF6 associate with transforming growth
factor β–activated kinase (TAK1), TAK1 binding protein
1 (TAB1), and TAB2, which results in the activation of the
transcription factors AP-1 and NFκB. TAK1 is a member of
the MAPKKK family, and its activation directly leads to the
activation of the MAPK pathways (Fig. 20-3).
SIGNALING IN APOPTOSIS
Apoptosis is an evolutionarily conserved cell death
­pathway—components of this mechanism exist from
worms to humans.21 An apoptotic stimulus induces an ini­
tiation phase followed by an execution or commitment
phase and ending in a degradation phase.22 Characteristics
of the degradative phase of apoptosis are membrane bleb­
bing, mitochondrial dysfunction, cellular and cytoplasmic
shrinkage, chromosomal fragmentation and condensation,
and endonuclease activation resulting in the characteristic
180-bp nucleosomal DNA ladder.23 In contrast to necro­
sis, the apoptotic process does not induce an inflammatory
response because the cell forms an apoptotic body that is
either expelled into a lumen or phagocytosed by neigh­
boring cells,24 avoiding the release of subcellular antigens.
Apoptosis in mammals proceeds through two distinct path­
ways—an “extrinsic” pathway, which transduces an apop­
totic signal after the ligation of death receptors on the cell
 340 SCATIZZI  |  Signal Transduction
TLR ligand
TLR receptor
TIR domain
MyD 88
IRAK 4 IRAK 1
TRAF 6
TRAF 6
TAB2
Activation NF-κB TAK 1
pathway
TAB1
Activation of MAP
kinase pathways
IKK signal complex
Figure 20-3  Toll-like/interleukin-1 receptor signaling. After Toll-like
receptor (TLR) ligand binding, dimerization of receptors occurs, which
allows recruitment of MyD88 to the Toll/interleukin-1 receptor (TIR)
­domains of receptor cytoplasmic tail. MyD88 contains a death domain,
which allows for its interaction with IRAK proteins. The recruitment of
IRAK4 by MyD88 allows phosphorylation of IRAK1 by IRAK4. TRAF6 is
­recruited to the receptor complex by phosphorylated IRAK1. Phosphor-
ylated IRAK1 and TRAF6 dissociate from the receptor complex. ­ After
­degradation of IRAK1, TRAF6 forms a complex with TAK1, TAB1, and
TAB2. TAK1 is activated after TRAF6 ubiquitination and phosphorylates
the IKK complex and mitogen-activated protein kinases (MAPK). This
­activates the nuclear factor κB (NFκB) and MAPK signaling pathways, re-
sulting in the activation of multiple transcription factors.
surface, and an “intrinsic” pathway, in which mitochondria
play a crucial role. Caspases, an enzymatic family of cyste­
ine proteases (caspase 1 through 14) that cleave adjacent
to aspartate residues and regulate the degradative phase of
apoptosis.
DEATH RECEPTOR SIGNALING
The induction of apoptosis by the extrinsic pathway is ini­
tiated by binding of death ligands to its cognate receptor.
The TNF superfamily, including Fas, TRAIL RI (DR4) and
RII (DR5), and TNF-α receptor (TNFRI), are ubiquitously
expressed type I membrane receptors that share a homol­
ogous region (death domain). The ligands for the death
receptors are type II membrane proteins and include Fas
ligand (FasL), TRAIL, and TNF-α. Oligomerization of Fas
by binding to FasL induces the recruitment of an adapter
protein FADD and a cysteine protease procaspase 8 (see
later) to the C-terminus of Fas, forming the death-inducing
signaling complex.25
In contrast to Fas, TNF-α rarely induces apoptosis unless
NFκB activity is suppressed. After binding of TNF-α to
TNFR1, an adapter protein TRADD is recruited to the
C-terminus of TNFR1, and this recruits the serine/theon­
ine kinase RIP1, which is required for NFκB signaling, and
TRAF2, which is necessary for JNK signaling. The TRADD-
RIP1-TRAF2 complex is released from the cytoplasmic tail
of TNFR1 and forms an additional complex with caspase
8 and FADD in cells that are sensitive to TNF-α-induced
apoptosis.26 Aggregation or oligomerization of procaspase
8 leads to autocatalysis/activation. Active ­caspase 8 induces
the degradative phase of apoptosis through the activation
of caspases 3 and 7.25 FADD27 and caspase 828 are essential
for the transmission of the Fas-death signal because FADD-
­deficient or caspase 8–deficient embryonic fibroblasts are
insensitive to Fas-FasL-induced death. An additional
pathway of Fas-induced cell death involves the apoptotic
agonist Bid, a Bcl-2-family protein.29 Death receptor–
mediated apoptosis is blocked at the death-inducing signal­
ing complex by the naturally occurring dominant negative
caspase 8 protein termed Flice inhibitory protein (Flip).30
Two isoforms of Flip are produced owing to alternate splic­
ing, a larger, FlipL, and a smaller protein, FlipS. Flip binds to
procaspase 8 or FADD through the death effector domains
preventing the formation of the death-inducing signaling
complex. Mice deficient for Flip die in utero at embryonic
day 10.5 (E10.5),31 and embryonic fibroblasts isolated from
Flip-null mice are susceptible to all forms of death recep­
tor–mediated cell death, including Fas.31 These data show
that direct inhibition of Fas-induced apoptosis is mediated
by Flip.31
Bcl-2 FAMILY SIGNALING
The intrinsic pathway is regulated by the Bcl-2 protein
­family, which is divided into antiapoptotic (Bcl-2, Bcl-xL,
Mcl-1, A1/Bfl-1, and Bcl-w) and proapoptotic (Bax, Bak,
Bad, Bim/Bod, Bok/Mtd, Bik/Blk/Nbk, Bid, Hrk/DP5, Bmf,
Noxa, PUMA/Bbc3) members.32 Bcl-2-related proteins con­
tain Bcl-2-homology (BH1 through BH4) domains, which
are crucial for homodimer and heterodimer formation
between the family members.33 Although the antiapoptotic
Bcl-2-like proteins contain all four (or at least three) BH
domains, the proapoptotic Bcl-2–related proteins are sub­
divided into two categories—the multi-BH domain (BH1
through BH3, e.g., Bak, Bax) and the BH3-only proteins
(e.g., Bad, Bim).34 Studies using peptides that correspond to
the BH3 domains have shown that BH3-only proteins also
are subdivided into two categories based on their ability to
induce apoptosis.35 Bid and Bim are sufficient to sequester
antiapoptotic Bcl-2 family members and induce oligomer­
ization of Bak and Bax, permeabilization of liposomes, and
release of cytochrome c.35 In contrast, Bad, Bmf, Hrk, Noxa,
and PUMA are sensitizers for apoptosis because they are
able to bind only to the antiapoptotic Bcl-2 members and
require Bid or Bim to induce the death response.35-40
Many Bcl-2 family members are localized to the mito­
chondrial outer membrane (and certain other intracellular
membranes), suggesting that mitochondrial dysfunction is
involved in apoptosis.22,29,32 During intrinsic apoptosis sig­
naling, the integrity of the outer mitochondrial membrane is
lost, leading to the dissipation of the transmembrane poten­
tial through the opening of mitochondrial permeability
transition pores22 and release of apoptogenic mitochon­
drial intermembrane proteins, such as cytochrome c.41,42 In
the cytoplasm, cytochrome c binds to the adapter protein
Apaf-1, which causes aggregation and activation of the ini­
tiator caspase 9. Caspase 9 activates the effector caspases 3
and 7,43 which cause the downstream degradative events in
apoptosis.44 Apoptosis signaling through the intrinsic path­
way is inhibited by overexpression of any of the Bcl-2-like
prosurvival members or by loss of both multi-BH domain
proteins Bak and Bax.45
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
p53, CELL CYCLE ARREST, AND APOPTOSIS
p53 is a transcription factor that has been ascribed prop­
erties of a tumor suppressor. p53 induces various cellular
responses, including differentiation, cell cycle arrest, DNA
repair, and apoptosis.46 p53 stimulates cell cycle arrest or
DNA repair or both by inducing the transcription of p21
and GADD45, whereas p53 activates the apoptosis cascade
through the induction of proapoptotic Bcl-2 proteins, such
as Bax, PUMA, and Noxa. The mechanism that controls
which pathway is activated by p53 is poorly understood.
One possibility is that binding affinity of p53 to specific cell
cycle or apoptotic promoters may be a crucial determinant
and is related to the level of p53 after cellular stress.47 Alter­
natively, post-translational modifications to p5348 or recruit­
ment of cofactors by p53 may decide the fate of the cell.49
Dysregulation of cell cycle arrest, DNA repair, and apoptosis
have been suggested to contribute to chronic inflammatory
diseases.
SUMMARY
There has been a substantial increase in recent years in
understanding of the signal transduction pathways that are
activated in inflammatory and autoimmune diseases. This
increased understanding has led to the development of
small molecules to inhibit each pathway, including NFκB,
JNK, and p38. Blocking these signaling pathways repre­
sents a major potential therapeutic approach to rheumatic
­diseases.
REFERENCES
  1. Murphy LO, Blenis J: MAPK signal specificity: The right place at the
right time. Trends Biochem Sci 31:268-275, 2006.
2. Qi M, Elion EA: MAP kinase pathways. J Cell Sci 118:3569-3572,
2005.
3. Firestein GS, Manning AM: Signal transduction and transcription
factors in rheumatic disease. Arthritis Rheum 42:609-621, 1999.
  4. Nishimoto S, Nishida E: MAPK signalling: ERK5 versus ERK1/2.
EMBO Rep 7:782-786, 2006.
  5. Davis RJ: Signal transduction by the JNK group of MAP kinases. Cell
103:239-252, 2000.
  6. Ashwell JD: The many paths to p38 mitogen-activated protein kinase
activation in the immune system. Nat Rev Immunol 6:532-540, 2006.
  7. Ransone LJ, Verma IM: Nuclear proto-oncogenes fos and jun. Annu
Rev Cell Biol 6:539-557, 1990.
8. Firestein GS: NF-kappaB: Holy Grail for rheumatoid arthritis?
Arthritis Rheum 50:2381-2386, 2004.
9. Karin M: Nuclear factor-kappaB in cancer development and pro-
gression. Nature 441:431-436, 2006.
10. Gilmore TD: Introduction to NF-kappa B: players, pathways, perspec­
tives. Oncogene 25:6680-6684, 2006.
11. Malek S, Chen Y, Huxford T, et al: IkappaBbeta, but not IkappaBal­
pha, functions as a classical cytoplasmic inhibitor of NF-kappaB dimers
by masking both NF-kappaB nuclear localization sequences in resting
cells. J Biol Chem 276:45225-45235, 2001.
12. Malek S, Huxford T, Ghosh G: Ikappa Balpha functions through direct
contacts with the nuclear localization signals and the DNA binding
sequences of NF-kappaB. J Biol Chem 273:25427-25435, 1998.
13. Kovalenko A, Wallach D: If the prophet does not come to the moun­
tain: Dynamics of signaling complexes in NF-kappaB activation. Mol
Cell 22:433-436, 2006.
14. Pasparakis M, Luedde T, Schmidt-Supprian M: Dissection of the
NF-kappaB signalling cascade in transgenic and knockout mice. Cell
Death Differ 13:861-872, 2006.
15. Li Q, Estepa G, Memet S, et al: Complete lack of NF-kappaB activity in
IKK1 and IKK2 double-deficient mice: additional defect in neurulation.
Genes Dev 14:1729-1733, 2000.
341
16. Senftleben U, Cao y, Xiao G, et al: Activation by IKKalpha of a sec­
ond, evolutionary conserved, NF-kappa B signaling pathway. Science
293:1495-1499, 2001.
17. Walker JG, Smith MD: The Jak-STAT pathway in rheumatoid
arthritis. J Rheumatol 32:1650-1653, 2005.
18. Ernst M, Inglese M, Waring P, et al: Defective gp130-mediated signal
transducer and activator of transcription (STAT) signaling results in
degenerative joint disease, gastrointestinal ulceration, and failure of
uterine implantation. J Exp Med 194:189-203, 2001.
19. Akira S, Takeda K: Toll-like receptor signalling. Nat Rev Immunol
4:499-511, 2004.
20. Medzhitov R: Toll-like receptors and innate immunity. Nat Rev
Immunol 1:135-145, 2001.
21. Ameisen JC: The origin of programmed cell death. Science 272:
1278-1279, 1996.
22. Zamzami N, Brenner C, Marzo I, et al: Subcellular and submito­
chondrial mode of action of Bcl-2-like oncoproteins. Oncogene 16:
2265-2282, 1998.
23. Yang E, Korsmeyer SJ: Molecular thanatosis: A discourse on the BCL2
family and cell death. Blood 88:386-401, 1996.
24. Thompson CB: Apoptosis in the pathogenesis and treatment of dis­
ease. Science 267:1456-1462, 1995.
25. Green DR: Apoptotic pathways: The roads to ruin. Cell 94:695-698,
1998.
26. Micheau O, Tschopp J: Induction of TNF receptor I-mediated apop-
tosis via two sequential signaling complexes. Cell 114:181-190,
2003.
27. Yeh WC, Pompa JL, McCurrach M, et al: FADD: Essential for embryo
development and signaling from some, but not all, inducers of apopto­
sis. Science 279:1954-1958, 1998.
28. Varfolomeev EE, Schuchmann M, Luria V, et al: Targeted disruption
of the mouse Caspase 8 gene ablates cell death induction by the TNF
receptors, Fas/Apo1, and DR3 and is lethal prenatally. Immunity
9:267-276, 1998.
29. Green DR, Reed JC: Mitochondria and apoptosis. Science 281:
1309-1312, 1998.
30. Irmler M, Thome M, Hahne M, et al: Inhibition of death receptor
signals by cellular FLIP. Nature 388:190-195, 1997.
31. Yeh WC, Itie A, Elia AJ, et al: Requirement for Casper (c-FLIP) in
regulation of death receptor-induced apoptosis and embryonic devel­
opment. Immunity 12:633-642, 2000.
32. Adams JM, Cory S: The Bcl-2 protein family: arbiters of cell sur-
vival. Science 281:1322-1326, 1998.
33. Kroemer G: The proto-oncogene Bcl-2 and its role in regulating apop­
tosis. Nat Med 3:614-620, 1997.
34. Opferman JT, Korsmeyer SJ: Apoptosis in the development and main­
tenance of the immune system. Nat Immunol 4:410-415, 2003.
35. Galonek HL, Hardwick JM: Upgrading the BCL-2 network. Nat Cell
Biol 8:1317-1319, 2006.
36. Letai A, Bassik MC, Walensky LD, et al: Distinct BH3 domains either
sensitize or activate mitochondrial apoptosis, serving as prototype can­
cer therapeutics. Cancer Cell 2:183-192, 2002.
37. Certo M, DelGaizo Moore V, Nishino M, et al: Mitochondria primed
by death signals determine cellular addiction to antiapoptotic BCL-2
family members. Cancer Cell 9:351-365, 2006.
38. Chen L, Willis SN, Wei A, et al: Differential targeting of prosurvival
Bcl-2 proteins by their BH3-only ligands allows complementary apop­
totic function. Mol Cell 17:393-403, 2005.
39. Willis SN, Chen L, Dewson G, et al: Proapoptotic Bak is sequestered by
Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins.
Genes Dev 19:1294-1305, 2005.
40. Kuwana T, Bouchier-Hayes L, Chipor JE, et al: BH3 domains of BH3-
only proteins differentially regulate Bax-mediated mitochondrial
membrane permeabilization both directly and indirectly. Mol Cell
17:525-535, 2005.
41. Yang J, Liu X, Bhalla K, et al: Prevention of apoptosis by Bcl-2: Release
of cytochrome c from mitochondria blocked. Science 275:1129-1132,
1997.
42. Kluck RM, Bossy-Wetzel E, Green DR, et al: The release of cyto­
chrome c from mitochondria: A primary site for Bcl-2 regulation of
apoptosis. Science 275:1132-1136, 1997.
43. Kuida K, Haydar TF, Kuan CY, et al: Reduced apoptosis and cyto­
chrome c-mediated caspase activation in mice lacking caspase 9. Cell
94:325-337, 1998.
44. Rao L, White E: Bcl-2 and the ICE family of apoptotic regulators:
Making a connection. Curr Opin Genet Dev 7:52-58, 1997.
 342 SCATIZZI  |  Signal Transduction

45. Lindsten T, Ross AJ, King A, et al: The combined functions of pro­ 48. Brooks CL, Gu W: p53 ubiquitination: Mdm2 and beyond. Mol
apoptotic Bcl-2 family members bak and bax are essential for normal Cell 21:307-315, 2006.
development of multiple tissues. Mol Cell 6:1389-1399, 2000. 49. Mihara M, Erster S, Zaika A, et al: p53 has a direct apoptogenic role at
46. Vousden KH, Woude GF: The ins and outs of p53. Nat Cell Biol the mitochondria. Mol Cell 11:577-590, 2003.
2:E178-E180, 2000.
47. Inga A, Storici F, Darden TA, et al: Differential transactivation by
the p53 transcription factor is highly dependent on p53 level and pro­
moter target sequence. Mol Cell Biol 22:8612-8625, 2002.
 21
KEY POINTS
Eicosanoid biosynthesis is catalyzed by cyclooxygenases and
lipoxygenases.
Conversion of the endoperoxide intermediate prostaglandin
H2 requires activity of specific terminal synthases.
Eicosanoid receptors have been identified.
Eicosanoids regulate inflammatory and immune responses.
Eicosanoid synthesis is modified by administration of
­precursor fatty acids.
The addition of oxygen to arachidonic acid and other
polyunsaturated fatty acids not bound to membrane
phospholipids in nearly all human cell types results in
formation of several classes of bioactive products termed
eicosanoids. These include prostaglandins (PGs), prostacy-
clin, thromboxanes (TXs), leukotrienes (LTs), and lipox-
ins. All of these compounds are crucial to the regulation of
immunity and inflammation, among other physiologic and
pathologic processes. Although eicosanoids are derived
from C20 polyunsaturated fatty acids (eicosa = 20),
only a small percentage of these polyenoic acids form
the eicosanoids: dihomogamma linolenic acid (DGLA),
which is 8,11,14-eicosatrienoic acid; arachidonic acid
(AA), which is 5,8,11,14-eicosatetraenoic acid; and
eicosapentaenoic acid (EPA), which is 5,8,11,14,17-EPA
(Fig. 21-1).
Two groups of fatty acids are essential to the body: the
omega-6 series derived from linoleic acid (18:2 n-6) and
the omega-3 series derived from α-linolenic acid (18:3
n-3). The n refers to the number of carbon atoms from
the methyl (omega) end of the fatty acid chain to the
first double bond (i.e., omega-3 and omega-6 designa-
tions). Using this notation, 18 refers to the number of
carbon atoms in the fatty acid. The degree of unsatura-
tion (the number of double carbon-carbon bonds) follows
the number of carbon atoms. Fatty acids are metabolized
by an alternating sequence of desaturation (i.e., removal
of two hydrogens) and elongation (i.e., addition of two
carbons). Membrane phospholipids are the main storage
site for polyunsaturated fatty acids and are particularly
rich in eicosanoid precursors, which are located at the
sn-2 position (Fig. 21-2).Because mammalian cells can-
not interconvert n-3 and n-6 fatty acids, the composition
of membrane phospholipids is determined by exogenous
sources of fatty acids.
Prostaglandins,
Leukotrienes, and
Related Compounds
Robert B. Zurier
BIOSYNTHESIS OF EICOSANOIDS
PHOSPHOLIPASES
Phospholipase A2 (PLA2) in lysosomes or bound to cell
membranes catalyzes the breaking of the sn-2 bond, facili-
tating release of AA or other polyunsaturated fatty acids
(see Fig. 21-2). The enzyme is crucial to regulation of
eicosanoid synthesis because it is in the nonesterified state
that the polyunsaturated precursors enter into the cascades
leading to eicosanoid formation. Only a scant amount of
oxidation at carbon 15 of AA occurs catalytically when
the fatty acid is still covalently bound as part of a phospho-
lipid.1 Lysophospholipids “left over” after the action of PLA2
are direct precursors of platelet-activating factor (PAF), a
potent mediator of inflammation, which is generated by
acylation (addition of fatty acid) in the open sn-2 position
of the lysophospholipid. One member of the PLA2 super-
family, lipoprotein PLA2, may serve as a marker for cardio-
vascular risk.2
Four distinct types of PLA2 activities hydrolyze fatty
acids esterified at the sn-2 position: Secretory PLA2
(sPLA2) has small disulfide cross-linked proteins that
require Ca2+ in mM for optimal activity. Cytoplasmic
PLA2 (cPLA2) has larger proteins requiring Ca2+ in μM,
which are AA-selective and can deacylate diacylphospho-
lipids completely, preventing accumulation of potentially
toxic lysophospholipids. Ca2+-independent PLA2 (iPLA2)
exhibits specificity for plasmalogen substrates. Finally, PAF
acetylhydrolase (PAF-PLA2) has a series of isozymes spe-
cific for short chains.2
Under basal conditions, AA is liberated by iPLA2, then
reincorporated into cell membranes (reacylated), and is
unavailable for appreciable eicosanoid biosynthesis. The
acylase enzymes competitively inhibit the cyclooxygen-
ase (COX) isoenzymes. After receptor activation and cell
stimulation, intracellular Ca2+ levels increase, and the
Ca2+-dependent cPLA2 liberates arachidonate at a rate that
exceeds the rate of reacylation, leading to arachidonate
metabolism by COX isoenzymes. Initial formation of PGE2
seems to be due to preferential coupling between cPLA2,
COX-1, and cytosolic PGH-PGE isomerase. More intense
inflammation leads to participation of secreted sPLA2 and
amplification of PGE2 biosynthesis by inducible COX-2. It
is an oversimplification to regard availability of AA as the
sole rate-limiting step in cellular eicosanoid biosynthesis.
The coordinated action of phospholipases and the restricted
expression and altered activation of COX isoenzymes also
are important.3
Phospholipase C (PLC) hydrolyzes the polar head group
(e.g., inositol, choline) from phospholipids to yield diacyl­
glycerol (DAG) and the polar head group. Direct protein
343
 344 ZURIER  |  Prostaglandins, Leukotrienes, and Related Compounds

OMEGA 6 (n-6) FATTY ACIDS OMEGA 3 (n-3) FATTY ACIDS

COOH COOH

CH3 CH3
Linoleic acid (18:2) Alpha linolenic acid (18:3)

Delta-6 desaturase

COOH COOH

CH3 CH3
Gamma linolenic acid (18:3) 6,9,12,15 Octadecatetraenoic acid (18:4)

Elongase

Cyclooxygenase
COOH PGE1 COOH

CH3 LTC3 CH3

Dihomogamma linolenic acid (20:3) Lipoxygenase Stearadonic acid (20:4)

Delta-5 desaturase

COOH COOH
CH3 CH3
Arachidonic acid (20:4) Eicosapentaenoic acid (20:5)

Figure 21-1  Metabolic pathways

of essential fatty acids. The pathways
are ones of progressive desaturation
alternating with elongation. Eico- Cyclooxygenase Lipoxygenase Cyclooxygenase Lipoxygenase
sanoid precursors include dihomo-
gamma linolenic acid, arachidonic
acid, and eicosapentaenoic acid. PG,
PGE2, PGI2, TXA2 LTA4, LTB4, LTC4 PGE3, TXA3 LTB5
prostaglandins; LT, leukotriene; TX,
thromboxane.

isolation and molecular-cloning studies have revealed
­multiple PLC isozymes in mammalian tissues. Phosphati-
dylinositol-PLC occurs in cytosolic (cPLC) and secreted
(sPLC) forms and can be divided into three major classes
(PLC-β, PLC-γ, and PLC-δ) based on substrate specificity.
PLC with specificity for phosphatidylinositol and phosphor-
ylated phosphatidylinositol is a key component of phos-
phatidylinositol-mediated signaling pathways. DAG is an
activator of protein kinase C (PKC), and rapid production
of this lipid by phosphatidylinositol-PLC hydrolysis of the
phosphorylated phosphatidylinositol pool is a primary step
in signaling. Further AA is made available by the sequen-
tial actions of diglyceride lipase and monoglyceride lipase.4
PLC with activity on phosphatidylcholine also has been
identified. Peripheral blood monocytes from patients with
rheumatoid arthritis (RA) exhibit greater PLA2 and PLC
activity than cells from healthy volunteers. The greatest
increases in enzyme activity were seen in cells from patients
with the most severe, persistent, proliferative disease, not in
cells from patients with the most active disease at the time
the cells were studied.5
Hydrolysis of phospholipids by phospholipase D (PLD)
produces phosphatidic acid (PA) and the respective polar
head groups. The capacity of cells to interconvert PA and
DAG through the action of specific cellular phosphatases
and kinases (Fig. 21-3) suggests that AA release from DAG
and a variety of intracellular signaling and protein-traffick-
ing events may be regulated by PLD activity. PLD may be
activated after or independent of PLC activation.
The tetraenoic precursor (AA) is the most abundant of
the three precursor fatty acids in cells of individuals who
eat usual Western-style diets. Metabolites of AA constitute
the “2” series (dienoic) PGs (two double bonds in the mol-
ecule), and the metabolic pathway has acquired the famil-
iar name AA cascade. Figure 21-4 illustrates the COX and
5-lipoxygenase pathways of the cascade.
CYCLOOXYGENASE PATHWAY
The first step in the biosynthesis of the “prostanoids” (e.g.,
PGs, TXs, prostacyclin) is catalyzed by the bifunctional PG
endoperoxide synthase isozyme (PGHS)-1 (COX-1) and
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
Lysophospholipid
Diacylglycerol PLC
O Polar Head Group
CH2 O P O C H2
CH2 N+(CH3)3
CH2 CH O
O O
O O
PLA2
CH3
Arachidonic
Acid
R
Figure 21-2  Arachidonic acid release from phospholipid. Shown here
is phosphatidylcholine, the major membrane storage site for polyunsatu-
rated fatty acids. PLA2, phospholipase A2; PLC, phospholipase C.
PGHS-2 (COX-2). To form the characteristic five-carbon
ring structure (TXs contain a six-member ring), the precur-
sor fatty acids must have double bonds at carbons 8, 11,
and 14 (numbering from the carboxyl group). When a mol-
ecule of oxygen is inserted across carbons 9 and 11, ring
closure occurs enzymatically across C8 and C12, creating
the unstable PG endoperoxide PGG. Subsequent peroxida-
tion yields PGH with formation of the cyclopentane ring.
PGH serves as the common precursor for PGs, prostacy-
clin, and TXs that are formed under the influence of ter-
minal synthases (see Fig. 21-4). In addition to the activity
of phospholipases, regulation of PG synthesis also occurs
at the level of PGHS gene expression. PGHS levels are
increased by interleukin (IL)-1, platelet-derived growth
factor, and epidermal growth factor, agents that increase
PG synthesis.
Cell membranes constitute the source of substrate AA
and the site of action of eicosanoid-forming enzymes. PG
synthesis also can form at lipid bodies, which are non–
membrane-bound, lipid-rich cytoplasmic inclusions that
develop in cells associated with inflammation. Lipid bod-
ies isolated from human monocytes express PGHS activity,
are reservoirs of arachidonyl phospholipids, and can func-
tion as domains of PG synthesis during an inflammatory
reaction.6
PGHS, the well-known target of nonsteroidal anti-
inflammatory drugs (NSAIDs), exists in two isoforms. These
isoforms are similar in terms of amino acid identities (about
60%), catalytic properties, and substrate specificity, but they
differ in their genomic regulation.7
345
PLC IP3 Ca2+
PIP2
DAG PKC
PIP Pi
PI DAG kinase PA hydrolase
PA
Phosphatidylcholine
PLD Choline
Figure 21-3  Reactions catalyzed by phospholipases C and D,
i­ llustrating interconversion of diacylglycerol (DAG) and phosphatidic
acid (PA).
Regulation of Cyclooxygenase-1 Expression
COX-1 is preferentially expressed constitutively at high
levels in selected cells, including endothelium, monocytes,
platelets, renal collecting tubules, and seminal vesicles.
Because the expression level of the enzyme does not vary
greatly, it has been difficult to study its transcriptional regu-
lation. The gene has a TATA-less promoter that contains
multiple start sites for transcription. It also is known that
Sp1/Cis regulatory elements in the promoter bond the Sp1
transcription factor to induce COX-1 gene expression. In
addition, COX-1 (COX-2) splice variants may function in
tissue-specific normal and pathologic processes and may rep-
resent new targets for therapy.8 The localization of COX-1
in nearly all tissues under basal conditions suggests that
its major function is to provide eicosanoids for physiologic
regulation. This is seen clearly in platelets that do not have
nuclei and cannot produce an inducible enzyme on activa-
tion. Rather, TXs are produced constitutively so that plate-
let aggregation can be completed.
Regulation of Cyclooxygenase-2 Expression
The regulated formation of eicosanoids implies that cells
have an appreciable ability to amplify the rate and amount of
eicosanoid synthesis. Several processes contribute to that reg-
ulation, including silencing of sPLA2 expression by COX-2
and autoinactivation (“suicide inactivation”) of COX-2 and
other oxygenases and synthases. In addition, the COX-2
transcript contains at least 12 copies of the AUUUA RNA
motif, which makes it unstable and subject to rapid degra-
dation. Factors that regulate COX-2 expression are specific
for the physiologic processes involved. Expression of COX-2
in the macula densa in the kidney depends on luminal salt
concentrations. Transcriptional activation of the COX-2
gene by mediators of inflammation such as IL-1β and tumor
necrosis factor (TNF)-α is likely regulated by the transcrip-
tion factors nuclear factor κB (NFκB) and C/EBP. Perhaps
the most crucial of the several demonstrated regulatory
sequences in the 5′ flanking regions of the COX-2 gene is
the ATF/CRE, a site that is activated by the transcriptional
activator protein-1 (AP-1) and the cyclic adenosine mono-
phosphate (cAMP) regulatory binding protein. Depending
on cell and tissue specificity, several signaling pathways
(kinases, Rho, cyclic guanosine monophosphate, wnt) and
transcription factors (NFκB, AP-1, nuclear factor of acti-
vated T cells (NFAT)) are involved in COX-2 expression.9
 346 ZURIER  |  Prostaglandins, Leukotrienes, and Related Compounds

9 7 5 3 1 COOH
8 6 4 2 COOH
Arachidonic Acid
10 CH3
CH3
12 14 16 18 20
11 13 15 17 19
Prostanoic Acid. The basic structure of a 20-carbon fatty Cyclooxygenase
acid, with C8-C12 closed to form a five-membered ring. +2 O2

O COOH

O CH3

O O

Dioxygenase

COOH

O CH3
PGG2
O
OOH

Prostaglandin
Endoperoxide
Prostacyclin Synthase OH PGD2
COOH COOH
O COOH
Prostacyclin Isomerase
O CH3
Synthase CH3
O
OH Isomerase O OH
CH3
OH OH Thromboxane
Synthase OH
Isomerase
COOH COOH
PGH2
PGF2α
CH3 CH3
O O OH OH OH
HHT
O
COOH COOH
MDA O PGE2
CH3 CH3
TXA2 O
OH OH OH

Figure 21-4  Cyclooxygenase pathway of arachidonic acid metabolism. PG, prostaglandins; MDA, malondialdehyde; TX, thromboxane.

COX-1 and COX-2 effect a balance in several physiologic
and pathologic situations. Of particular interest are their
actions in kidney and stomach. During times of low blood vol-
ume, the kidney releases angiotensin and other factors to main-
tain blood pressure by systemic vasoconstriction. Angiotensin
also provokes PG synthesis in the kidney. COX-1, expressed
in vessels, glomeruli, and collecting ducts, produces vasodilat-
ing PGs, which maintain renal plasma flow and glomerular
filtration during conditions of systemic vasoconstriction. In
the antrum of the stomach, COX-1 leads to production of
PGs, which increase gastric blood flow and mucus secretion.
Inhibition of COX-1 by NSAIDs prevents these protective
mechanisms and results in renal ischemia and damage and
gastric ulcers (mainly antral) in susceptible individuals. These
observations have led to development of NSAIDs that selec-
tively inhibit COX-2 and spare COX-1. AA gains access to
the active site of the COX via a hydrophobic channel, and
access is blocked by insertion of an acetyl residue on Ser 530
in COX-1 and Ser 516 in COX-2. The irreversibility of the
interaction and the unique expression of COX-1 in the anu-
cleate platelet is the reason for the clinical efficacy of low-dose
aspirin. Nonacetylated NSAIDs compete with arachidonate
for the active site and can interfere with the sustained effects
of aspirin. Although the structures of COX isozymes are simi-
lar, COX-2 is characterized by a side-pocket extension to the
hydrophobic channel, which is where the selective COX-2
inhibitors localize.7
The major adverse effects of NSAIDs, gastroduodenal
injury and impaired renal function, are caused by inhibi-
tion of COX-1, whereas the analgesic and anti-inflamma-
tory activities of NSAIDs rest in part on their ability to
inhibit COX-2. COX-2 also seems to have a regulating role,
however, in renal, brain, gastrointestinal, ovarian, and bone
function. COX-2 also is expressed in endothelial cells, and
inhibition suppresses prostacyclin synthesis by endothelial
cells.10 COX-2 acts in the initiation and the resolution of
inflammation. Its expression increases transiently early in the
course of carrageenan-induced pleurisy in rats. Later in the
response, COX-2 is expressed at even higher levels, leading
to synthesis of PGD2 and its dehydration product 15-deoxy-
δ12,14-PGJ2 (15δPGJ2). Early expression of COX-2 is asso-
ciated with production of inflammatory PGs, whereas the
later peak results in production of PGs that suppress inflam-
mation.11 That inflammation occurs in COX-2 knockout
mice12 reminds us, as Lewis Thomas stated,13 “inflammation
will take place at any cost.”
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
Cyclooxygenase-3
Acetaminophen, similar to NSAIDs, suppresses pain and
fever. It is not an anti-inflammatory agent, and its mech-
anism of action—despite its extensive use—has not been
apparent. The finding that acetaminophen inhibits COX
activity more in canine brain homogenates than in spleen
homogenates gave rise to the concept that variants of the
COX enzyme exist that are differentially sensitive to acet-
aminophen. A distinct COX isoenzyme, COX-3, is made
from the COX-1 gene (splice variant), is expressed in canine
brain, and has been characterized in rat cerebral endothe-
lial cells.14 In humans, COX-3 mRNA is expressed as an
approximately 5.2-kb transcript most abundant in cerebral
cortex and heart. COX-3 (COX-1b) possesses glycosyl-
ation-dependent COX activity that is selectively inhibited
by analgesic or antipyretic drugs, such as acetaminophen
and phenacetin.
Prostaglandin Synthases
Conversion of the endoperoxide intermediate PGH2 to
PGs requires the activity of specific terminal synthases:
Hematopoietic PGD synthase (H-PGDS) catalyzes the
isomerization of PGH2 to PGD2 in immune and inflamma-
tory cells, cytosolic PGE synthase (cPGES) is responsible
for constitutive expression of PGE2, and microsomal PGE
synthase (mPGES-1) induces PGE in response to inflam-
matory stimuli. At least 10 enzymes convert PG precursors
into biologically active PGs.15 Suppression of PG synthase
activity might be considered as an alternative strategy that
would fall between global blockade by inhibition of COX
and blockade of a single eicosanoid receptor.16
PRODUCTS OF THE CYCLOOXYGENASE
PATHWAY
PROSTAGLANDINS
The basic structure of all PGs is a “prostanoic acid” skel-
eton, a 20-carbon fatty acid with a five-membered ring at
C8 through C12 (see Fig. 21-4, inset). The term prostaglan-
dins is employed widely, but should be used to describe only
the oxygenation products that contain the five-membered
carbon ring. A family of acidic lipids found first in human
seminal fluid, PGs were misnamed because it was thought
they were produced in the prostate gland rather than in the
seminal vesicles.17-19 The alphabetic PG nomenclature (e.g.,
PGE, PGF, PGD) is related to the chemical architecture
of the cyclopentane ring. PGE and PGF differ only in the
presence of a ketone or hydroxyl function at C9 (see Fig.
21-4). These compounds are made by a variety of cells (e.g.,
PGE2 and PGD2 by isomerases, PGF2α by a reductase). In
the nomenclature, a subscript numeral after the letters indi-
cates the degree of unsaturation in the alkyl and carboxylic
acid side chains. The numeral 1 indicates the presence of a
double bond at C13-C14 (PGE1), 2 marks the presence of
an additional double bond at C5-C6 (PGE2), and 3 denotes
a third double bond at C17-C18 (PGE3).
PGs are produced on demand and seem to exert their
effects on the cell of origin or nearby structures. Abun-
dant experimental evidence supports the view that PGs
347
p­ articipate in development of the inflammatory response.
Administration to rats of a monoclonal antibody to PGE2
prevents carrageenan-induced pain and inflammation.20
PGs are probably better at potentiating the effects of other
mediators of inflammation than they are at inducing inflam-
mation directly. PGE compounds and intermediate hydro-
peroxides of AA increase pain sensitivity to bradykinin and
histamine. The effects of PGE are cumulative, depending on
concentration and time. Even very small amounts of PGs,
if allowed to persist at the site of injury, may in time cause
pain.
PGE2 stimulates bone resorption,21 and its 13,14-dihy-
dro derivative is nearly as potent, which is of interest
because derivatives of the biologically active PGs are usu-
ally assumed not to be of functional significance. Addition
of serum to the culture medium stimulates bone resorption,
a process that is complement dependent and PG mediated.
The mechanism may help explain bone erosion in joints of
patients with RA, in which complement is activated, and
PGE2 concentrations are high. The observation that PGE1
can stimulate bone formation22 suggests that PGs physi-
ologically participate in coordination of bone formation
and resorption. Many effects of IL-1 and TNF-α on cells are
associated with stimulation of PG production and inflam-
mation. Cartilage explants from osteoarthritis patients
express COX-2 (but not COX-1) and release 50 times more
PGE2 in culture than cartilage from healthy subjects and
18 times more PGE2 than normal cartilage stimulated with
cytokines plus lipopolysaccharide. Explants from osteoar-
thritis patients release IL-1β, whereas normal cartilage does
not express mRNA for pro-IL-1β or release IL-1β spontane-
ously. It seems that in osteoarthritis—and probably in RA—
upregulation of cartilage IL-1β and subsequent production
of PGE2 leads to cartilage degradation.23
Mast cells, often overlooked as important in ­inflammatory
responses, are seen in large numbers in synovium from
patients early in the course of RA. PGD2, the major PG
formed by mast cells, also can mediate histamine release
from mast cells exposed to anti-IgE antibody. PGJ2, formed
from the dehydration of PGD2, seems to function as a brake
on the inflammatory response. It reduces macrophage activa-
tion, reduces nitric oxide production from stimulated cells,
and induces apoptosis in tumor cell lines. PGJ2 is metabo-
lized to 15-deoxy-δ12,14 PGJ2 and δ12 PGJ2, which also are
biologically active.24
PROSTACYCLIN
Prostacyclin, discovered in 1976,25 has been purified, and the
cDNA for prostacyclin synthase has been cloned. In addi-
tion to a cyclopentane ring, a second ring is formed by an
oxygen bridge between carbons 6 and 9. It is generated from
PGH2 by a distinct prostacyclin synthase, a 56-kD mem-
ber of the cytochrome P-450 superfamily of enzymes found
predominantly in endothelial and vascular smooth muscle
cells.15 Production of prostacyclin can be stimulated by
thrombin or generated by transfer of PGH2 from platelets
(the endoperoxide steal), contact with activated leukocytes,
or stretching of the arterial wall. It is a powerful vasodila-
tor and inhibits platelet aggregation through activation of
adenylate cyclase, which leads to an increase in intracellular
cAMP. It is metabolized rapidly (half-life in plasma is less
 348 ZURIER  |  Prostaglandins, Leukotrienes, and Related Compounds
than one circulation time) to the more stable, less biologi-
cally active 6-keto-PGF1α. The enzymatic products of its
conversion—2,3-dinor-6 keto-PGF1α and 6,15-di-keto-
2,3-dinor PGF1α—also are chemically stable and have very
little biologic activity. They are the major metabolites of
prostacyclin excreted in urine, in which they can be assayed
as indicators of prostacyclin generation.
Prostacyclin generated in the vessel wall has antiplate-
let and vasodilator actions, whereas TXA2 generated by
platelets from the same precursors induces platelet aggrega-
tion and vasoconstriction. These two eicosanoids represent
biologically opposite poles of a mechanism for regulating
the interaction between platelets and the vessel wall and
of formation of hemostatic plugs and intra-arterial thrombi.
Given the central role of platelets in inflammatory reac-
tions, an appropriate prostacyclin-TX balance is important
to regulation of inflammation. The balance may be altered
in patients with antiphospholipid antibody syndrome; in
patients treated with cyclosporine; and in patients treated
with NSAIDs, most especially patients treated with selec-
tive COX-2 inhibitors. Although COX-2 inhibitors reduce
recurrence of colorectal adenomas, they increase the risk
for cardiovascular events, such as myocardial infarction and
stroke.26
Intravascular infusion of prostacyclin also reduces some
of the clinical changes associated with pulmonary embo-
lism. The instability of prostacyclin makes it cumbersome
to administer therapeutically. Nonetheless, it has been
used with limited success to treat peripheral vascular dis-
ease, including Raynaud’s phenomenon. New therapeu-
tic approaches for treatment of pulmonary hypertension
include prostacyclin analogues, such as epoprostenol, bera-
prost, and iloprost.27 In addition to its vasodilator effects,
prostacyclin suppresses endothelial cell proliferation. Pros-
tacyclin analogues might prove useful as adjunct treatment
for some cancers.
THROMBOXANES
The endoperoxide PGH2 can be converted into TXs after
the action of the enzyme TX synthase, a microsomal 60-kD
member of the cytochrome P-450 family, which is quite
active in the platelet. The gene that encodes the enzyme
has been cloned. TXs contain a six-member oxane ring
instead of the cyclopentane ring of the PGs. TX synthase
converts PGH2 into equal amounts of TXA2 and 12L-
hydroxy-5,8,10-heptadecatrienoic acid. TXA2 stimulates
platelet activation, contributes to intravascular aggregation
of platelets, and contracts arteriolar and bronchiolar smooth
muscles. It is hydrolyzed rapidly (half-life is 30 seconds) to
the inactive, stable, measurable product, TXB2; its actions
are limited to the microenvironment of its release.
The extraordinary rapidity with which platelets adhere
to damaged tissue, aggregate, and release potent biologically
active materials suggests that the platelet is well suited to
be a cellular trigger for the inflammatory process. Efforts
directed at suppression of TX synthesis and platelet aggre-
gation may result in limitation of inflammatory responses,
especially in coronary arteries. Inhibition of platelet aggre-
gation may be important to the anti-inflammatory effects of
aspirin and other NSAIDs. Long-term administration of low
doses of aspirin (40 mg/day—the lowest dose predicted to
cause total inhibition of TX formation in serum, according
to mathematic modeling) has inhibitory effects on platelet
function ex vivo that are indistinguishable from the effects
caused by giving 325 mg/day of aspirin.28 Nonetheless, the
response to aspirin can vary among individuals. It has been
suggested29 that serum TXB2 levels be monitored to ensure
the efficacy of aspirin therapy. Even low-dose aspirin has
some depressive effect on prostacyclin synthesis, however.
This means recovery of vascular prostacyclin production
may be more difficult to attain in elderly patients.30
Selective inhibition of TX synthase represents an
approach that may be put into effect without depress-
ing prostacyclin formation. The endoperoxide steal seems
to function in vivo after administration of a TX synthase
inhibitor. Antagonists of the receptors shared by endoper-
oxide and TXA2 have been developed, and these agents
inhibit platelet aggregation in patients who are recalcitrant
to TX synthase inhibition. More specific inhibition of TX
action may become possible now that TX receptors have
been cloned and characterized.31
LIPOXYGENASE PATHWAYS
In contrast to the COX pathway, in which stable products
have three atoms of oxygen covalently attached to AA from
2 moles of molecular oxygen, lipoxygenases insert a single
oxygen atom into the molecular structure of AA. Separate
lipoxygenases exist in certain cells and have strict structural
requirements for their substrates. Three major mammalian
lipoxygenases exist that insert their oxygen atoms into the
5, 12, or 15 position of AA, with formation of a new double
bond and hydroperoxy group. The hydroperoxy fatty acids
(hydroxyperoxy-eicosatetraenoic acid [HPETE]) can be
reduced by peroxidases in the cell to yield the corresponding
hydroxy fatty acids (hydroxy-eicosatetraenoic acid [HETE]).
The exclusive lipoxygenase product of the human platelet is
12-HPETE, which on reduction of the hydroperoxy group
yields 12-HETE. In contrast, the human neutrophil makes
predominantly 5-HPETE, but when high concentrations
of AA are added, 15-lipoxygenase can be shown. Lipoxy-
genases that act on AA are found in the cytosol fraction of
cells.
The human 5-lipoxygenase gene has been isolated and
characterized32,33 and produces a 78-kD enzyme. In myeloid
cells, the 5-lipoxygenase pathway leads to formation of the
biologically active LTs (Fig. 21-5) that were originally found
in leukocytes and that contain three conjugated double
bonds (trienes). Cell activation leads to translocation of
5-lipoxygenase from cytosol to the nuclear membrane, where
it encounters the 18-kD, 5-lipoxygenase-activating pro-
tein. AA also is translocated to 5-lipoxygenase-­activating
protein for presentation to 5-lipoxygenase. The ability of
macrophages and dendritic cells to respond appropriately
during innate immune responses is likely regulated by
5-lipoxygenase and 12-lipoxygenase.34
The unstable HPETE is the initial metabolite of each
lipoxygenase pathway. HPETE is reduced to the more stable
HETE or is converted by 5-lipoxygenase to LTA4. LTA4 can
be converted to LTB4 (in neutrophils and macrophages) or
conjugated with reduced glutathione to form LTC4 (in eosino-
phils, mast cells, endothelial cells, and macrophages). In con-
trast to lipoxygenase, which is mainly distributed in myeloid
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION 349

COOH
Arachidonic Acid
CH3

15-Lipoxygenase 5-Lipoxygenase 12-Lipoxygenase

COOH
15-HPETE CH3 12-HPETE

15-HETE +O2 +H 12-HETE

COOH
5-HPETE OOH
CH3

LTA4 5-HETE
O OH
COOH COOH

CH3 CH3
Glutathione
Epoxide S-Transferase S-R
Hydrolase LTC,D,E
COOH
OH OH
OH
COOH CH3

CH3
CH2CHCONHCH2COOH
LTB4 LTC4: R=
NHCOCH2CH2CHCOOH
γ Glutamyl
Transpeptidase
NH2
Dipeptidase CH2CHCONHCH2COOH
LTD4: R=

NH2
Peptidase

CH2CHCONHCH2COOH Figure 21-5  5-Lipoxygenase path-

LTE4: R= way of arachidonic acid metabolism.
LT, leukotriene; HETE, hydroxy-eicosa-
NH2 tetraenoic acid; HPETE, hydroperoxy-
eicosatetraenoic acid.

cells, LTA4 hydrolase (5,12-dihydroxy-eicosatetraenoic acid),
a zinc-requiring enzyme that converts LTA4 to LTB4, is widely
distributed. From the cDNA sequence, it was suggested that
mRNA for LTA4 may have a short half-life, which could
account for the properties of extremely rapid production and
shutdown of LTB4 and other eicosanoid biosynthesis.
LTA4 can be exported from the cell of origin and converted
in other cells by LTA4 hydrolase to LTB4. This variation on
the endoperoxide steal—perhaps better called transcellular
metabolism—also applies to conversion of LTA4 to LTC4
by LTC4 synthase, a glutathione-S-transferase.35 Although
human endothelial cells do not produce terminal products of
the 5-lipoxygenase system, they do generate LTC4 from LTA4
provided by neutrophils. LTC4 and its products, LTD4 and
LTE4, constitute the biologic mixture previously known as
slow-reacting substance of anaphylaxis. LTD4 and LTE4 arise
from LTC4 after sequential removal of γ-glutamic acid and
glycine from LTC4. The enzyme γ-glutamyl transpeptidase is
present in many cells as part of a complex enzymatic system
involved in glutathione biosynthesis and amino acid trans-
port. In many systems, the major sulfidopeptide LT has been
reported to be LTD4, rather than the precursor LTC4. Removal
of glycine from LTD4 results in LTE4 with concomitant loss of
a significant amount of biologic activity. The principal route
of inactivation of LTB4 is by omega oxidation.
PRODUCTS OF THE LIPOXYGENASE PATHWAYS
The biologic effects of compounds produced in the lipoxy-
genase pathway indicate their importance in inflammatory
­diseases.36 They are the major mediators of inflammation
formed by the oxygenation of AA and are implicated as key
mediators in several diseases, including inflammatory bowel
disease, psoriasis, bronchial asthma, and RA.
 350 ZURIER  |  Prostaglandins, Leukotrienes, and Related Compounds
5-HETE and 5-HPETE stimulate the generation of super-
oxide in human neutrophils. These compounds also augment
intracellular calcium levels, facilitating PKC-dependent
activation of a superoxide generating system of neutrophils.
LTB4 increases adherence of leukocytes to endothelial cells,
a response that is augmented by exposure of the endothelial
cells to TNF-α. LTB4 does not seem to have a direct vascu-
lar contractile action because it is inactive in the hamster
cheek pouch preparation and several other microvascula-
ture systems. In rabbit skin, administration of LTB4 with a
vasodilator PG induces plasma exudation, which suggests
that LTB4 may facilitate enhanced vascular permeability.
Increased venule permeability does occur in response to
LTC4, LTD4, and LTE4. LTB4 is a potent chemotactic factor
for neutrophils and is weakly chemotactic for eosinophils.
LTB4, and to a lesser extent 5-HETE, enhance migration of
T lymphocytes in vitro. Synovial cells produce 5-HETE, but
do not seem to produce significant amounts of LTB4. Mac-
rophages that invade the synovium in RA patients generate
substantial quantities of 5-lipoxygenation and 15-lipoxy-
genation products, however, including LTB4. In addition to
the local signs of inflammation induced by products of the
lipoxygenase pathway, these compounds may contribute to
the pain, tenderness, and aching common in RA patients.
LTB4 also seems to serve an immunoregulatory function. It
stimulates differentiation of competent CD8+ T lympho-
cytes from precursors lacking the CD8 marker. LTB4 also
stimulates interferon-γ and IL-2 production by T cells and
biosynthesis of IL-1 by monocytes.37
Synovial cell proliferation and endothelial cell prolif-
eration are central to propagation of the rheumatoid joint
lesion. LTB4 and the cysteinyl LTs act as growth or differen-
tiation factors for numerous cell types in vitro. These com-
pounds also increase proliferation of fibroblasts when PG
synthesis is inhibited,38 findings that emphasize the impor-
tance of interactions between the COX and lipoxygenase
pathways, and that suggest limitations to NSAID therapy
for RA patients.
Strategies for inhibiting production or antagonizing the
actions of LTs include development of selective LT receptor
antagonists and inhibition of the production of LT by block-
ing the action of 5-lipoxygenase. Inhibition of enzymes distal
in the LT cascade, such as LTA4 hydrolase,39 also is a prom-
ising strategy for development of anti-inflammatory drugs.
A compound that inhibits binding of 5-lipoxygenase to
5-lipoxygenase-activating protein exhibits anti-inflamma-
tory effects in animal models. Lipoxygenase inhibitors have
not been useful for treatment of RA patients. New com-
pounds, mainly resulting from natural products chemistry,
seem more promising.40 In addition, the existing agents
may be aiming at the wrong target. Fibroblasts—such as
synovial cells—do not make much LTB4, but they do make
12-HETE, which is a growth factor, through a cytochrome
P-450 pathway.41 Cytochrome P-450 inhibitors may be more
to the therapeutic point. Some inhibitors are more active
when activated by exposure to light.42 These inhibitors
might prove useful as topical agents for treatment of inflam-
matory skin disease.
Lipoxygenase activities do not lead solely to production
of mediators of inflammation. DGLA is converted by
15-lipoxygenase into 15-HETE, which is incorporated
into DAG and exerts anti-inflammatory effects partly by
interfering with PKC-β activity. A lipoxygenase product
of ­linoleic acid, 13-hydroxyoctadecadienoic acid, also sup-
presses inflammation and cell proliferation by means of a
similar mechanism.43 EPA is converted by lipoxygenase
into 15-hydroxyeicosapentaenoic acid, which also exhibits
anti-inflammatory properties.44
LIPOXINS
Another large family of AA metabolites arises from the
sequential action of 5-lipoxygenases and 15-lipoxygen-
ases. Addition of 15-HPETE and 15-HETE to human
leukocytes results in formation of a pair of oxygenated
products containing a unique conjugated tetraene.
One compound (lipoxin A4 [LXA4]) was identified as
5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic acid, and
the other proved to be its positional isomer (lipoxin B4
[LXB4]), 5D-14,15-trihydroxy-6,8,10,12-eicosatetrae-
noic acid (Fig. 21-6). Because both of these compounds
can arise through an interaction between lipoxygenase
pathways, the trivial name lipoxins (i.e., lipoxygenase
interaction products) was introduced. Platelet 12-lipoxy-
genase can transform neutrophil LTA4 to lipoxins. The
complete stereochemistry and multiple routes of biosyn-
thesis for the biologically active LXA4 and LXB4 have
been ­determined.45
That macrophages of rainbow trout generate ­ lipoxins
rather than LTs or PGs as their major products of AA
metabolism indicates that lipoxins have a long evolu-
tionary history. LTs and lipoxins can be generated in
parallel in fish. In humans, the process has diverged to a
two-cell system. Biosynthesis of eicosanoids by transcel-
lular and cell-cell interactions is recognized as an impor-
tant way to generate and amplify lipid-derived mediators.
Lipoxins can be generated within the vascular lumen
during platelet-leukocyte interactions and at mucosal
surfaces via leukocyte–epithelial cell interactions. In
humans, lipoxins are formed in vivo during multicellu-
lar responses, such as inflammation, atherosclerosis, and
in asthma. These tetraene-containing products serve
as stop signals in that they prevent leukocyte-mediated
tissue injury. A major problem in joints of patients with
RA is that inflammation usually does not resolve. Lipox-
ins and aspirin-induced 15-epilipoxins are endogenous
components of events governing resolution of inflamma-
tion. Aspirin acetylation of COX-2 in endothelial cells
suppresses PG synthesis, but leads to generation of 15R-
HETE from AA, which is transformed to 15-epilipoxin
by leukocytes in a transcellular biosynthetic route involv-
ing vascular endothelial cells or epithelial cells. These
15-epilipoxins exhibit anti-inflammatory and antiprolif-
erative actions in vitro and in vivo. Stable analogues of
LXA4 and of aspirin-triggered lipoxin also suppress inflam-
mation in animal models.46 These observations may lead
to the development of new anti-inflammatory drugs.
Induction by interferon of particular genes seems
­important to the pathogenesis of systemic lupus erythema-
tosus. A stable synthetic analogue of LXA4 suppresses sev-
eral interferon-induced genes and reduces kidney damage in
a murine model of immune-mediated nephritis.47
Lipoxins block human polymorphonuclear ­leukocyte che-
motaxis, but stimulate monocyte chemotaxis and ­adherence.
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION 351

COOH ISOEICOSANOIDS
Arachidonic Acid
CH3 Isoeicosanoids, isomers of enzymatically derived ­eicosanoids,
are derived from auto-oxidation of polyunsaturated fatty
15-Lipoxygenase acids, including omega-3 fatty acids.48 They include mem-
bers of the F, D, and E isoprostanes, isothromboxanes, and
isoleukotrienes. Analysis of the isoprostanes indicates that
COOH they reflect lipid peroxidation in vivo. Theoretically, 64
CH3 F-type isoprostanes may be formed, although few have
OOH been characterized. As more isoprostanes are identified, the
nomenclature probably will continue to change. One F-type
5-Lipoxygenase isoprostane, isoprostane F2α III (formerly 8-isoprostaglan-
din F2α) has been studied in detail because of its biologic
activity in vitro. Isoprostane F2α III (Fig. 21-7) is a potent
COOH vasoconstrictor and may function as a mitogen, with actions
CH3 that are blocked by TX receptor antagonists. Although iso-
OH prostanes may act as ligands at TX or PG receptors (8,12-­
O2
isoprostane F2α III activates the PGF2α receptor), they also
may activate specific isoprostane receptors.

OOH Isoprostanes
COOH
What all isoprostanes have in common, and what distin-
CH3
guishes them from the PGs, is the fact that the top (α) and
OH
the bottom (ω) side chains are always syn—that is, crowded
together on the same face of the cyclopentane ring. The
minimal requirement for generation of an isoprostane is a
polyunsaturated fatty acid with three contiguous methylene-
O
COOH interrupted double bonds, a requirement met by dozens of
CH3 naturally occurring polyunsaturated fatty acids. Fatty acids
formed from docosahexaenoic acid might be important
OH
markers for brain disorders. In contrast to conventional,
enzymatically derived PGs that are formed intracellularly
and released immediately, isoprostanes are formed in the cell
HO OH OH membrane, cleaved by phospholipases, circulate in plasma,
COOH and are excreted in urine. They are increased in several dis-
COOH
OH eases, including acute respiratory distress syndrome, in which
CH3 CH3 polymorphonuclear leukocytes generate reactive oxygen spe-
OH OH cies that damage pulmonary epithelium. The immune cells
in inflamed tissues are exposed to reactive oxygen interme-
Lipoxin A4 Lipoxin B4
diates produced by neutrophils and other phagocytic cells.
Oxidants also are generated as mediators in intracellular sig-
Figure 21-6  Lipoxin biosynthesis. The lipoxins result from the naling pathways by cytokines such as IL-1β and TNF-α.
­sequential action of 15-lipoxygenase and 5-lipoxygenase on arachidonic
acid.
Isoprostanes are likely to be proved to be important
in inflammatory conditions, such as vasculitis and RA.
Because isoprostanes are released preformed, their produc-
Monocytes do not release mediators of ­ inflammation in tion is not blocked by NSAIDs, which suppress metabolism
response to lipoxins, however, and ­ lipoxins are converted of free AA. It is possible that inflammation unresponsive to
rapidly by monocytes to inactive compounds. This selec- NSAIDs may yield to inhibition of isoprostanes. In keeping
tive effect on chemotaxis suggests that lipoxins can play with the notion of eicosanoids as regulators, however, some
a role in wound healing. LXA4 antagonizes LTD4-induced isoprostanes suppress release of mediators of inflammation
vasoconstriction in vivo and blocks binding of LTD4 to its from macrophages.49
receptors on mesangial cells. LXA4 suppresses LTB4-induced
plasma leakage and leukocyte migration and blocks LTB4-
ENDOCANNABINOIDS
induced neutrophil inositol triphosphate generation and
calcium mobilization, but not superoxide anion generation. Groups of naturally occurring members of the eicosanoid
Conversely, LXA4 activates PKC and is more potent in this superfamily that can activate cannabinoid receptors and
regard than DAG and AA. LXA4 seems to be specific for are derivatives of long-chain fatty acids have been referred
the γ subspecies of PKC. These results indicate that lipoxins to as endocannabinoids. One of the most important endo-
may regulate the actions of vasoconstrictor LTs and suggest cannabinoids is anandamide (from the Sanskrit word for
that LXA4 may be an important modulator of intracellular “bliss”), the amide conjugate of AA and ethanolamine
signal transduction. ­(arachidonoyl ethanolamide) (Fig. 21-8). Anandamide is
 352 ZURIER  |  Prostaglandins, Leukotrienes, and Related Compounds
HO
HO OH
Figure 21-7  Isoprostane F2α struc-
tures. Jagged lines indicate that stereo- III
chemistry is uncertain.
CONHCH2CH2OH
Anandamide
Figure 21-8  Chemical structure of anandamide (arachidonoyl etha-
nolamide).
not stored in cells. Rather, it is synthesized rapidly in response
to stimuli in the manner of PGs and LTs. Anandamides and
other endocannabinoids, such as 2-arachidonylglycerol and
virodamine, seem to modulate immune responses.50,51
Discovery of naturally occurring and synthetic analogues
and metabolites of anandamide suggest that a family of such
biologically active substances exists. The polyunsaturated
amides dihomogammalinolenoyl (20:3 n-6) and adrenoyl
(22:4 n-6) ethanolamides have been found in mammalian
brain.52 It is likely that n-3 fatty acid ethanolamides also
exist in mammalian tissues. That anandamide can enhance
its own synthesis in macrophages suggests the presence
of a rapid response to counter excessive inflammatory or
immune responses. Anandamide is converted by COX-2
(but not by COX-1) into PGE2 or PGF2α ethanolamide
directly, without going through free AA.52 These novel PGs
(“prostamides”) are pharmacologically active.53 Because
anandamide is a substrate for COX-2, inhibitors of COX-2
may reduce anandamide metabolism with a subsequent
increase in concentration of the anandamide. A combina-
tion of anandamide with ibuprofen produced synergistic
analgesia in rats.54 In addition, anandamide is metabolized
by fatty acid amide hydrolase. Compounds that inhibit fatty
acid amide hydrolase and maintain anandamide levels may
be useful anti-inflammatory agents. A naturally occurring
lipoamino acid, N-arachidonylglycine, is also a substrate for
COX-2, giving rise to amino acid conjugates of the PGs.55
EICOSANOID RECEPTORS
For many years, it was thought that the lipophilic
­eicosanoids—in contrast to the peptide molecules for which
receptors were characterized routinely—simply “diffused”
into cell membranes or were carried in by a binding protein.
The isolation and cloning of eicosanoid receptors changed
that thinking.56
COOH
Arachidonic Acid
HO OH
COOH COOH
HO
IV
PROSTAGLANDIN RECEPTORS
PGs exert most of their actions through G protein–­coupled
receptors. They also bind to peroxisome proliferator-
­activated receptors (PPARs) in vitro, but it is not clear
that PPARs mediate PG effects in vivo. Receptors for the
COX products are designated P receptors, depending on the
prostanoid that has most affinity for them. These receptors
include the PGD receptor (DP); four subtypes of the PGE
receptor (EP1 through EP4); the PGF receptor (FP); the
PGI receptor (IP); and the TX receptor (TP). The IP, DP,
EP2, and EP4 receptors mediate increases in cellular cAMP,
whereas TP, FP, and EP1 receptors induce calcium mobiliza-
tion. EP2, EP4, and IP regulate macrophage cytokine pro-
duction in a similar manner. As might be expected, signaling
through these receptors is more complicated. Although
PGI2 analogues are ligands for IP and increase cAMP, high
concentrations of PGI2 analogues activate phospholipase C
and induce calcium mobilization.
It does seem clear that modification of immune cell and
surrounding cell functions by prostanoids during immune or
inflammatory responses is influenced by the different reper-
toire of PG receptors expressed on these cells. Knowledge
of the roles of the P receptors in physiologic and pathologic
processes has been advanced by experiments with mice
deficient in the receptors (knockout mice). As noted previ-
ously, PGE can suppress or induce bone formation. EP2 and
EP4 knockout mice exhibit impaired osteoclastogenesis and
inflammation-induced bone resorption. The receptor medi-
ating PGE-induced bone formation is unknown. In animal
models, acute inflammation and pain are completely absent
in IP-deficient mice. The profile of PG formation changes as
the inflammatory response evolves to a more chronic state,
so other receptors are probably involved. It is unlikely that
blockade of one receptor would block completely an inflam-
matory response. More encouraging is the fact that PGs par-
ticipate in allodynia, a pain response to a usually nonpainful
stimulus. Knowing the contribution of P receptors to allo-
dynia might lead to better treatment of neuropathic pain
and myofascial pain syndromes such as fibromyalgia.
The availability of cloned P receptors should facilitate
development of more effective receptor-active compounds.
The PGI2 analogue iloprost is useful for treatment of
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
peripheral vascular disease and pulmonary hypertension.
Although iloprost binds to IP with high affinity, it also binds
EP1 and EP3. It may be that targeting activation, blockade,
or both of a single P receptor or a specific set of P recep-
tors would provide advantages over compounds that work
“upstream,” such as the COX-2 inhibitors or traditional
NSAIDs. The implications for therapy derived from this new
knowledge are clear and exciting, but prostanoid analogues
with selective binding properties need to be developed.
Some progress has been made, and it seems that deletion
of P receptors, with the exception of EP4, is not associated
with serious problems of fetal development or physiologic
function in animals.
LEUKOTRIENE RECEPTORS
Less is known about the surface receptors for LTs. Receptors
for LTB4 (LTB4 R-1 and LTB4 R-2) and for the cysteinyl LTs
also exert their actions through transmembrane spanning
G protein–coupled protein receptors.57 High-affinity LTB4
receptors transduce chemotaxis and adhesion responses,
whereas low-affinity receptors are responsible for secretion
of granule contents and superoxide generation. The recep-
tor for cysteinyl LTs includes two subtypes—Lys LT1 and Cys
LT2—that have been identified pharmacologically, although
their molecular structures are unknown. Most actions of the
cysteinyl LTs are mediated by Cys LT1. More than a dozen
chemically distinct, specific, and selective antagonist drugs
that block the binding of LT to Cys LT1 have been identi-
fied. Clinical use of these compounds has mainly been in the
treatment of asthma. A 5-lipoxygenase-specific inhibitor
reduced whole-blood LTB4 production, but did not suppress
synovitis in a 4-week trial of RA patients.58 A challenge in
designing “antireceptor therapy” is the genetic variation in
G protein–coupled receptors that can be associated with
disease.59 Adding to the complexity is the fact that vari-
ants may result in altered predisposition to disease, rather
than manifestation of the disease. Each variant provides an
opportunity to understand receptor function, such as recy-
cling or desensitizartion, enhancing the potential for devel-
opment of therapy.
LIPOXIN RECEPTORS
Lipoxins can act at their own specific receptors for LXA4
and LXB4, and LXA4 can interact with a subtype of LTD4
receptors. Lipoxins also can act at intracellular targets
within their cell of origin or after uptake by another cell.
The cDNA for the seven-transmembrane-spanning, G pro-
tein–coupled LXA4 receptor named ALX has been cloned
and characterized. Its signaling involves a novel polyisopre-
nyl-phosphate pathway that regulates phospholipase D.60
Lipoxin actions are cell type–specific. The monocyte and
neutrophil LXA4 receptors are identical at the cDNA level,
but they evoke different responses, and the LXA4 receptor
on endothelial cells seems to be a structurally distinct form.
Nuclear Receptors
PPARs are members of the nuclear receptor family of tran-
scription factors, a large and diverse group of proteins that
mediate ligand-dependent transcriptional activation and
353
repression. PPARs were first cloned as nuclear ­receptors that
mediate the effects on gene transcription of synthetic com-
pounds called peroxisome proliferators. Interest in PPARs
increased dramatically when they were shown to be acti-
vated by medically relevant compounds, including NSAIDs
and PGD2 and its metabolite 15-deoxy-δ12,14 PGJ2.61 Most
information available on a potential role of PPARs in inflam-
mation relates to PPARγ. Upregulation of PPARγ reduces
expression of several mediators of inflammation, raising
the possibility that PPARγ ligands may be therapeutic for
diseases characterized by inflammation. The metabolite
15-deoxy-δ12,14 PGJ2 also can exhibit anti-inflammatory
activity in a PPARγ-independent manner, however. PPARα
is expressed mainly in tissues that have a high fatty acid
catabolism, including liver and the immune system. LTB4 is
an activator and natural ligand of PPARα.62 Activation of
PPARα results in induction of genes involved in fatty acid
oxidation pathways that degrade fatty acids and derivatives,
including LTB4. A feedback mechanism is established that
controls inflammation. Experiments with PPAR knockout
(−/−) mice indicate that PPARα suppresses LTB4-induced
inflammation. PPARs are being considered as therapeutic
targets for a wide range of immune-mediated diseases char-
acterized by chronic inflammation.63
Platelet-Activating Factor
PAF, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine, is a
potent mediator of inflammation that causes neutrophil acti-
vation, increased vascular permeability, vasodilation, and
bronchoconstriction in addition to platelet activation. PAF
is formed by a smaller number of cell types than the eico-
sanoids—mainly leukocytes, platelets, and endothelial cells.
Because of the extensive distribution of these cells, however,
the actions of PAF can manifest in virtually every organ sys-
tem. In contrast to the two long-chain acyl groups present
in phosphatidylcholine, PAF contains a long-chain alkyl
group joined to the glycerol backbone in an ether linkage
at position 1 and an acetyl group at position 2 (Fig. 21-9).
PAF represents a family of phospholipids because the alkyl
group at position 1 can vary in length from 12 to 18 carbons.
PAF, similar to the eicosanoids, is not stored in cells. Rather,
it is synthesized when cells are stimulated, at which time the
composition of the alkyl group may change. The immediate
effects of PAF seem to be mediated through cell surface G pro-
tein–coupled receptors, whereas long-term responses depend
on intracellular—probably nuclear—receptor ­activation.64
Despite the potent inflammatory effects of PAF, its
­inhibition in animal models does not lead to marked sup-
pression of inflammatory responses. Plasma PAF acetylhy-
drolase, an enzyme that hydrolyzes PAF, may be a particularly
O CH2 O(CH2)xCH3
CH2 C O CH O
CH2 O P O CH2 CH2 N+(CH3)3
O
-
Figure 21-9  Chemical structure of platelet-activating factor.
 354 ZURIER  |  Prostaglandins, Leukotrienes, and Related Compounds
i­mportant terminator of PAF-induced tissue injury and
may find a place among strategies designed to suppress
­inflammation.65 In addition, PAF production is suppressed
by adenosine,66 which may account for some of the thera-
peutic efficacy of methotrexate. Given the involvement of
PAF in immediate-hypersensitivity reactions and inflamma-
tion, further search for PAF antagonists is warranted.
Eicosanoids as Regulators
of Inflammation and Immune
Responses
The role of PGs in the inflammatory process is not as well
defined as previously supposed because the stable PGs PGE
and PGI2 have anti-inflammatory and inflammatory effects.67
PGJ and lipoxins seem to act as brakes to protect against
runaway inflammatory responses. Even LTB4 seems capable
of modulating inflammation and immune responses.37 The
observation that PGE1 inhibits platelet aggregation led to
the notion that COX products of AA metabolism might
have anti-inflammatory activity. As it becomes clearer that
NSAIDs have anti-inflammatory effects other than interfer-
ence with COX production and subsequent PG ­inhibition,
the potential protective effects of PGs are being ­considered.
PGE1 has remained an orphan among the eicosanoids,
mainly because of a long-held notion that not enough of
it is made by human cells to be of use, and that its biologic
effects are no different from the effects of PGE2 and PGI2.
Contrary to popular belief, PGE1 is found in physiologically
important amounts in humans. Lost in the vast literature on
the “AA cascade” are the early observations of Bygdeman and
­Samuelsson,68 who found (using bioassay) the concentration of
PGE1 in human seminal plasma (16 μg/mL) to be higher than
PGE2 (13 μg/mL), PGE3 (3 μg/mL), PGF1α (2 μg/mL), and
PGF2α (12 μg/mL). Karim and associates69 found PGE1 to be
the sole PGE in the human thymus. PG immunoassays usually
do not distinguish between PGE1 and PGE2. To identify PGE1,
it must first be separated from PGE2 by thin-layer or high-per-
formance liquid chromatography. When such methods have
been used, PGE1 has been identified consistently in platelets,
leukocytes, macrophages, vas deferens, oviducts, uterus, heart,
and skin.70 Evidence from in vitro and in vivo experiments
indicates that PGs, notably PGE compounds, can suppress
diverse effector systems of inflammation. PGE can enhance
and diminish cellular and humoral immune responses, obser-
vations that reinforce a view of these compounds as regulators
of cell function. These actions of eicosanoids depend on the
stimulus to inflammation, the predominant eicosanoid pro-
duced at a particular time in the host response, and the profile
of eicosanoid-receptor expression.71,72
As noted,11,45,47,48,60 PGJ, lipoxins, and epilipoxins facili-
tate resolution of inflammation. These compounds are
referred to collectively as “resolvins.”
Modulation of Eicosanoid
Synthesis by Administration
of Precursor Fatty Acids
The relationship between essential fatty acids and PGs was
discovered simultaneously and independently by van Dorp
and coworkers73 and Bergstrom and associates.74 Both groups
reported that AA was converted to PGE2, and shortly
thereafter they showed that PGE1 is formed from DGLA.75
Attempts to modulate eicosanoid production have been
directed at providing fatty acids other than AA as substrates
for oxygenation enzymes in an effort to generate a unique
eicosanoid profile with immunosuppressive and anti-inflam-
matory effects.44,76 The fatty acids themselves, by virtue of
their incorporation into signal-transduction elements, also
have effects that are independent of eicosanoid effects on
cells involved in inflammation and immune responses.77
Experiments directed at suppression of TX synthesis,
enhancement of prostacyclin production, and inhibition
of platelet aggregation have been done in an effort to limit
inflammatory responses. EPA is not found in appreciable
amounts in cells from individuals who eat a Western-style
diet. Fish oil lipids, rich in EPA (20:5 n-3), inhibit forma-
tion of COX products (e.g., TXA2, PGE2) derived from
AA, and the newly formed TXA3 has much less ability
than TXA2 to constrict vessels and aggregate platelets. Pro-
duction of PGI2 (prostacyclin) by endothelial cells is not
reduced appreciably by increased EPA content, and the
physiologic activity of newly synthesized PGI3 is added to
that of PGI2. Administration of fish oil to humans leads
to reduced production of LTB4 by means of 5-lipoxygen-
ase in stimulated neutrophils and monocytes and induces
EPA-derived LTB5, which is far less biologically active than
LTB4. Fish oil also reduces production of IL-1β, TNF-α, and
PAF by activated blood monocytes. Fish oil supplements in
the treatment of RA for 6 to 12 months result in significant
reductions in number of tender joints and time of morning
stiffness compared with the same measures done at baseline.
Fish oil treatment allowed patients to reduce or stop NSAID
treatment.78 Human endothelial cells treated with aspirin
in vitro convert EPA to anti-inflammatory lipoxins; these
novel compounds also are found in inflammatory exudates
from animals administered aspirin and fish oil.45
The other “alternative” eicosanoid precursor fatty acid,
DGLA (20:3 n-6), also can be increased by administration
of certain plant-seed oils, notably oils extracted from the
seeds of Oenothera biennis (evening primrose) and Boragio
officinalis (borage), which contain relatively large amounts
of γ-linolenic acid (GLA). GLA is converted to DGLA,
the immediate precursor of PGE1, an eicosanoid with
known anti-inflammatory and immunoregulating proper-
ties. Administration of GLA to volunteers and RA patients
results in increased production of PGE1 and reduced produc-
tion of the inflammatory eicosanoids PGE2, LTB4, and LTC4
by stimulated peripheral blood monocytes. In addition to
competing with AA for oxidative enzymes, DGLA cannot
be converted to inflammatory LTs. Rather, it is converted by
means of 15-lipoxygenase to a 15-hydroxy-DGLA, which
has the capacity to inhibit 5-lipoxygenase and 12-lipoxy-
genase activities. DGLA should have anti-inflammatory
actions because of its capacity to reduce synthesis of oxy-
genation products of AA through the COX and the lipoxy-
genase pathways.79
In addition to their roles as precursors of eicosanoids,
essential fatty acids are important for the maintenance of
cell membrane structure and function and protect the gastric
mucosa from NSAID-induced injury. DGLA can modulate
immune responses in an eicosanoid-independent man-
ner. DGLA suppresses IL-2 production by human periph-
eral blood monocytes in vitro, suppresses ­ proliferation of
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
IL-2-dependent human peripheral blood and synovial tis-
sue T lymphocytes, and reduces expression of activation
markers on T lymphocytes directly in a manner that is
independent of its conversion to eicosanoids. Oral admin-
istration of oils enriched in GLA, but not administration of
oils enriched in linoleic acid (the parent n-6 fatty acid) or
α-linolenic acid (the parent n-3 fatty acid) reduce prolif-
eration of human lymphocytes activated through the T cell
receptor ­complex.80
Addition to peripheral blood mononuclear cells in vitro
or administration of GLA in vivo reduces secretion of IL-1β
and TNF-α from stimulated human cells. GLA also reduces
autoinduction of IL-1β in human monocytes, preserving
the protective effects of IL-1β, while suppressing excessive
production of the cytokine.81,82 IL-1β and TNF-α are impor-
tant polypeptide mediators of inflammation and joint tis-
sue injury in patients with RA, and both cytokines have
become targets of therapy for patients with RA. GLA sup-
presses acute and chronic inflammation, including arthritis,
in several animal models, and randomized, double-blind,
placebo-controlled trials of GLA in patients with RA and
active synovitis indicate that GLA treatment results in
statistically significant and clinically relevant reduction
in signs and symptoms of disease activity compared with
baseline and placebo.83 In addition, treatment with GLA
reduced the need for NSAID and corticosteroid therapy.
EPA suppresses conversion of DGLA to AA, and a
­combination of EPA-enriched and GLA-enriched oils
exhibits synergy in its capacity to reduce synovitis in animal
models.84 A combination of GLA and EPA may be useful
therapy for RA patients. Continued study of the eicosanoids
and their precursor fatty acids should delineate mechanisms
by which these lipids influence the function of cells that par-
ticipate in immune responses and inflammatory reactions.
REFERENCES
  1. Brash AR: Specific lipoxygenase attack on arachidonic acid and
­linoleate esterified in phosphatidylcholine: Precedent for an
­alternative mechanism in activation of eicosanoid biosynthesis. Adv
­Prostaglandin Thromboxane Leukot Res 15:197, 1985.
  2. Zalewski A, Nelson JJ, Hegg L, et al: LpPLA2: A new kid on the block.
Clin Chem 529:1645, 2006.
  3. Farooqui AA, Horrocks LA: Signaling and interplay mediated by
phospholipases A2, C, and D in LA-n-1 cell nuclei. Reprod Nutr Dev
45:613, 2005.
  4. Hasham SN, Pillarisetti S: Vascular lipases, inflammation, and
­atherosclerosis. Clin Chim Acta 372:179, 2006.
  5. Bomalaski JS, Clark MA, Zurier RB: Enhanced phospholipase ­activity
in peripheral blood monocytes from patients with rheumatoid ­arthritis.
Arthritis Rheum 29:312, 1986.
  6. Bozza PT, Yu W, Penrose JF, et al: Eosinophil lipid bodies: Specific,
inducible intracellular sites for enhanced eicosanoid formation. J Exp
Med 186:909, 1997.
7. Smith WL, DeWitt DL, Garavito RM: Cyclooxygenases: Structural,
cellular, and molecular biology. Annu Rev Biochem 69:145, 2000.
  8. Roos KL, Simmons DL: Cyclooxygenase variants: The role of
­alternative splicing. Biochem Biophys Res Commun 338:62, 2005.
  9. Telliez A, Furman C, Pommery N, Henichart J-P. Mechanisms ­leading
to COX-2 expression and COX-2 induced tumorigenesis: ­ Topical
­therapeutic strategies targeting COX-2 expression and activity.
­Anticancer Agents Med Chem 6:187, 2006.
10. Debey S, Meyer-Kirchrath J, Schror K: Regulation of cyclooxygenase-2
expression in iloprost in human vascular smooth muscle cells: Role of
transcription factors CREB and ICER. Biochem Pharmacol 65:979,
2003.
355
11. Morris T, Stables M, Gilroy DW: New perspectives on aspirin and
the endogenous control of acute inflammatory resolution. Sci World J
6:1048, 2006.
12. Dinchuk JE, Car BD, Focht RJ, et al: Renal abnormalities and an
altered inflammatory response in mice lacking cyclooxygenase II.
Nature 378:406, 1995.
13. Thomas L: The Lives of a Cell. New York, Penguin, 1995.
14. Snipes JA: Cloning and characterization of cyclooxygenase-1b (puta-
tive Cox-3) in rat. J Pharm Exp Ther 313:668, 2005.
15. Wu KK, Liou JY: Cellular and molecular biology of prostacyclin syn-
thase. Biochem Biophys Res Commun 338:45, 2005.
16. Zeilhofer HU, Brune K: Analgesic strategies beyond the inhibition of
cyclooxygenases. Trends Pharmacol Sci 27:467, 2006.
17. Kurzrock R, Lieb CC: Biochemical studies of human semen, II:
The action of semen on the human uterus. Proc Soc Exp Biol Med
28:268, 1930.
18. von Euler US: On the specific vasodilating and plain muscle stimu-
lating substances from accessory genital glands in man and certain
animals (prostaglandin and vesiglandin). J Physiol (Lond) 88:213,
1936.
19. Bergstrom S, Ryhage R, Samuelsson B: The structure of prostaglan-
dins E1, F1, and F2. Acta Chem Scand 16:501, 1962.
20. Ferreira SH: Prostaglandins, aspirin-like drugs and analgesia. Nat New
Biol 240:200, 1972.
21. Raisz LG: Pathogenesis of osteoporosis: Concepts, conflicts, and pros-
pects. J Clin Invest 115:3318, 2005.
22. Marks SC, Miller SC: Prostaglandins and the skeleton: The legacy and
challenges of two decades of research. Endocr J 1:337, 1993.
23. Abramson SB, Yazici Y: Biologics in development for rheumatoid
arthritis: Relevance to osteoarthritis. Adv Drug Deliv Rev 58:212,
2006.
24. Herlong JL, Scott TR: Positioning prostanoids of the D and J series in
the immunopathogenic scheme. Immunol Lett 102:121, 2006.
25. Moncada S, Gryglewski R, Bunting S, et al: An enzyme isolated
from arteries transforms prostaglandin endoperoxides to an unstable
substance that inhibits platelet aggregation. Nature 263:633, 1976.
26. Bertagnolli MM, Eagle CJ, Zauber AG, et al: Celecoxib for the preven-
tion of sporadic colorectal adenomas. N Engl J Med 355:873, 2006.
27. Bresser P, Pepke-Zaba J, Jais X, et al: Medical therapies for chronic
thromboembolic pulmonary hypertension: An evolving treatment
paradigm. Proc Am Thorac Soc 3:594, 2006.
28. Remuzzi G, Fitzgerald GA, Patrono C: Thromboxane synthesis and
action within the kidney. Kidney Int 41:1483, 1992.
29. Sciulli MG, Renda Capone ML, et al: Heterogeneity in the suppres-
sion of platelet cyclooxygenase-1 activity by aspirin in coronary heart
disease. Clin Pharmacol Ther 80:115, 2006.
30. Oates JA, Fitzgerald G, Branch RA, et al: Clinical implications of
prostaglandin and thromboxane A2 formation. N Engl J Med 219:761,
1988.
31. Shankar H, Kahner B, Kunapuli SP: G-protein dependent platelet sig-
naling: Perspectives for therapy. Curr Drug Targets 7:1253, 2006.
32. Osher E, Weisinger G, Limor R, et al: The 5 lipoxygenase system in
the vasculature: emerging role in health and disease. Mol Cell Endo-
crinol 252:201, 2006.
33. Chang WC, Chen BK: Transcription factor Sp1 functions as an anchor
protein in gene transcription of human 12(S)-lipoxygenase. Biochem
Biophys Res Commun 338:117, 2005.
34. Kuhn H, O’Donnell VB: Inflammation and immune regulation by
12/15 lipoxygenases. Prog Lipid Res 45:334, 2006.
35. Folco G, Murphy RC: Eicosanoid transcellular biosynthesis: From
cell-cell interactions to in vivo tissue responses. Pharmacol Rev
58:375, 2006.
36. Brash AR: Lipoxygenases: Occurrence, functions, catalysis, and acqui-
sition of substrate. J Biol Chem 274:23679, 1999.
37. Haeggstrom JZ, Wetterholm A: Enzymes and receptors in the leuko­
triene cascade. Cell Mol Life Sci 59:742, 2002.
38. Baud L, Perez J, Denis M, et al: Modulation of fibroblast prolifera-
tion by sulfidopeptide leukotrienes: Effect of indomethacin. J Immunol
138:1190, 1987.
39. Haeggstrom JZ, Kull F, Rudberg PC, et al: Leukotriene A4 hydrolase.
Prostaglandins Other Lipid Mediat 68-69:495, 2002.
40. Jankun J, Aleem AM, Malgorzewicz S, et al: Synthetic curcuminoids
modulate the arachidonic metabolism of human platelet 12-lipoxy-
genase and reduce sprout formation of human endothelial cells. Mol
Cancer Ther 5:1371-1382, 2006.
 356 ZURIER  |  Prostaglandins, Leukotrienes, and Related Compounds
41. Nieves D, Moreno JJ: Hydroxyeicosatetraenoic acids released through
cytochrome P450 pathway regulate 3T6 fibroblast growth. J Lipid Res
47:2681-2689, 2006.
42. Herre S, Schadendorf T, Ivanov T, et al: Photoactivation of the 12/15
lipoxygenase pathway. Chembiochemistry 7:1089, 2006.
43. Mani I, Iversen L, Ziboh VA: Upregulation of nuclear PKC and MAP-
kinase during hyperproliferation of guinea pig epidermis: Modulation
by 13-(s) hydroxyoctadecadienoic acid (13-HODE). Cell Signal
10:143, 1998.
44. Calder PC: Polyunsaturated fatty acids and inflammation. Prostaglan-
dins Leukotr Fatty Acids 75:197, 2006.
45. Serhan CN, Cish CB, Brannon J, et al: Anti-microinflammatory
lipid signals generated from dietary N-3 fatty acids via cyclo-
oxygease-2 and transcellular processing: A novel mechanism for
NSAID and N-3 PUFA therapeutic actions. J Physiol Pharmacol
51:643, 2000.
46. Chiang N, Serhan CN: Cell-cell interaction in the transcellular
­biosynthesis of novel omega-3 derived lipid mediators. Methods Mol
Biol 341:227, 2006.
47. Ohse T, Ota T, Godson C, et al: Modulation of interferon induced
genes by lipoxin analogue in anti-glomerular basement membrane
nephritis. J Am Soc Nephrol 15:919, 2004.
48. Lawson JA, Kim S, Powell WS, et al: Oxidized derivatives of omega-3
fatty acids: Identification of IPF3alpha-VI in human urine. J Lipid Res
47:2515-2524, 2006.
49. Milne G, Morrow JD: Isoprostanes and related compounds: Update
2006. Antioxid Redox Signal 8:1379, 2006.
50. Burstein S: The cannabinoid acids: Nonpsychoactive derivatives
with therapeutic potential. Pharmacol Ther 82:87, 1999.
51. Rockwell CE, Snider NT, Thompson JT, et al: Interleukin-2
­suppression by 2-arachidonylglycerol is mediated through peroxisome
proliferator activated receptor gamma independently of cannabinoid
receptorrs 1 and 2. Mol Pharmacol 70:101, 2006.
52. Burstein SH, Rossetti RG, Yagen B, et al: Oxidative metabolism of
anandamide. Prostaglandins Other Lipid Mediat 61:29, 2000.
53. Ross RA, Craib SJ, Stevenson LA, et al: Pharmacological character-
ization of the anandamide cyclooxygenase metabolite: Prostaglandin
E2 ethanolamide. J Pharmacol Exp Ther 301:900, 2002.
54. Guindon J, DeLean A, Beaulieu P: Local interactions between
­anandamide, an endocannabinoid, and ibuprofen, a nonsteroidal
antiinflammatory drug, in acute and inflammatory pain. Pain 21:85,
2006.
55. Kohno M, Hasegawa H, Inoue A, et al: Identification of N-arachido-
nylglycine as the endogenous ligand for the orphan G-protein-coupled
receptor GPR18. Biochem Biophys Res Commun 347:827, 2006.
56. Hata AN, Breyer RM: Pharmacology and signaling of prostaglandin
receptors: Multiple roles in inflammation and immune modulation.
Pharmacol Ther 103:147, 2006.
57. Sharma JN, Mohammed LA: The role of leukotrienes in the patho-
physiology of inflammatory disorders: Is there a case for revisiting
leukotrienes as therapeutic targets? Inflammopharmacology 14:99,
2006.
58. Weinblatt ME, Kremer JM, Coblyn JS, et al: Zileuton, a 5-lipoxygen-
ase inhibitor in rheumatoid arthritis. J Rheumatol 19:1537, 1992.
59. Thompson MD, Burnham WM, Cole DE: The G-protein coupled
receptors: Pharmacogenetics and disease. Crit Rev Clin Lab Sci
42:311, 2005.
60. Chiang N, Serhan CN, Dahlen S-E, et al: The lipoxin receptor
ALX: Potent ligand-specific and stereoselective actions in vivo. Phar-
macol Rev 58:463, 2006.
61. Ricote M, Li AC, Willson TM, et al: The peroxisome-proliferator-
activated receptor-gamma is a negative regulator of macrophage acti-
vation. Nature 391:79, 1998.
62. Devchand PR, Keller H, Peters JM, et al: The PPARα-leukotriene B4
pathway to inflammation control. Nature 384:39, 1996.
63. Sundararajan S, Jiang Q, Heneka M, et al: PPARgamma as a therapeu-
tic target in central nervous system diseases. Neurochem Int 49:136,
2006.
64. Zhu T, Gobeil F, Vazquez-Tello A, et al: Intracrine signalling through
lipid mediators and their cognate nuclear G-protein coupled receptors:
A paradigm based on PGE2, PAF, and LPA1 receptors. Can J Physiol
Pharmacol 84:377, 2006.
65. Zimmerman GA, McIntyre TM, Prescott SM, et al: The platelet acti-
vating factor signaling system and its regulators in syndromes of inflam-
mation and thrombosis. Crit Care Med 30(Suppl 5):S294, 2002.
66. Flamand N, Lefebvre J, Lapointe G, et al: Inhibition of platelet acti-
vating factor biosynthesis by adenosine and histamine in human
neutrophils: Involvement of cPLA2alpha and reversal by lysoPAF.
J Leukoc Biol 79:1043, 2006.
67. Zurier RB: Prostaglandins, fatty acids, and arthritis. In Cummingham-
Rundles S (ed): Nutrient Modulation of the Immune Response. New
York, ­Marcel Dekker, 1993, p 201.
68. Bygdeman M, Samuelsson B: Quantitative determination of
­prostaglandins in human semen. Clin Chim Acta 10:566, 1964.
69. Karim SMM, Soindler M, Williams ED: Distribution of prostaglandins
in human tissues. Br J Pharmacol Chemother 31:340, 1967.
70. Horrobin DF: The roles of essential fatty acids in the development
of diabetic neuropathy and other complications of diabetes mellitus.
Prostaglandins Leukot Essent Fatty Acids 31:181, 1988.
71. Tilley SL, Coffman TM, Koller BH: Mixed messages: ­ Modulation
of inflammation and immune responses by prostaglandins and
­thromboxanes. J Clin Invest 108:15, 2001.
72. Brash AR: Arachidonic acid as a bioactive molecule. J Clin Invest
107:7700, 2001.
73. van Dorp DA, Beer Thuis RK, Nugteren DH: The biosynthesis of
prostaglandins. Biochim Biophys Acta 90:204, 1964.
74. Bergstrom S, Daniellson H, Samuelsson B: The enzymatic formation
of prostaglandin E2 from arachidonic acid. Biochim Biophys Acta
90:207, 1964.
75. Bergstrom S, Daniellson H, Klenberg D, et al: The enzymatic
­conversion of essential fatty acids into prostaglandins. J Biol Chem
239:4006, 1964.
76. Calder PC, Zurier RB: Polyunsaturated fatty acids and rheumatoid
arthritis. Curr Opin Clin Nutr Metabol Care 4:115, 2001.
77. Vassilopoulos D, Zurier RB, Rossetti RG, et al: Gammalinolenic acid
and dihomogammalinolenic acid suppress the CD3 mediated signal
transduction pathway in human T cells. Clin Immunol Immunopathol
83:237, 1997.
78. Kremer JM: Effects of modulation of inflammatory and immune
­parameters in patients with rheumatic and inflammatory disease
receiving dietary supplementation of n-3 and n-6 fatty acids. Lipids
31:S253, 1996.
79. DeLuca P, Rothman D, Zurier RB: Marine and botanical lipids
as immunomodulatory and therapeutic agents in the treatment of
­rheumatoid arthritis. Rheum Dis Clin North Am 21:759, 1995.
80. Rossetti RG, Seiler CM, DeLuca P, et al: Oral administration
of ­ unsaturated fatty acids: Effects on human peripheral blood T
lympho­cyte proliferation. J Leukoc Biol 62:438, 1997.
81. Furse RK, Rossetti RG, Zurier RB: Gamma linolenic acid, an
­unsaturated fatty acid with antiinflammatory properties, blocks
­amplification of IL-1β production by human monocytes. J Immunol
167:490, 2001.
82. Furse RK, Rossetti RG, Seiler CM, et al: Oral administration of ­gamma
linolenic acid, an unsaturated fatty acid with antiinflammatory prop-
erties, modulates interleukin-1β production by human monocytes.
J Clin Immunol 22:83, 2002.
83. Zurier RB, Rossetti RG, Jacobson EW, et al: Gamma linolenic acid
treatment of rheumatoid arthritis: A randomized, placebo-controlled
trial. Arthritis Rheum 39:1808, 1996.
84. Tate G, Mandell BF, Karmali RA, et al: Suppression of monosodium
urate induced inflammation by diets enriched with gamma-linolenic
acid and eicosapentaenoic acid. Arthritis Rheum 31:1543, 1988.
 22
KEY POINTS
Leukocyte recruitment through the vessel wall into the
synovium is a crucial process in the pathogenesis of arthritis.
A number of cell adhesion molecules are involved in
leukocyte extravasation.
Chemokines and their receptors are involved in the chemotaxis
of neutrophils, lymphocytes, and monocytes into tissues.
Angiogenesis, the formation of new vessels, is involved in
inflammation and tumor progression.
A number of soluble and cell-bound factors, including
chemokines and adhesion receptors, may stimulate or inhibit
angiogenesis.
Specific targeting of leukocyte adhesion, chemokines, or
angiogenesis, primarily by the use of agents with multiple
actions, may be useful for the future management of arthritis.
Many cell types, including inflammatory leukocytes, endo-
thelial cells (ECs), and synovial fibroblasts, as well as soluble
mediators and cell adhesion molecules (CAMs), are involved
in cell trafficking into inflammatory sites in diseases such as
rheumatoid arthritis (RA).1-6 In arthritis, leukocyte ingress
into the synovium occurs by leukocyte adhesion to ECs
and then by transendothelial migration (Fig. 22-1).2-4 The
chemotaxis of these cells is regulated mainly by chemotac-
tic mediators termed chemokines.7-10 The formation of blood
vessels, termed angiogenesis, is a key event underlying tissue
inflammation, which perpetuates the recruitment of leu-
kocytes.8,11 Several CAMs interacting with soluble inflam-
matory mediators, such as cytokines and chemokines, are
involved in leukocyte extravasation and angiogenesis.2,3,8,10,11
In this chapter, we use the example of inflammatory syno-
vitis to describe the role of vascular ECs in the pathogenesis of
rheumatic diseases. The role of CAMs and chemokines is also
discussed, followed by a description of the two key processes:
leukocyte recruitment and angiogenesis. Some aspects of the
regulatory mechanisms and the clinical importance of adhe-
sion and angiogenesis research are discussed as well.
MORPHOLOGY AND FUNCTION
OF THE VASCULAR ENDOTHELIUM
IN INFLAMMATION
It is now clear that ECs are not just passive bystanders but are
very active players in inflammation. The vascular endothe-
lium undergoes morphologic changes, including vasodilation
and increased permeability (leakage), during the evolu-
tion of ­ synovitis.5,6,12 Vasodilation itself may promote leak-
age. Increased endothelial permeability results from several
Cell Recruitment
and Angiogenesis
ZoltÁn Szekanecz  • Alisa E. Koch
mechanisms, including EC contraction and retraction, leu-
kocyte- or anti-EC antibody–mediated vascular injury, and
EC regeneration.5,6,12
ECs secrete several mediators resulting in vasodilation,
including prostacyclin, nitric oxide, and platelet-activating
factor.12,13 Thrombin, histamine, and leukotriene C4 may
support the synthesis of these vasodilators.5,6,13
The morphologic basis for increased vascular permeabil-
ity and leakage is EC retraction and contraction, resulting in
wider intercellular gaps.12 Several mediators, including hista-
mine, serotonin, complement factors (C3a, C5a), bradykinin,
leukotrienes, platelet-activating factor, and anti-EC anti-
bodies, may promote vascular leakage.5,6,12,13 The gaps form as
a result of EC contraction mediated by vasoactive agents.12
Cytoskeletal reorganization leading to EC retraction may be
regulated by proinflammatory cytokines such as interleukin-1
(IL-1), tumor necrosis factor-α (TNF-α), or interferon-γ
(IFN-γ). Treatment of cultured EC monolayers with any of
these cytokines results in EC retraction and increased vas-
cular permeability lasting for days.5,6,14 Thus, the mechanism
of EC retraction, which is long term and is influenced by
cytokines, is different from that of EC contraction, which
occurs more rapidly and is rather histamine dependent. Fur-
ther, whereas EC retraction is an example of EC stimulation,
EC contraction is a result of EC activation.5,6,12,13
Among other soluble mediators, anti-EC antibodies
have been detected in several autoimmune and inflamma-
tory diseases, including RA, lupus, vasculitis, and others.5,6,15
Moreover, the production of anti-EC antibodies has been
correlated with some markers of clinical activity in these
disorders.15 Circulating anti-EC antibodies may be markers
of vascular damage.5,6,15
Leukocyte-EC interactions (described later) may them-
selves trigger EC injury and thus vascular leakage. Briefly,
the key mediators in this process are reactive oxygen inter-
mediates and matrix metalloproteinases (MMPs) produced
by inflammatory leukocytes.5,6 The outcome of leukocyte-
mediated EC injury depends on activated leukocytes,
because nonactivated neutrophils extravasate without
affecting endothelial permeability.5,6
Vascular regeneration occurring after injury or during
angiogenesis may also be associated with leakage.5,6,8,11,16
Endothelial regeneration may occur without the formation
of new blood vessels, when regeneration is accompanied by
increased capillary permeability.5,6
INTERCELLULAR ADHESION MOLECULES
Adhesion of peripheral blood leukocytes to ECs leads to the
process of leukocyte transendothelial migration into inflam-
matory sites1-4 (see Fig 22-1). For example, in arthritis, the
357
 358 SZEKANECZ  |  Cell Recruitment and Angiogenesis

Rolling Activation Firm adhesion Transendothelial Subendothelial

migration migration

Lymphocyte SLex, CIA,and other Cytokine, β1, β2, and β7 PECAM-1, β2 β1 and β2
Sialylated fucosylated chemokine, and integrins integrins; β1 and β7 integrins; CD44
structures chemoattractant integrins (?)
L-selectin receptors

Endothelial
P-selectin Chemokines (e.g., ICAM-1; PECAM-1
L-selectin Ligand IL-8, MCP-1, ICAM-2 ICAM-1
E-selectin MIP-1β) VCAM-1; VCAM-1
CD34 PAF MAdCAM-1 (?)
MadCAM-1 PECAM-1
E-selectin

Tissue Histamine Cytokines Chemokines

Cytokines Extracellular
Thrombin
(e.g., GM-C5F-IL-8) (e.g., TNF-α, Chemoattractants matrix
Oxidants
LPS Chemoattractants IL-1, IFN-γ, Components
Leukotrienes (e.g., C5a, FMLP) IL-4) Chemokines
Cytokines Chemokines Chemoattractants
(e.g., IL-1, TNF-α) (e.g., IL-8, MCP-1)

Figure 22-1  The process of leukocyte extravasation into the synovium.

cascade of events begins with the adhesion of neutrophils,
lymphocytes, and monocytes to the specialized, fenestrated
synovial ECs.2,3 Microvessels resembling high endothelial
venules, found primarily in lymphoid organs and involved
in the physiologic “homing” of lymphocytes, are also pres-
ent in synovial tissue2 (see Fig. 22-1). Thus, inflammatory
leukocyte recruitment to the synovium and to other tissues
may be considered “pathologic homing.”1-4
EC adhesion to synovium-infiltrating leukocytes or to
extracellular matrix components is mediated by adhesion
receptors and their ligands. These CAMs have been classi-
fied into a number of distinct superfamilies, including integ-
rins, selectins, immunoglobulins, cadherins, and others2-4,17,18
(Table 22-1).
The selectin superfamily includes E-, P- and L-selectin. All
selectins contain a lectin-like extracellular N-terminal domain,
an epidermal growth factor (EGF)–like motif, and two to nine
moieties related to complement regulatory proteins. E- and
P-selectin are expressed by ECs, while L-selectin is expressed
mainly by leukocytes.2-4,17-19 ­During leukocyte transendothelial
migration, selectins mediate the initial tethering and rolling of
leukocytes, which enable subsequent firm adhesion mediated
mostly by integrins.4,18-20
E-selectin is a good marker for cytokine-induced EC acti-
vation.2,19 Ligands for E-selectin, such as E-selectin ligand-1
and P-selectin ligand-1, contain sialylated glycan motifs,
such as sialyl Lewis-X..2,3,17,19 E-selectin has been associated
with RA synovitis.2,3,17 There is abundant expression of
E-selectin in RA synovial tissues and increased production
of soluble E-selectin in RA synovial fluids.2-4
P-selectin is constitutively present on the membrane
of EC Weibel-Palade bodies.2,19 Proinflammatory cytokines
upregulate P-selectin expression on ECs within seconds.
Thus, P-selectin is involved in the very early phases of
leukocyte-EC adhesion.21 P-selectin is expressed by syno-
vial ECs.2-4
L-selectin is absent from ECs but present on most leu-
kocytes. L-selectin serves as a lymphocyte homing recep-
tor, where it mediates the physiologic recirculation of
naive lymphocytes through specialized high endothelial
venules.17-19 In addition, L-selectin may be involved in
leukocyte-EC interactions underlying arthritis.2,3 In the
synovium, L-selectin is expressed by synovial lining cells
and sublining leukocytes.2,3,22
Integrins are αß heterodimers and are classified into sub-
families with respect to their common ß subunits. Each of
the common ß chains is associated with one or more a sub-
units.2-4,17 Each subunit contains a long extracellular domain,
a transmembrane region, and a cytoplasmic domain. The
last may be connected to the actin cytoskeleton and thus
may trigger signal transduction events.17 Cell adhesion to the
extracellular matrix is mediated mainly by ß1, while intercel-
lular adhesion is facilitated through both ß1 and ß2 integrins
binding to CAMs belonging to the immunoglobulin super-
family.4,17 Both ß1 and ß3 integrins are expressed on ECs.2,3,17
Integrin-mediated adhesion pathways have been impli-
cated in leukocyte-EC interactions during inflammation.
For example, ß1 and ß2 integrins are involved in animal
models of arthritis.2,3,23 Abundant expression of EC integ-
rins was described in human synovitis.2-4 Without going into
detail, the α1, α2, α3, α4, α5, α6, αV, αL, αM, αX, ß1, ß2,
ß3, ß4, ß5, and ß7 integrin subunits have been detected on
various cells in the inflamed synovium.2-4
The immunoglobulin superfamily is a group of transmem-
brane glycoproteins containing one or more immunoglobulin-
like motifs of 60 to 100 amino acids.17 Vascular cell adhesion
molecule-1 (VCAM-1) is a member of this superfamily.
VCAM-1 is constitutively expressed on resting ECs; how-
ever, its expression is strongly upregulated by proinflammatory
cytokines.2,3,17 There is abundant VCAM-1 ­expression in the
inflamed synovium.2-4,24 Soluble VCAM-1 has been detected
in RA sera and synovial fluid samples.2,3,25
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION 359

Table 22-1  Relevant Members of the Selectin,   was originally isolated from synovial ECs. The expression
Integrin, Immunoglobulin, and Cadherin Adhesion   of VAP-1 is increased in RA.2,29 Endoglin is a receptor for
Molecule Superfamilies transforming growth factor-ß (TGF-ß). Endoglin is involved
Adhesion Receptors Ligands in EC adhesion and is expressed in synovitis.2,3 Cadherins
mediate homophilic, calcium-dependent adhesion.4,17 The
Selectins
cytoplasmic tail of cadherins interacts with ß-catenin,
L-selectin (CD62L, LAM-1) Sialylated carbohydrates,   forming a link between the cadherin-catenin complex
GlyCAM-1
E-selectin (CD62E, ELAM-1) Sialyl-Lewis-X
and the cytoskeleton. Cadherins are primarily involved in
P-selectin (CD62P, PADGEM) Sialyl-Lewis-X embryogenesis; however, E-cadherin, N-cadherin, and cad-
herin-11 are present in the inflamed synovium as well.4,17,31
Integrins
JAM-1, JAM-2, and JAM-3, as well as CD99, have also
α1β1 (VLA-1) Laminin, collagen been ­ implicated in leukocyte migration through endothe-
α2β1 (VLA-2) Laminin, collagen lial junctions.5,6,30 ICAM-3 is a leukocyte CAM, which is a
α3β1 (VLA-3) Laminin, collagen, fibronectin
α4β1 (VLA-4) Fibronectin, VCAM-1 known ligand for LFA-1. It is absent from most types of ECs.
α5β1 (VLA-5) Fibronectin However, it is present on some RA synovial ECs.2,26
α6β1 (VLA-6) Laminin
αLβ2 (LFA-1, CD11a/CD18) ICAM-1, ICAM-2, ICAM-3, JAM-A
αMβ2 (Mac-1, CD11b/CD18) ICAM-2, iC3b CHEMOKINES AND CHEMOKINE
αXβ2 (CD11c/CD18) iC3b, fibrinogen RECEPTORS
αEβ7 E-cadherin
α4β7 Fibronectin, VCAM-1, MadCAM-1 Chemokines are small proteins exerting chemotactic activ-
Immunoglobulins ity toward immune cells.7-9,32,33 Chemokines drive leuko-
cytes to inflamed tissues.33 Chemokines have been classified
ICAM-1 (CD54) LFA-1, Mac-1
ICAM-2 LFA-1
into supergene families with respect to their structure.7-9,32
ICAM-3 LFA-1 According to the location of cysteine (C) residues, these
VCAM-1 α4β1, α4β7 families are designated as CXC, CC, C, and CX3C chemo-
MadCAM-1 α4β7, L-selectin kines, and the four respective chemokine receptor groups
CD2 LFA-3 are CXCR, CCR, CR, and CX3CR7-9,32 (Table 22-2). More
PECAM-1 (CD31) PECAM-1, αVβ3
than 50 chemokines and 19 chemokine receptors have been
Cadherins identified.7-9,32 Some years ago, a new nomenclature was
E-cadherin (cadherin-1) E-cadherin introduced whereby chemokines are considered ligands of
N-cadherin (cadherin-2) N-cadherin chemokine receptors, and each chemokine has been desig-
Cadherin-11 Cadherin-11 nated CXCL, CCL, XCL or CX3CL1.9,32
See text for abbreviations. Recently, chemokine–chemokine receptor pairs have
also been categorized according to their function: some
of them are primarily homeostatic (also called ­constitutive,
Intercelluar adhesion molecule-1 (ICAM-1) is a ligand for housekeeping, or lymphoid), and others are inflammatory
the ß2 integrins LFA-1 (αLß2), Mac-1 (αMß2), and αXß2.4,17,26 (or inducible), although these functions often overlap.9,33
ICAM-1 expression on ECs can be induced by IL-1, TNF-α, Generally, all chemokines involved in the pathogen-
and IFN-γ.12 The ICAM-1–ß2 integrin–dependent adhesion esis of arthritis can be considered inflammatory. Homeo-
pathway is crucial in inflammation; patients with leukocyte- static chemokines generally play a role in physiologic
adhesion deficiency syndrome who have mutations in the ­lymphocyte recruitment and the development of lym-
ß2 integrin subunit show minimal inflammatory response.27 phoid ­ tissues. However, some homeostatic chemokines
ICAM-1 is highly expressed on ECs in inflammatory sites, have also been implicated in inflammation-associated
such as in RA synovium.2-4,24 Again, high levels of soluble B cell migration.9,33
ICAM-1 have been detected in the synovia of RA patients.2,3 Most CXC chemokines chemoattract neutrophils.34 How-
Other CAMs involved in leukocyte-EC adhesion under- ever, platelet factor-4 (PF-4)/CXCL4 and IFN-γ-inducible
lying inflammation include LFA-3, CD44, vascular adhe- 10-kD protein (IP-10)/CXCL10 recruit lymphocytes and
sion proteins (VAP-1 and VAP-2), endoglin, E-cadherin, monocytes.7,34 In addition, some CXC chemokines promote
N-cadherin, cadherin-11, junctional adhesion molecules angiogenesis, while others inhibit it (discussed later).8 IL-8/
(JAMs), platelet-endothelial cell adhesion molecule-1 CXCL8, epithelial-neutrophil activating protein-78 (ENA-
(PECAM-1, CD31), CD99, and possibly ICAM-3.2-4,17,22,28-30 78)/CXCL5, growth-regulated oncogene α (Groα)/CXCL1,
Both LFA-3 and its counterreceptor, CD2, are members connective tissue–activating peptide III (CTAP-III)/CXCL7,
of the immunoglobulin superfamily. LFA-3 is present on granulocyte chemotactic protein-2 (GCP-2)/CXCL6, IP-10/
ECs, and the CD2–LFA-3 adhesion pathway is involved in CXCL10, PF-4/CXCL4, monokine induced by IFN-γ (Mig)/
T cell adhesion to ECs in various inflammatory responses.2,3,28 CXCL9, stromal cell–derived factor-1 (SDF-1)/CXCL12,
LFA-3 is present on synovial ECs and on lining and sublin- B cell–activating chemokine-1 (BCA-1)/CXCL13, and,
ing cells.28 PECAM-1, another member of the immunoglob- recently, CXCL16 have been implicated in arthritis.7-9 These
ulin superfamily, mediates homotypic adhesion as well as chemokines were detected in the synovial fluid or synovial
heterotypic adhesion by recognizing the αVß3 integrin.2,3,17 tissue of RA patients.7-9 Thus, these chemokines may be con-
PECAM-1 is a marker of activated ECs, and it is expressed sidered inflammatory.7-9,33
in RA synovium.2,17 CD44 is a receptor for hyaluronate17 and CC chemokines stimulate monocyte chemotaxis, but some
is expressed on activated ECs in inflammatory sites.2,22 VAP-1 members of this subclass may also recruit lymphocytes.35
 360 SZEKANECZ  |  Cell Recruitment and Angiogenesis

Table 22-2  Chemokine Receptors and Their Most   chemokines implicated in synovitis, plays a crucial role in
Relevant Ligands inflammation and angiogenesis. CXCR3 is a receptor for
Chemokine Receptor Chemokine Ligand
angiostatic CXC chemokines.7-9,39 Chemokine receptors
have also been associated with various subtypes of inflam-
CXC Chemokine Receptors matory response. It has been suggested that CXCR3 and
CXCR1 IL-8/CXCL8 CCR5 are involved in T helper type 1 (Th1) diseases such
CXCR2 IL-8/CXCL8, ENA-78/CXCL5, Groα/ as RA, whereas CCR3, CCR4, and CCR8 have been impli-
CXCL1, CTAP-III/CXCL7
CXCR3 IP-10/CXCL10, PF-4/CXCL4, Mig/
cated in asthma-associated T helper type 2 (Th2) inflam-
CXCL9, ITAC/CXCL11 mation.7,33 Several CXC and CC chemokine receptors, as
CXCR4 SDF-1/CXCL12 well as XCR1 and CX3CR1, are expressed in the arthritic
CXCR5 BCA-1/CXCL13 synovium.7-9
CXCR6 CXCL16
CXCR7 SDF-1/CXCL12, ITAC/CXCL11
CC Chemokine Receptors PROCESS OF LEUKOCYTE RECRUITMENT
CCR1 MIP-1α/CCL3, RANTES/CCL5, Inflammatory leukocyte adhesion to ECs occurs in a
MCP-3/CCL7, MPIF-1/CCL23 sequence of well-regulated steps. An early, weak adhe-
CCR2 MCP-1/CCL2, MCP-3/CCL7 sion, termed “rolling,” occurs first; this event is mediated
CCR3 RANTES/CCL5, MCP-2/CCL8
CCR4 TARC/CCL17, MDC/CCL22 mainly by selectins and their ligands. This is followed by
CCR5 MIP-1α/CCL3, MIP-1β/CCL4, leukocyte activation, which is dependent on interactions
RANTES/CCL5 between chemokine receptors expressed on leukocytes and
CCR6 MIP-3α/CCL20 proteoglycans on ECs. Activation-dependent, firm adhesion
CCR7
CCR8
MIP-3β/CCL19, SLC/CCL21
I-309/CCL1
involves mostly α4ß1 integrin–VCAM-1, LFA-1–ICAM-1,
CCR9 TECK/CCL25 and JAM-integrin interactions, as well as the secretion of
CCR10 CTACK/CCL27, MEC/CCL28 numerous chemokines described earlier. Chemokines may
C Chemokine Receptors also upregulate integrin expression via phosphatidylino-
sitol 3-kinase (PI3K)–mediated pathways (discussed in
XCR1 Lymphotactin/XCL1
more detail later). Transendothelial migration or diapedesis
CX3C Chemokine Receptors involving integrins occurs when chemokines bind to endo-
CX3CR1 Fractalkine/CX3CL1 thelial heparan sulfate. Chemokines preferentially attract
Other
EC-bound leukocytes1,18,20 (see Fig. 22-1).
DARC Duffy antigen, some CC and CXC
chemokines ANGIOGENESIS: ROLE OF CHEMOKINES
See text for abbreviations. AND ADHESION RECEPTORS
Angiogenesis, the formation of new blood vessels, is patho-
logically enhanced in a number of inflammatory diseases,
Among these chemokines, monocyte chemoattractant such as RA and psoriasis, as well as in malignancies.8,11 The
protein-1 (MCP-1)/CCL2, macrophage inflammatory protein- outcome of such angiogenic diseases is dependent on the
1a (MIP-1a)/CCL3, MIP-3a/CCL20, RANTES/CCL5, balance or imbalance between angiogenic mediators and
Epstein-Barr virus–induced gene 1 ligand chemokine (ELC)/ angiostatic factors (Fig. 22-2). Several cytokines, growth
CCL19, SLC/CCL21 and, recently, chemokine-like factor- factors, chemokines, certain CAMs, and other mediators
1 (CKLF1) have been implicated in inflammatory mecha- can modulate neovascularization. Angiogenesis inhibition
nisms underlying synovitis.7-9 by blocking the action of angiogenic mediators, or by the
The C chemokine family contains two members: lym- administration of angiostatic compounds, may be useful for
photactin/XCL1 and single C motif 1ß (SCM-1ß)/XCL2.36 suppressing various inflammatory processes, such as arthri-
­Lymphotactin/XCL1 has been detected on T cells in RA.7,8,36 tis8,11,40,41 (Table 22-3). Here, we focus on the role of chemo-
The CX3C subset contains a single member: fractalkine/ kines and CAMs in angiogenesis, because they are crucial
CX3CL1.7,8,37 Fractalkine is a mononuclear cell chemoat- in leukocyte recruitment. Other angiogenic and angiostatic
tractant, but is also serves as a CAM.7,8,37 This chemokine factors, such as growth factors, cytokines, proteases, and
has been detected in RA synovial fluid and tissue samples.37 others, are included in Table 22-3.
Fractalkine/CX3CL1 is also angiogenic.8 It has been impli- In general, the angiogenic or angiostatic action of che-
cated in the development of accelerated atherosclerosis,38 mokines depends on whether their structure contains the
which is the primary cause of death in RA patients. ELR amino acid motif or not.39 ELR-containing chemokines,
There is a redundancy between the CXC and CC che- such as IL-8/CXCL8, ENA-78/CXCL5, Groα/CXCL1, and
mokine receptors and their ligands (see Table 22-2). For CTAP-III/CXCL7, stimulate neovascularization. In con-
example, CXCR2, CCR1, and CCR3 have numerous che- trast, the ELR-lacking PF-4/CXCL4, IP-10/CXCL10, and
mokine ligands. In contrast, CXCR6, CCR8, and CCR9 Mig/CXCL9 inhibit angiogenesis.8,9,39 However, an excep-
are specific receptors for their respective single ligands.7-9 tion to the rule is SDF-1/CXCL12, which is angiogenic
There may be a relationship between a certain chemokine even though it lacks the ELR sequence.7-9,11
receptor and the function of its ligand. Single-ligand recep- Among CXC chemokines, IL-8/CXCL8 is a major
tors, such as CCR8 or CCR9, bind mostly to homeostatic ­regulator of angiogenesis in RA.7-9,11,39 In addition, ENA-78/
chemokines. In contrast, CXCR2, a receptor for most CXC CXCL5, CTAP-III/CXCL7, and Groα/CXCL1 also stimulate
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION 361

Angiogenic factors Macrophage Fibroblast Angiostatic factors

Chemokines Chemokines
Adhesion molecules Cytokines
Matrix components Lumen Matrix components
Cytokines Protease inhibitors
Growth factors DMARDs
Proteases Anti-TNF agents
Others Others
Endothelium

Lumen

Angiogenesis
Figure 22-2  Imbalance between angiogenic and angiostatic factors in the inflamed synovium. DMARD, disease-modifying antirheumatic drug; 
TNF, tumor necrosis factor.

­neovascularization in RA.7-9,11 All these chemokines have Table 22-3  Angiogenic and Angiostatic Factors  
been detected in the blood or synovia of RA patients.7-9 in Rheumatoid Arthritis
SDF-1/CXCL12 is a specific ligand for CXCR4. SDF-1/ Factor Mediators Inhibitors
CXCL12, despite lacking the ELR motif, promotes neovas-
Chemokines IL-8/CXCL8, ENA-78/ PF-4/CXCL4, IP-10/
cularization.8,42 This chemokine induces EC chemotaxis CXCL5, Groα/CXCL1, CXCL10, Míg/
in vitro and angiogenesis in mice in vivo.42 Thus, SDF-1 CTAP-III/CXCL7,   CXCL9, SLC/CCL21
may be the first angiogenic CXC chemokine that lacks SDF-1/CXCL12,  
the ELR motif. Circulating human CD34+ cells expressing MCP-1/CCL2, SLC/
the vascular endothelial growth factor-2 (VEGF-2) recep- CCL21, MPIF/CCL23,
fractalkine/CX3CL1
tor have been identified and characterized as EC precur-
sor cells.43 Virtually all CD34+/VEGF-2 receptor–positive Matrix   Type I collagen,   Thrombospondin,
molecules fibronectin, laminin, RGD sequence
cells express CXCR4 and migrate in response to SDF-1/ heparin, heparan
CXCL12.42,43 sulfate
In contrast, IP-10/CXCL10 exerts proinflammatory but Cell adhesion   β1 and β3 integrins,   RGD sequence  
antiangiogenic effects in RA.7-9,11 However, a recent study molecules E-selectin, P-selectin, (integrin ligand)
suggests that the effect of VEGF on ECs may be mediated, CD34, VCAM-1,  
in part, by IP-10/CXCL10.44 IP-10/CXCL10 inhibits VEGF- endoglin, PECAM-1,
VE-cadherin, Ley/H,
induced EC motility.45 Thus, VEGF and IP-10/CXCL10 may MUC-18
form an autocrine regulatory loop in angogenesis.44,45 The
Growth factors VEGF, bFGF, aFGF,   TGF-β*
ELR-lacking Mig/CXCL9 and PF-4/CXCL4 are also angio- PDGF, EGF, IGF-1,  
static.8,11,39 All these chemokines have been implicated in HIF-1, TGF-β*
the pathogenesis of RA.7-9,11 Cytokines TNF-α, IL-6*, IL-15, IL-18 IL-4, IL-6*, IFN-α, IFN-γ
Among CXC chemokine receptors, CXCR2 recognizes
Proteases MMPs, plasminogen TIMPs, plasminogen
the most important proinflammatory and proangiogenic activators activator inhibitors
CXC chemokines described earlier.7-9 CXCR2 is expressed
Others Angiogenin,   DMARDs, infliximab,
in the RA synovium.7-9 The role of the SDF-1/CXCL12 substance P,   etanercept, angio-
receptor CXCR4 in neovascularization and the migra- prolactin statin, endostatin
tion of EC progenitor cells has already been discussed.42,43
See text for abbreviations.
It is generally believed that SDF-1/CXCL12 has a single *Mediators with both pro- and antiangiogenic effects.
receptor, CXCR4; recently, however, CXCR7, an alternative
receptor for this chemokine and for IFN-inducible T cell a
chemoattractant (I-TAC)/CXCL11, has been implicated in neovascularization was associated with binding to its recep-
angiogenesis.46 Because the angiostatic IP-10/CXCL10 and tor, CCR2.8,47 MPIF-1/CCL23 has been implicated in the
Mig/CXCL9 bind to CXCR3, this receptor may be involved migration of vascular ECs and the angiogenesis-associated
in chemokine-mediated angiogenesis inhibition.8,11 production of matrix metalloproteinases.48 With regard to
There is little evidence of the role of CC chemokines angiogenesis-suppressing CC chemokines, secondary lym-
in angiogenesis. MCP-1/CCL2 induces EC chemotaxis in phoid tissue chemokine (SLC/CCL21) exerts strong angio-
vitro and angiogenesis in vivo.8,47 MCP-1/CCL2–induced static and antitumor effects.49
 362 SZEKANECZ  |  Cell Recruitment and Angiogenesis
Fractalkine/CX3CL1 is involved in neovascularization
and the pathogenesis of atherosclerosis.8,37,38 Fractalkine/
CX3CL1 is abundantly produced in RA.37
DARC cannot be grouped into one of the four classic
chemokine receptor subclasses (see Table 22-2). DARC
binds the Duffy blood group antigen, as well as some CXC
and CC chemokines. Recently, DARC has been detected
in RA synovial ECs, and it is involved in tumor-associated
angiogenesis.9,50
Extracellular matrix macromolecules and CAMs mediate
adhesive interactions between ECs during ­neovascularization.11
Among extracellular matrix components, type I collagen,
fibronectin, heparin, laminin, and tenascin promote angio-
genesis.11,40 Some endothelial CAMs—including soluble
E-selectin; soluble P-­selectin; the L-selectin ligand CD34;
soluble VCAM-1; some endothelial ß1, ß3, and ß5 integrins;
PECAM-1 (CD31); endoglin (CD105); and some cadher-
ins—have been implicated in angiogenesis.8,11,51-55 Among
glycoconjugates with adhesive properties, Ley/H promotes
neovascularization.56 MUC-18 (CD146) has also been
implicated in synovial angiogenesis.57 As described earlier,
most of these extracellular matrix components and CAMs
play an important role in cell adhesion and migration under-
lying synovial inflammation.1-4
There may be interactions between chemokines and
CAMs during angiogenesis. For example, MCP-1/CCL2
acts via the upregulation of the Ets-1 transcription factor,
and activation of Ets-1 also involves integrin-dependent
adhesive mechanisms.47
REGULATION OF
LEUKOCYTE-ENDOTHELIAL
ADHESION AND ANGIOGENESIS
Leukocyte-EC interactions depend on numerous factors (see
Fig. 22-1). Physical factors, such as altered shear stress, stim-
ulate the neutrophil adhesion to EC.21 The state of leukocyte
activation is also important. For example, resting neutrophils
readily adhere to E-selectin and VCAM-1 but not to ICAM-1,
while activated neutrophils adhere to ICAM-1 as well.5,6,12
TNF-α, IL-1, and IFN-γ may upregulate EC CAM expres-
sion and stimulate leukocyte-EC interactions.5,6,12 As
described earlier, chemokines may also act in concert with
integrins.47 ECs themselves produce a number of inflam-
matory mediators.5,6 Certain CAMs can also cross-talk
with each other: for example, E- and P-selectin stimulate
the adhesive activity of ß2 integrins on neutrophils.58 The
cross-talk between selectins and integrins is crucial for the
transition from rolling to firm adhesion.18,20 Finally, intercel-
lular contact itself may result in increased cytokine release
and CAM expression.59 These mechanisms may synchronize
the adhesive events described earlier, as well as the steps of
neovascularization.
REGULATION OF CHEMOKINE
PRODUCTION DURING LEUKOCYTE
RECRUITMENT
There is a temporal regulation of chemokine and chemo-
kine receptor production in the inflamed synovium. We
have assessed the temporal expression of CXC and CC
chemokines in the sera and joint homogenates of rats with
adjuvant-induced arthritis (AIA). The production of ENA-
78/CXCL5 and MIP-1a/CCL3 was increased very early,
before the appearance of clinical symptoms. In contrast,
MCP-1/CCL2 was involved in the later phase of AIA.60
When we assessed the temporal regulation of chemokine
receptors in rat AIA, CCR1 exhibited high constitutive
expression on macrophages throughout the disease course,
whereas CCR2 expression on ECs was downregulated dur-
ing progression of the disease.61
Proinflammatory cytokines regulate chemokine produc-
tion in the arthritic synovium.7-10 For example, TNF-α, IL-1,
IL-6, IL-15, and IL-18 may enhance chemokine production,
while others, such as IL-4, may suppress it.7-10 These data
suggest that the Th1-Th2 balance or imbalance may influ-
ence the local chemokine pattern in the synovium.9,33
CD4+/CD28– T cells resemble a functionally end-
differentiated, nondividing, short-lived effector memory
T cell subpopulation. These cells, when adoptively trans-
ferred into human RA synovium–SCID (severe combined
immunodeficient) mouse chimeras, coexpressed the CCR5,
CCR7, and CXCR4 chemokine receptors and migrated in
response to RANTES/CCL5 and SDF-1/CXCL12.62
Toll-like receptors (TLRs) may also be involved in the regu-
lation of chemokine function. TLR2 ligands activate synovial
fibroblasts. Peptidoglycan, a TLR2 ligand, stimulates IL-8/
CXCL8, Groα/CXCL1, MCP-1/CCL2, MIP-1a/CCL3, and
RANTES/CCL5 mRNA expression by these fibroblasts.63
There may be distinct histologic patterns of synovitis
in RA, which may be associated with different chemokine
profiles. There are at least two distinguishable histologic
types: some synovial biopsy samples show diffuse lymphoid
infiltrates, while others are classified as “follicular synovitis,”
showing the formation of germinal center–like structures.
Serum levels of IL-8/CXCL8, MCP-1/CCL2, and RAN-
TES/CCL5 were significantly higher in the follicular type
than in the diffuse histologic variant.64
The molecular mechanisms and signaling pathways of
chemokine-induced CAM expression have recently been
described. Briefly, an atypical protein kinase C, PKC- ξ,
has been identified. Treatment of cells with chemokines
induces PKC- ξ activity through its interaction with PI3K.
This leads to increased cell surface integrin expression via
additional signaling steps.1
INHIBITION OF CELL ADHESION,
CHEMOKINES, AND ANGIOGENESIS:
FUTURE PERSPECTIVES
IN ANTIRHEUMATIC THERAPY
The inhibition of cell adhesion and migration, angiogen-
esis, and chemokines with the use of specific antibodies or
purified ligands has provided an important perspective on
the molecular pathogenesis of RA. Some of these strategies
may be included in the future therapy of arthritis.65
In anti-CAM trials, an anti–human ICAM-1 antibody
(enlimomab) was used to treat refractory RA, and many
patients reported improvement in their status. A tran-
sient increase in the number of circulating T cells after the
administration of the antibody suggested that leukocyte
extravasation into the synovium was inhibited.2,65,66 Unfor-
tunately, repeated administration of this antibody resulted in
diminished efficacy and frequent adverse events suggestive
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
of neutrophil activation and immune complex formation.66
In addition, anti–ICAM-1 and anti–ß2 integrin antibodies
prevented the development of arthritis in rats and rabbits,
respectively.2,3,65 Two anti-CAM strategies, efalizumab (anti–
LFA-1) and alefacept (LFA-3-Ig fusion protein), have been
used to treat inflammatory diseases, primarily psoriasis.4,67,68
Natalizumab (anti–α4 integrin) has been tried in multiple
sclerosis and Crohn’s disease,4,69 and a monoclonal antibody
to the α4ß7 integrin was administered to patients with ulcer-
ative colitis.4,70 These and other anti-CAM strategies may be
useful in other inflammatory conditions, including RA.2-4,65
Chemokines and chemokine receptors can be targeted
in a number of ways. In humans, disease-modifying anti-
rheumatic drugs (DMARDs) and anti-TNF biologics, cur-
rently used in the treatment of RA, may indirectly influence
chemokine production. For example, sulfasalazine and
sulfapyridine inhibited chemokine production by cultured
RA synovial explants and ECs, respectively.7,9 Infliximab
reduced synovial expression of IL-8/CXCL8 and MCP-1/
CCL2 in RA patients. Decreased chemokine release was
associated with diminished inflammatory cell ingress into
the synovium.71 Treatment of RA patients with infliximab
or etanercept resulted in the sustained retention of CXCR3+
T cells in the circulation, indicating clearance of these cells
from the synovium.72
Regarding direct chemokine inhibition in animal models
of arthritis, antibodies to IL-8/CXCL8 prevented arthritis in
rabbits.73 A neutralizing polyclonal anti–ENA-78/CXCL5
antibody was administered intravenously to rats using the
AIA model. The antibody injected before the onset of
arthritis attenuated the severity of the disease. This anti-
body also prevented the ingress of IL-1–expressing leuko-
cytes into the synovium.74 IP-10/CXCL10 and PF-4/CXCL4
have also been targeted in several ways.7-9,65 Recently, an
anti-CXCL16 monoclonal antibody suppressed murine
collagen-induced arthritis (CIA).75 Passive immunization
of mice with anti–MIP-1a/CCL3 postponed the onset and
decreased the severity of CIA.9 Antibodies to MCP-1/CCL2
reduced ankle swelling and prevented the recruitment of
111
In-labeled T cells into the rat synovium.7,9 An antibody to
fractalkine/CX3CL1 reduced synovial leukocyte infiltration
and bone erosion and ameliorated murine CIA.76
Recent studies have addressed the use of combined che-
mokine blockade. For example, a combination of MCP-
1/CCL2 and Groα/CXCL1 inhibition resulted in more
pronounced arthritis suppression than did MCP-1/CCL2
blockade alone in a murine AIA model.77 However, there
may be increased toxicity using combined strategies.9
Regarding chemokine receptor targeting, an oral CXCR2
antagonist inhibited IL-8/CXCL–induced rabbit arthri-
tis.9,65 AMD3100, a CXCR4 antagonist, inhibited CIA in
IFN-γ–deficient mice.9,65 Several CCR1 and CCR2 antag-
onists have been developed in recent years, and some of
them have undergone human trials.9,65,78 Met-RANTES, a
CCR1/CCR5 antagonist, suppressed leukocyte infiltration
of the joint and the development of murine CIA.9,65 When
anti-CCR2 antibodies were administered during the initia-
tion of murine CIA, clinical symptoms improved markedly,
whereas blockade during the later stages of the disease aggra-
vated both clinical and histologic signs of arthritis.9 Hence,
CCR blockade using antibodies or other inhibitors may be a
promising therapy in the future.
363
There have been a limited number of human antichemo-
kine studies. There was one trial using an anti–IL-8/CXCL8
antibody in RA, but results were not published, and the
development of this compound was terminated.9 Numer-
ous small-molecule CCR1 antagonists have been devel-
oped.78 In a 2-week phase Ib study, one of these inhibitors
decreased the number of synovial macrophages. One third
of the patients also fulfilled the ACR20 criteria for improve-
ment.79 Some CCR2 inhibitors have also entered clinical
trials.9,65
Angiogenesis can be inhibited by either blocking the
action of angiogenic mediators or using angiostatic com-
pounds. Focusing on leukocyte recruitment, CAMs,
chemokines, and chemokine receptors involved in neovas-
cularization may be targeted in several ways. These antiad-
hesive and antichemokine strategies were discussed earlier.
A number of currently used antirheumatic agents such as
dexamethasone, gold salts, chloroquine, sulfasalazine, meth-
otrexate, azathioprine, cyclophosphamide, leflunomide,
thalidomide, minocycline, anti-TNF agents, and possibly
cyclosporine A suppress angiogenesis.8,11,41 For example, in-
fliximab treatment reduced synovial VEGF expression and
vascularity.11 Future antiangiogenic therapy, which also con-
trols synovial inflammation in RA, may target growth factors
or cytokines. There have been attempts to target VEGF, and
a number of synthetic VEGF and VEGF receptor inhibitors
and anti-VEGF antibodies are under development.80
It is likely that multipotent rather than specific immu-
notherapy may be useful to control inflammation and the
progression of RA. For example, DMARD treatment or
anti–TNF-α targeting has multiple beneficial effects in RA,
including the inhibition of chemokine production, CAM
expression, and neovascularization.8-11 Thus, the roles of che-
mokines, CAMs, and angiogenesis in RA are overlapping
and may be useful for future targeting.
SUMMARY
In this chapter we have discussed the putative role of
leukocyte-EC adhesion, chemokines, and angiogenesis
in leukocyte recruitment underlying the pathogenesis of
inflammatory synovitis. The concepts are exemplary of
ongoing processes in a variety of rheumatic disorders.
A number of CAMs are involved in this process. These
CAMs interact with soluble inflammatory mediators, such
as cytokines and chemokines. The presence of various CAM
pairs and the existence of distinct steps of rolling, activa-
tion, adhesion, and migration account for the diversity
and specificity of leukocyte-EC interactions. Chemokines
and their receptors drive inflammatory leukocytes into the
synovium. A number of soluble and cell-bound factors may
stimulate or inhibit angiogenesis. The outcome of inflamma-
tory and other “angiogenic diseases,” such as various forms
of arthritis, depends on the imbalance between angiogenic
and angiostatic mediators. Some CAMs and chemokines
are also involved in neovascularization. There have been
several attempts to therapeutically interfere with their cel-
lular and molecular mechanisms. Specific targeting of leu-
kocyte adhesion, CAMs, chemokines, chemokine receptors,
and angiogenesis, primarily by using agents with multiple
actions, may be useful for the future management of a vari-
ety of inflammatory rheumatic diseases.
 364 SZEKANECZ  |  Cell Recruitment and Angiogenesis
REFERENCES
1. Imhof BA, Aurrand-Lions M: Adhesion mechanisms regulating the
migration of monocytes. Nat Rev Immunol 4:432-444, 2004.
2. Szekanecz Z, Szegedi G, Koch AE: Cellular adhesion molecules in
rheumatoid arthritis: Regulation by cytokines and possible clinical
importance. J Invest Med 44:124-135, 1996.
  3. Szekanecz Z, Koch AE: Leukocyte-endothelial adhesion molecules in
the rheumatoid synovium. Autoimmun Rev 3(Suppl 2):32-33, 2004.
  4. Agarwal S.K., Brenner M.B.: Role of adhesion molecules in synovial
inflammation. Curr Opin Rheumatol 18:268-276, 2006.
  5. Szekanecz Z, Koch AE: Endothelial cells in inflammation and angio-
genesis. Curr Drug Targ 4:319-323, 2005.
  6. Szekanecz Z, Koch AE: Vascular endothelium and immune responses:
Implications for inflammation and angiogenesis. Rheum Dis Clin
North Am 30:97-114, 2004.
  7. Szekanecz Z, Kim J, Koch AE: Chemokines and chemokine receptors
in rheumatoid arthritis. Semin Immunol 399:1-7, 2002.
  8. Szekanecz Z, Koch AE: Chemokines and angiogenesis. Curr Opin
Rheumatol 13:202-208, 2001.
  9. Koch AE: Chemokines and their receptors in rheumatoid arthritis.
Arthritis Rheum 52:710-721, 2005.
10. Szekanecz Z, Strieter RM, Koch AE: Cytokines in rheumatoid arthri-
tis: Potential targets for pharmacological intervention. Drugs Aging
12:377-390, 1998.
11. Szekanecz Z, Gáspár L, Koch AE: Angiogenesis in rheumatoid arthritis.
Front Biosci 10:1739-1753, 2005.
12. Cotran RS, Pober JS: Cytokine-endothelial interactions in inflamma-
tion, immunity and vascular injury. J Am Soc Nephrol 1:225-235, 1990.
13. Brenner BM, Troy JL, Ballermann BJ: Endothelium-dependent vas-
cular responses: Mediators and mechanisms. J Clin Invest 84:1373-
1378, 1989.
14. Brett J, Gerlach H, Nawroth P, et al: Tumor necrosis factor/cachectin
increases permeability of endothelial cell monolayers by a mechanism
involving regulatory G proteins. J Exp Med 169:1977-1991, 1989.
15. Westphal JR, Boerbooms AMT, Schalkwijk CJM, et al: Anti-
­endothelial cell antibodies in sera of patients with autoimmune
­diseases: Comparison between ELISA and FACS analysis. Clin Exp
Immunol 96:444-449, 1994.
16. Schoefl G: Studies on inflammation. III. Growing capillaries. Vir-
chows Arch A337:97-100, 1963.
17. Springer T.A.: Adhesion receptors of the immune system. Nature
346:425-433, 1990.
18. Springer TA: Traffic signals for lymphocyte recirculation and leuko-
cyte emigration: The multistep paradigm. Cell 76:301-314, 1994.
19. Patel KD, Cuvelier SL, Wiehler S: Selectins: Critical mediators of
leukocyte recruitment. Semin Immunol 14:73-81, 2002.
20. Butcher EC: Leukocyte-endothelial cell recognition: Three (or more)
steps to specificity and diversity. Cell 67:1033-1036, 1991.
21. Lawrence MB, Springer TA: Leukocytes roll on a selectin at physi-
ologic flow rates: Distinction from and prerequisite for adhesion
through integrins. Cell 65:859-873, 1991.
22. Johnson BA, Haines GK, Harlow LA, et al: Adhesion molecule expres-
sion in human synovial tissue. Arthritis Rheum 36:137-146, 1993.
23. Jasin HE, Lightfoot E, Davis LS, et al: Amelioration of antigen-
induced arthritis in rabbits treated with monoclonal antibodies to
leukocyte adhesion molecules. Arthritis Rheum 35:541-549, 1992.
24. Koch AE, Burrows JC, Haines GK, et al: Immunolocalization of leu-
kocyte and endothelial adhesion molecules in human rheumatoid and
osteoarthritic synovial tissue. Lab Invest 64:313-320, 1991.
25. Wellicome SM, Kapahi P, Mason JC, et al: Detection of a circulating
form of vascular cell adhesion molecule-1: Raised levels in rheumatoid
arthritis and systemic lupus erythematosus. Clin Exp Immunol 92:
412-418, 1993.
26. Szekanecz Z, Haines GK, Lin TR, et al: Differential distribution of
intercellular adhesion molecules (ICAM-1, ICAM-2 and ICAM-3),
and the MS-1 antigen in normal and diseased human synovia. Arthritis
Rheum 37:221-231, 1994.
27. Anderson R, Springer TA: Leukocyte adhesion deficiency. Annu Rev
Med 38:175-190, 1978.
28. Hale LP, Martin ME, McCollum DE, et al: Immunohistologic analysis
of the distribution of cell adhesion molecules within the inflammatory
synovial microenvironment. Arthritis Rheum 32:22-30, 1989.
29. Salmi M, Kalimo K, Jalkanen S: Induction and function of vas-
cular adhesion protein-1 at sites of inflammation. J Exp Med 178:
2255-2260, 1993.
30. Cunningham SA, Arrate MP, Rodriguez JM, et al: A novel protein
with homology to the junctional adhesion molecule: Characterization
of leukocyte interactions. J Biol Chem 275:34750-34756, 2000.
31. Valencia X, Higgins JM, Kiener HP, et al: Cadherin-11 provides spe-
cific cellular adhesion between fibroblast-like synoviocytes. J Exp Med
200:1673-1679, 2004.
32. Zlotnik A, Yoshie O: Chemokines: A new classification system and
their role in immunity. Immunity 12:121-127, 2000.
33. Moser B, Loetscher P: Lymphocyte traffic control by chemokines.
Nat Immunol 2:123-128, 2001.
34. Walz A, Kunkel SL, Strieter RM: C-X-C chemokines—an overview.
In Koch AE, Strieter RM (eds): Chemokines in Disease. Austin, Tex,
RG Landes, 1996, pp 1-25.
35. Taub DD: C-C chemokines—an overview. In Koch AE, Strieter RM
(eds): Chemokines in Disease. Austin, Tex, RG Landes, 1996, pp 27-54.
36. Bazan JF, Bacon KB, Hardiman G, et al: A new class of membrane
bound chemokine with a X3C motif. Nature 385:640-644, 1997.
37. Ruth JH, Volin MV, Haines GK III, et al: Fractalkine, a novel che-
mokine in rheumatoid arthritis and rat adjuvant-induced arthritis.
Arthritis Rheum 44:1568-1581, 2001.
38. Lesnik P, Haskell CA, Charo IF: Decreased atherosclerosis in CX3CR1
–/– mice reveals a role for fractalkine in atherogenesis. J Clin Invest
111:333-340, 2003.
39. Strieter RM, Polverini PJ, Kunkel SL, et al: The functional role
of the ELR motif in CXC chemokine-mediated angiogenesis. J Biol
Chem 270:27348-27357, 1995.
40. Folkman J, Klagsbrun M: Angiogenic factors. Science 235:442-448,
1987.
41. Auerbach W, Auerbach R: Angiogenesis inhibition: A review. Phar-
macotherapy 63:265-311, 1994.
42. Nanki T, Hayashida K, El-Gabalawy HS, et al: Stromal cell-derived
factor-1-CXC chemokine receptor 4 interactions play a central role in
CD4+ T-cell accumulation in rheumatoid arthritis synovium. J Immu-
nol 165:6590-6598, 2000.
43. Peichev M, Naiyer AJ, Pereira D, et al: Expression of VEGFR-2 and
AC133 by circulating human CD34+ cells identifies a population of
functional endothelial precursors. Blood 95:952-958, 2000.
44. Boulday G, Haskova Z, Reinders ME, et al: Vascular endothelial
growth factor-induced signaling pathways in endothelial cells that
mediate overexpression of the chemokine IFN-gamma-inducible pro-
tein of 10 kDa in vitro and in vivo. J Immunol 176:3098-3107, 2006.
45. Bodnar RJ, Yates CC, Wells A: IP-10 blocks vascular endothelial
growth factor-induced endothelial cell motility and tube formation
via inhibition of calpain. Circ Res 98:617-625, 2006.
46. Burns JM, Summers BC, Wang Y, et al: A novel chemokine recep-
tor for SDF-1 and I-TAC involved in cell survival, cell adhesion and
tumor development. J Exp Med 203:2201-2213, 2006.
47. Stamatovic SM, Keep RF, Mostarica-Stojkovic M, et al: CCL2 regu-
lates angiogenesis via activation of Ets-1 transcription factor. J Immu-
nol 177:2651-2661, 2006.
48. Son KN, Hwang J, Kwon BS, et al: Human CC chemokine CCL23
enhances expression of matrix metalloproteinase-2 and invasion
of vascular endothelial cells. Biochem Biophys Res Commun 340:
498-504, 2006.
49. Vicari AP, Ait-Yahia S, Chemin K, et al: Antitumor effects of the
mouse chemokine 6Ckine/SLC through angiostatic and immunologi-
cal mechanisms. J Immunol 165:1992-2000, 2000.
50. Wang J, Ou ZL, Hou YF, et al: Enhanced expression of Duffy antigen
receptor for chemokines by breast cancer cells attenuates growth and
metastasis potential. Oncogene 25:7201-7211, 2006.
51. Koch AE, Halloran MM, Haskell CJ, et al: Angiogenesis mediated
by soluble forms of E-selectin and vascular cell adhesion molecule-1.
Nature 376:517-519, 1995.
52. Morbidelli L, Brogelli L, Granger HJ, et al: Endothelial cell migration
is induced by soluble P-selectin. Life Sci 62:7-11, 1998.
53. Brooks PC, Clark RA, Cheresh DA: Requirement of vascular integrin
alpha v beta 3 for angiogenesis. Science 264:569-571, 1994.
54. Horak ER, Leek R, Klenk N, et al: Angiogenesis, assessed by plate-
let/endothelial cell adhesion molecule antibodies, as indicator of
node metastases and survival in breast cancer. Lancet 340:1120-1124,
1992.
55. Szekanecz Z, Haines GK, Harlow LA, et al: Increased synovial expres-
sion of transforming growth factor (TGF)-ß receptor endoglin and
TGF-ß1 in rheumatoid arthritis: Possible interactions in the patho-
genesis of the disease. Clin Immunol Immunopathol 76:187-194,
1995.
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
56. Halloran MM, Carley WW, Polverini PJ, et al: Ley/H: An endothe-
lial-selective, cytokine-inducible, angiogenic mediator. J Immunol
164:4868-4873, 2000.
57. Neidhart M, Wehrli R, Bruhlmann P, et al: Synovial fluid CD146
(MUC18), a marker for synovial membrane angiogenesis in rheuma-
toid arthritis. Arthritis Rheum 42:622-627, 1999.
58. Lo SK, Lee S, Ramos RA, et al: Endothelial-leukocyte adhesion
molecule 1 stimulates the adhesive activity of leukocyte integrin
CR3 (CD11b/CD18, Mac-1) on human neutrophils. J Exp Med 173:
1493-1500, 1991.
59. Bombara MP, Webb DL, Conrad P, et al: Cell contact between T cells
and synovial fibroblasts causes induction of adhesion molecules and
cytokines. J Leukoc Biol 54:399-406, 1993.
60. Szekanecz Z, Halloran MM, Volin MV, et al: Temporal expression
of inflammatory cytokines and chemokines in rat adjuvant-induced
arthritis. Arthritis Rheum 43:1266-1277, 2000.
61. Haas CS, Martinez RJ, Attia N, et al: Chemokine receptor expres-
sion in rat adjuvant-induced arthritis. Arthritis Rheum 52:3718-3730,
2005.
62. Zhang X, Nakajima T, Goronzy JJ, et al: Tissue trafficking patterns
of effector memory CD4+ T cells in rheumatoid arthritis. Arthritis
Rheum 52:3839-3849, 2005.
63. Pierer M, Rethage J, Seibl R, et al: Chemokine secretion of rheuma-
toid arthritis synovial fibroblasts stimulated by Toll-like receptor 2
ligands. J Immunol 172:1256-1265, 2004.
64. Klimiuk PA, Sierakowski S, Latosiewicz R, et al: Histological pat-
terns of synovitis and serum chemokines in patients with rheumatoid
arthritis. J Rheumatol 32:1666-1672, 2005.
65. Szekanecz Z, Koch AE: Therapeutic inhibition of leukocyte recruit-
ment in inflammatory diseases. Curr Opin Pharmacol 4:423-428,
2004.
66. Kavanaugh AF, Schulze-Koops H, Davis LS, et al: Repeat treatment
of rheumatoid arthritis patients with a murine anti-ICAM-1 mono-
clonal antibody. Arthritis Rheum 40:849-853, 1997.
67. Gordon KB, Papp KA, Hamilton TK, et al: Efalizumab for patients
with moderate to severe plaque psoriasis: AA randomized controlled
trial. JAMA 290:3073-3080, 2003.
365
68. Krueger GG, Ellis CN: Alefacept therapy produces remission for patients
with chronic plaque psoriasis. Br J Dermatol 148:784-788, 2003.
69. Sandborn WJ, Colombel JF, Enns R, et al: Natalizumab induction
and maintenance therapy for Crohn’s disease. N Engl J Med 353:
1912-1925, 2005.
70. Feagan BG, Greenberg GR, Wild G, et al: Treatment of ulcerative
colitis with a humanized antibody to the α4ß7 integrin. N Engl J Med
352:2499-2507, 2005.
71. Nissinen R, Leirisalo-Repo M, Peltomaa R, et al: Cytokine and che-
mokine receptor profile of peripheral blood mononuclear cells during
treatment with infliximab in patients with active rheumatoid arthri-
tis. Ann Rheum Dis 63:681-687, 2004.
72. Aeberli D, Seitz M, Juni P, et al: Increase of peripheral CXCR3 posi-
tive T lymphocytes upon treatment of RA patients with TNF-alpha
inhibitors. Rheumatology (Oxford) 44:172-175, 2005.
73. Akahoshi T, Endo H, Kondo H, et al. Essential involvement of inter-
leukin-8 in neutrophil recruitment in rabbits with acute experimental
arthritis induced by lipopolysaccharide and interleukin-1. Lympho-
kine Cytokine Res 13:113-116, 1994.
74. Halloran MM, Woods JM, Strieter RM, et al: The role of an epithelial
neutrophil-activating peptide-78-like protein in rat adjuvant-induced
arthritis. J Immunol 162:7492-7500, 1999.
75. Nanki T, Shimaoka T, Hayashida K, et al: Pathogenic role of
CXCL16-CXCR6 pathway in rheumatoid arthritis. Arthritis Rheum
52:3004-3014, 2005.
76. Nanki T, Urasaki Y, Imai T, et al: Inhibition of fractalkine ameliorates
murine collagen-induced arthritis. J Immunol 173:7010-7016, 2004.
77. Gong JH, Yan R, Waterfield JD, et al: Post-onset inhibition of murine
arthritis using combined chemokine antagonist therapy. Rheumatol-
ogy (Oxford) 43:39-42, 2004.
78. Pease JR, Horuk R: CCR1 antagonists in clinical development.
Expert Opin Investig Drugs 14:785-796, 2005.
79. Haringman JJ, Kraan MC, Smeets TJM, et al: Chemokine blockade
and chronic inflammatory disease: Proof of concept in patients with
rheumatoid arthritis. Ann Rheum Dis 62:715-721, 2003.
80. Shibuya M: VEGF-receptor inhibitors for anti-angiogenesis. Nippon
Yakurigaku Zasshi 122:498-503, 2003.
 23
KEY POINTS
Cytokines are peptides that have a fundamental role in
­communication within the immune system and in allowing the
immune system and host tissues cells to exchange information.
Cytokines act via binding to a cognate receptor which in turn
sends a signal to the recipient cell that leads to a change in
function or phenotype of that cell. Such signal cascades are
complex and allow the intebration of a variety of cytokine
signals to one cell at any given time.
Cytokines exist in broad families that are structurally
related but may contain rather diverse function, e.g., TNF/TNF
receptor superfamily, IL-1 superfamily.
Cytokine targeting has proved efficacious in a variety of
rheumatic diseases, particularly for TNF — many more cyto-
kines are currently under investigation as therapeutic targets,
or as therapeutic entities in their own right.
Immune function depends on the biologic activities of
n­umerous small glycoprotein messengers, termed cytokines.
Originally discovered and defined on the basis of their func-
tional activities, cytokines are now designated primarily
by structure. Typically, cytokines exhibit broad functional
activities that mediate not only effector and regulatory
i­mmune function, but also wider effects across a range of
tissues and biologic systems. As such, cytokines play a role
not only in host defense, but also in a variety of normal
ph­ysiologic and metabolic processes. The human genome
project has facilitated discovery of numerous cytokines,
p­osing considerable challenges in resolving their respective
and synergistic functions in complex tissues in health and
disease. Such understanding is, however, essential with the
advent of cytokine-targeting therapies in the clinic. This
chapter reviews general features of cytokine biology and the
cellular and molecular networks within which cytokines
operate; the focus is on the effector functions of cyto-
kines that are important in chronic inflammation and in
­rheumatic diseases.
CLASSIFICATION OF CYTOKINES
In the absence of a unified classification system, cy­tokines
are variously identified by numeric order of discovery
(currently interleukin [IL]-1 through IL-35), by a given
functional activity (e.g., tumor necrosis factor [TNF], gran-
ulocyte colony-stimulating factor), by kinetic or functional
role in inflammatory responses (early or late, innate or adap-
tive, proinflammatory or anti-inflammatory) (Fig. 23-1), by
primary cell of origin (monokine = monocyte derivation;
lymphokine = lymphocyte derivation), and, more recently,
by structural homologies shared with related molecules.
Cytokines
Iain B. Mcinnes
­Superfamilies of cytokines share sequence similarity and
exhibit homology and some promiscuity in their reciprocal
receptor systems. They do not exhibit functional similarity.
Cytokine superfamilies also contain important regulatory
cell membrane receptor-ligand pairs, reflecting evolution-
ary pressures that use common structural motifs in diverse
immune functions in higher mammals. The TNF/TNF
receptor superfamily1 contains immunoregulatory cytokines,
including TNF-α; lymphotoxins; and cellular ligands, such as
CD40L, which mediates B cell and T cell activation, and FasL
(CD95), which promotes apoptosis. Similarly, the IL-1/IL-1
receptor superfamily2 contains cytokines, including IL-1β,
IL-1α, IL-receptor antagonist, IL-18, and IL-33, which
mediate physiologic and host-defense function, but this
family also includes the Toll-like receptors, a series of mam-
malian pattern-recognition molecules with a crucial role in
recognition of microbial species early in innate responses.
ASSESSING CYTOKINE FUNCTION
IN VITRO AND IN VIVO
Although originally identified by bioactivity and quantified
by bioassay, most cytokines are now identified via homologous
receptor binding or sequence homology in gene databases.
They are quantified in biologic solutions by enzyme-linked
immunosorbent assay or by multiplex technology, the latter
allowing many (>25) cytokines to be measured in single,
small sample volumes (approximately 20 μL). Function is
thereafter assessed by identification of the cellular source of
cytokine, determination of native stimuli, characterization
of receptor distribution, and determination of function in
target cells. Experimental in vivo models use the addition
of neutralizing cytokine-specific antibodies or soluble recep-
tors (often as fragment crystallizable fusion or pegylated
proteins to enhance half-life and modulate functional inter-
action with leukocytes) to modulate cytokine function.
Genetically modified knockout mice (cytokine or receptor
rendered deficient by embryonic stem cell technology) or
transgenic mice (tissue/cell lineage-specific overexpression)
have proved particularly useful. Conditional gene-targeting
approaches (e.g., using the cre system) facilitate circumven-
tion of embryonic lethal deficiencies or allow kinetic evalu-
ation of the relative contribution of a cytokine throughout a
response. Cytokine function is assessed in vitro in primary or
transformed cell lines stimulated in the presence or absence
of recombinant cytokine or specific anticytokine antibody
or soluble receptor.
This general approach has proved crucial in rheu-
matic disease research. Studies in which cytokine addi-
tion and neutralization occurs in synovial tissue explants
or dis­aggregated cell populations, chondrocyte explants,
367
 368 Mcinnes  |  CYTOKINES
1 4 6 intact, can transmit signals to the target cell nucleus after
Stimulus Effector
2
3 function
e.g., 5 Membrane receptors may bind cell membrane cytokines
Microbe
Cell contact
DNA
Cytokine
Cytokine
Receptor
Antibody/IC
Sheer stress
Pressure
Figure 23-1  Overview of cytokine regulatory function. Numerous and
diverse stimuli (1) promote cytokine expression arising either from novel
gene expression (2) or from activation of preformed cytokine (3). Cytokine
proteins are thereafter expressed in the cytosol, on the cell membrane,
or in soluble form in the extracellular environment (4). Cytokines bind
to reciprocal receptors that reside either on the membrane of a target
cell or in the soluble phase (5). Membrane receptors, on cytokine ligation,
signal to the recipient cell nucleus (6) and drive novel gene expression
to promote effector function. Each phase of cytokine function offers rich
therapeutic potential. IC, immune complexes.
bone culture m­odels, skin, and renal tissue explants and
lines all have proved informative. Ex vivo methodologies
now include intracellular fluorescence activated cell sorter
methods, confocal and laser scanning microscopy, and
quantitative histologic evaluation using automated image
analysis. Such modalities, particularly when employed
in human therapeutic cytokine neutralization studies in
which inflammatory tissues are obtained through therapy,
are consid­erably advancing the understanding of basic and
pathogenetic cytokine function. Analysis of synovial biopsy
specimens obtained during infliximab, rituximab, IL-1Ra,
IL-10, and interferon (IFN)-β administration in rheumatoid
arthritis provides the strongest evidence for the success of
this approach.3,4
CYTOKINE RECEPTORS
Cytokine receptors exist in structurally related superfamilies
and comprise high-affinity molecular signaling complexes
that facilitate cytokine-mediated communication. Such
complexes often include heterodimeric or heterotrimeric
structures that use unique, cytokine-specific recognition
receptors together with common receptor chains shared
across a cytokine superfamily. Examples include the use
of the common γ chain receptor by IL-2, IL-4, IL-7, IL-9,
IL-15, IL-21, and glycoprotein 130 (gp130) by members of
the IL-6 family.5,6 Alternatively, distinct receptors may use
shared signaling domains. Homologous death domains are
found in many TNF-receptor family members. Similarly,
the IL-1 signaling domain is common to not only IL-1R,
but also to other IL-1R superfamily members, including
IL-18R, ST2, and the Toll-like receptors.2 Signaling path-
ways dependent on these are discussed in detail elsewhere.
It has been recognized more recently that unrelated cyto-
kine receptor systems exhibit close cross-communication
on the cell membrane, allowing a cell to integrate a variety
of external stimuli to optimize signaling pathways and the
cellular response in real-time in a changing environment.
Although best elucidated in the epidermal growth factor
receptor system, this also has been identified for members of
the common γ chain signaling family.
Cytokine receptors can operate via several mechanisms.
Membrane receptors, with intracellular signaling domains
soluble cytokine binding and promote effector function.
facilitating cross-talk between adjacent cells. Membrane-
bound and soluble cytokines may promote distinct receptor
function. TNF-α binds TNF-RI and TNF-RII with similar
affinity, but it has a slower rate of dissociation from TNF-RI.
Soluble TNF-α may dissociate rapidly from TNF-RII to
bind TNF-RI, promoting preferential signaling by the latter
(ligand passing).1 In contrast, during cell-cell contact, sta-
ble TNF-α/TNF-RI and TNF-α/TNF-RII complexes form,
allowing for differential signaling contribution by TNF-RI
and TNF-RII. Cytokine receptor/cytokine complexes also
may operate in trans, whereby component parts of the
ligand-receptor complex are derived from adjacent cells.
IL-15/IL-15Rα complexed on one cell may bind IL-15Rβ/γ
on another.7 Receptors also exist in soluble form, derived
either from alternative mRNA processing to generate
receptor-lacking transmembrane or intracellular domains
or from enzymatic cleavage of receptor from the cell sur-
face (e.g., sTNF-R, sIL-1R1). Soluble receptors may act to
antagonize cytokine function, regulating responses. Soluble
receptors also may preform complexes with cytokine to pro-
mote su­bsequent ligand-receptor assembly on the target cell
membrane and enhance function. Soluble receptors can
deliver cytokine to the cell membrane via ligand passing.
Finally, it is now recognized that some cytokines with the
capacity to be retained in the membrane may themselves
function as signaling molecules (reverse signaling).
REGULATION OF CYTOKINE EXPRESSION
Cytokines are synthesized in the Golgi and may traffic
through the endoplasmic reticulum to be released as soluble
mediators, or they may remain membrane bound, or they
may be processed into cytosolic forms that can traffic intra-
cellularly, even returning to the nucleus where they can act
as transcriptional regulators. Cytokines mediate autocrine
function either through release or membrane expression
and immediate receptor ligation on the source cell or intra-
cellularly within the source cell. Alternatively, cytokines
operate in a paracrine manner, allowing cellular communi-
cation beyond that facilitated by local cell-cell contact. The
distance and kinetics for effective function may be limited,8
however, by numerous factors, including physicochemical
considerations of the peptide structure itself, extracellular
matrix binding (e.g., to heparan sulfate), enzymatic degra-
dation (e.g., serine protease degradation of IL-18), or the
presence of soluble receptors (e.g., TNF-α/soluble TNF-RI
and TNF-RII, IL-2/soluble IL-2Rα) or novel cytokine-
binding proteins (e.g., IL-18/IL-18 binding protein) in the
inflammatory milieu.
Numerous factors promote cytokine expression in vivo
(Fig. 23-1), including cell-cell contact, immune complexes/
autoantibodies, local complement activation, microbial
sp­ecies and their soluble products, reactive oxygen and
nitrogen intermediates, trauma, sheer stress, ischemia,
ra­diation, ultraviolet light, extracellular matrix components,
DNA (mammalian or microbial), heat shock proteins, and
cy­tokines themselves in autocrine loops. Commonly used in
 Part 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
Hierarchy Late
Early
Kinetics
Anti
Inflammatory
potential Pro
Figure 23-2  Placing cytokines in functional context. Cytokines often
exhibit pleiotropic functions that vary throughout an immune response
and according to the nature of a stimulus. Cytokines could be envisaged
at one point in three-dimensional space, representing their hierarchic
capacity to contribute to an inflammatory lesion over time. A given cyto-
kine might have early proinflammatory function but late, net anti-inflam-
matory function within an evolving immune response. Similarly, at an
early stage, a cytokine might function atop a hierarchy, but later function
primarily as an effector downstream moiety. Cytokines may have roles in
innate and acquired responses that are discrete. A cytokine might have
distinct functions in different tissues and in response to different stimuli.
This has important implications for predicting the effect of cytokine tar-
geting in vivo.
vitro stimuli include many of these and chemical entities,
including phorbol esters, calcium ionophores, lectins (e.g.,
phytohemagglutinin), and receptor-specific antibodies such
as anti-CD3 and anti-CD28 for T cell activation or anti-
immunoglobulin and anti-CD40 for B cells.
Cytokine regulation within the cell can be usefully con-
sidered at several levels. Transcriptional regulation depends
on the recruitment of discrete transcription factors to the
cytokine promoter region. Transcription factor binding
allows for numerous signal pathways to regulate cytokine
expression across a range of stimuli. Several transcription
factors (e.g., nuclear factor κB [NFκB], activator protein-1
[AP-1], nuclear factor of activated T cell) are crucial in cyto-
kine production. Inhibition of NFκB activity using either
chemical inhibitors or adenoviral delivery of regulatory pep-
tides leads to amelioration of inflammatory synovitis in vivo
and in vitro.9 Sequence polymorphism within cytokine pro-
moters offers potential for differential cytokine expression
between individuals that could confer selective advantage
against infection, but also could increase susceptibility to, or
progression of, autoimmunity. This is best exemplified in the
TNF-α and IL-1 promoters.10,11 Single nucleotide polymor-
phisms in the TNF-α promoter region (e.g., −308) are asso-
ciated with altered TNF-α release on leukocyte stimulation
in vitro. Similarly, homozygotes for the A2 allele at +3954 in
the IL-1β gene produce more IL-1β with lipopolysaccharide
stimulation. Polymorphisms also exist in the IL-1Ra gene,
rendering the functional significance of individual single
nucleotide polymorphisms on IL-1 protein release difficult
to interpret. In general, the net effect of haplotypes may be
more important at the functional level, particularly when
their relevance to disease entities is considered.
Post-transcriptional regulation is important in deter-
mining longevity of cytokine expression. This regulation
may operate by promoting translational initiation, mRNA
369
s­ tability, and polyadenylation. AU-rich elements (AREs)
within the 5′ or 3′ untranslated regions (UTRs) of cytokine
mRNA are crucial for stability; 3′ UTR AREs downregulate
TNF expression such that transgenic knockin mice that lack
TNF-α AREs develop spontaneous inflammatory arthritis
and bowel disease.12 Regulatory proteins bind AREs to medi-
ate such effects. HuR and AUF1 exert opposing effects, stabi-
lizing and destabilizing ARE-containing transcripts.13 TIA-1
and TIAR have been identified as RNA recognition motif
family members14 that function as translational silencers.
Macrophages from TIA-1-deficient macrophages produce
excess TNF-α, whereas TIA-1-deficient lymphocytes exhibit
normal TNF-α release, suggesting distinctions in mRNA
regulation in discrete cell types.15 Alternatively, cytokines
may generate stable mRNA a priori to facilitate subsequent
rapid response in tissues. IL-15 mRNA 5′ UTR contains 12
AUG triplets that significantly reduce the efficiency of IL-15
translation. Deletion of this sequence permits IL-15 secre-
tion. IL-15 mRNA can produce a 48-amino acid signal pep-
tide that allows IL-15 release and a shorter 21-amino acid
signal peptide that targets intracellular distribution. IL-15
forms thus generated exhibit discrete functions.16
Post-translational regulation also modulates cytokine
expression via several mechanisms. Patterns of glycosyl-
ation are important for cytokine function and may regu-
late intracellular trafficking.16 Modified leader sequences
may alter intracellular trafficking of cytokines. Some cyto-
kines are translated without functional leader sequences.
Their secretion depends on nonconventional secretory
pathways that are so far poorly understood. IL-1β employs
a purine receptor–dependent pathway (P2X7) for cellular
release.17 Enzymatic activation of cytokines is common,
whereby nonfunctional promolecules are cleaved to gene­
rate functional subunits. Examples include the ­ cleavage
by caspase 1 of pro-IL-1β to generate active IL-1β and,
similarly, of pro-IL-18 to generate an active 18-kD
­species.18 This is an organized process sequentially and
in the orientation within the cell. IL-1 processing occurs
in a protein complex within the cytosol termed the
­inflammasome.
Alternative processing pathways for cytokines include
the serine proteases, proteinase 3 and elastase, and adamo-
lysin family members. Enzyme cleavage pathways oper-
ate within and outside cells, providing for extracellular
cytokine activation. Similarly, cell membrane enzymes
serve to cleave membrane-expressed cytokine. Members
of the adamolysin family regulate TNF-α release; TNF-
α-converting enzyme cleaves and mediates the release of
TN­F-α and its receptors.19 Extensive molecular machinery
exists to regulate tightly not only the production and stabil-
ity of cytokine mRNA, but also its translation and cellular
expression and distribution. At each level, opportunities
exist for intervention and therapeutic cytokine modulation.
EFFECTOR FUNCTION OF CYTOKINES
Cytokines possess pleiotropic and potent effector function
in acute and chronic inflammatory responses. The identity,
receptor specificity, and key effects of cytokines understood
to have particular importance in pathogenesis of human
autoimmunity and chronic inflammation are summarized in
Tables 23-1 through 23-8.
 370 Mcinnes  |  CYTOKINES

Table 23-1  Interleukin-1 Superfamily Cytokines with Roles in Rheumatic Disease

Cytokine Size (kD)* Receptors Major Cell Sources Key Functions
IL-1β 35 (pro) IL-1RI Monocytes; B cells; fibroblasts;   Fibroblast cytokine, chemokine, MMP, iNOS, PG
chondrocytes; keratinocytes release ↑
17 (active) IL-1RAcP Monocyte cytokine, ROI, PG ↑
IL-1RII (decoy) Osteoclast activation
Chondrocyte GAG synthesis ↓; iNOS, MMP, and
aggrecanase ↑
Endothelial adhesion-molecule expression
IL-1α 35 (pro)† IL-1RI Monocytes; B cells; PMNs;   Similar to IL-1β
epithelial cells; keratinocytes
17 (active) IL-1RAcP Autocrine growth factor (e.g., keratinocytes)
IL-1RII (decoy)
IL-1Ra 22 IL-1RI Monocytes Antagonize effects of IL-1β and IL-1α
IL-1RAcP
IL-1RII
IL-33 30 (pro) ST2L Epithelial cells; monocytes; smooth Promote Th2 cell activation, mast cell activation,
muscle cells; keratinocytes and cytokine production
18 (active) IL-1RAcP
IL-18 23 (pro) IL-18R Monocytes; PMNs; dendritic cells;   T cell effector polarization (Th1 with IL-12/Th2 with
platelets; endothelial cells IL-4)
18 (active) IL-18Rβα Chondrocyte GAG synthesis ↓; iNOS expression
NK activation; cytokine release; cytotoxicity
Monocyte cytokine release; adhesion molecule
expression
PMN activation; cytokine release; migration
Endothelial cells—proangiogenic
*Pro forms cleaved to active moieties by proteases, including caspase-1, calpain, elastase, and cathepsin G.
†Pro-IL-1α retains bioactivity before cleavage.

GAG, glycosaminoglycan; iNOS, inducible nitric oxide synthase; MMP, matrix metalloproteinase; NK, natural killer; PG, peptidoglycan; PMN, ­polymorphonuclear
neutrophil; ROI, reactive oxygen intermediates.

Table 23-2  Tumor Necrosis Factor Superfamily Cytokines* with Potential Role in Rheumatic Disease
Cytokine Size (kD) Receptors Major Cell Sources Selected Functions

TNF-α 26 (pro) TNF-RI (p55) Monocytes; T, B, NK cells; PMNs; eosinophils; Monocyte activation, cytokine, and PG ↑
mast cells; fibroblasts; keratinocytes; glial
cells; osteoblasts; smooth muscle
TNF-RII (p75) PMN priming, apoptosis, oxidative burst ↑
Endothelial cell adhesion molecule, cytokine release ↑;
fibroblast proliferation and collagen synthesis ↓
MMP and cytokine ↑
T cell apoptosis; clonal (auto)regulation; TCR dysfunction
Adipocyte FFA release ↑
Endocrine effects—ACTH, prolactin ↑; TSH, FSH, GH ↓
LTα 22-26 TNF-RI T cells; monocytes; fibroblasts; astrocytes;   Peripheral lymphoid development
myeloma; endothelial cells; epithelial cells
TNF-RII Otherwise similar bioactivities to TNF-α
RANK   35 RANK Stromal cells; osteoblasts; T cells Stimulates bone resorption via osteoclast maturation and
ligand activation
Modulation of T cell-DC interaction
OPG 55 RANKL Stromal cells, osteoblasts Soluble decoy receptor for RANKL
BLyS† 18-32 TACI Monocytes; T cells; DCs B cell proliferation, Ig secretion, isotype switching, survival
BCMA T cell costimulation
BLyS-R
APRIL — TACI Monocytes; T cells; tumor cells B cell proliferation
BCMA Tumor proliferation
*Additional members of importance include TRAIL, TWEAK, CD70, FasL, and CD40L. At least 18 members of the family are now described.
†Also called BAFF.

ACTH, adrenocorticotropic hormone; APRIL, a proliferation inducing ligand; BAFF, B cell activating factor belonging to the TNF family; BCMA, B cell matu-
ration protein; BLyS, B lymphocyte stimulator protein; DC, dendritic cell; FFA, free fatty acid; GAG, glycosaminoglycan; LT, lymphotoxin; MMP, matrix metal-
loproteinase; NK, natural killer; OPG, osteoprotegerin; PG, peptidoglycan; PMN, polymorphonuclear neutrophil; RANK, receptor activator of NFB ligand; TACI,
transmembrane activator and calcium modulator and cyclophilin ligand; TNF, tumor necrosis factor; TRAIL, TNF related apoptosis-inducing ligand; TWEAK,
TNF-like weak inducer of apoptosis.
 Part 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION 371

Table 23-3  Cytokines Associated Predominantly with Effector Function for T Cells*
Cytokine Size (kD) Receptors Major Cell Sources Key Functions
Type II Interferon
IFN-γ 20-25 IFNγR Th/c1 cells; NK cells; γδT cells;   Macrophage activation, DC APC function ↑
B cells; macrophage/DCs
Endothelial adhesion molecule ↑
MHC class II expression ↑
T cell growth ↓; opposes Th2 responses
Bone resorption ↓; fibroblast collagen synthesis
4a-Helix Family
IL-2 15 IL-2Rα Th/c cells; NK cells T cell division; maturation; cytokine release;
cytotoxicity
IL-2/15Rβ γ-chain NK cell cytokine release; cyotoxicity; monocyte
activation
Lymphocyte apoptosis ↓
IL-4 20 IL-4Rα/γ-chain Th/c cells (Th2); NK cells Th2 differentiation, maturation, apoptosis ↓
IL-4Rα/IL-13R1 B cell maturation; isotype switch (IgE)
Eosinophil migration, apoptosis ↓
Endothelial activation; adhesion molecule
expression
IL-5 25 monomer IL-5Rα Th/c2 cells; NK cells; mast cells; B cell differentiation; immunoglobulin  
epithelial cells production (IgA)
50 homodimer IL-5Rβ Eosinophil differentiation and activation
Th/c maturation
IL-17 Family†
IL-17A 20-30 IL-17R T cells (Th17); fibroblasts Chemokine release, fibroblast cytokine release,
MMP release ↑
Osteoclastogenesis; hematopoiesis
Chondrocyte GAG synthesis ↓
Leukocyte cytokine production ↑
IL-25 (IL-17E) 20-30 IL-17R Th2 cells Th2 cytokine release; B cell IgA and IgE synthe-
sis; eosinophilia; epithelial cell hyperplasia
*Additional T cell–derived cytokines of potential interest include IL-13 from Th2 and NK2 cells.
†IL-17 family also contains IL-17B, IL-17C, and IL-17F, the distinct functions of which are currently unclear.
APC, antigen presenting cell; DC, dendritic cell; GAG, glycosaminoglycan; IFN, interferon; MHC, major histocompatibility complex; MMP, matrix metallopro-
teinase; NK, natural killer; Th/c, T helper/cytotoxic.

CYTOKINES IN ACUTE INFLAMMATION

Cytokines operate at every stage in the crucial early events
that promote acute inflammation. Cells that make up the
innate immune response, including neutrophils, natural killer
cells, ­macrophages, mast cells, and eosinophils, all produce and
respond to cytokines generated within seconds of tissue insult.
Cytokines prime leukocytes for response to microbial and
­chemical stimuli; upregulate adhesion molecule expression on
migrating leukocytes and endothelial cells; and amplify the
release of reactive oxygen intermediates, nitric oxide, vasoac-
tive amines, and neuropeptides, and the activation of kinins and
arachidonic acid derivatives, prostaglandins, and leukotrienes,
which regulate cytokine release. Similarly, cytokines regulate the
expression of complement processing and membrane defense
molecules, scavenger receptors, NOD Like ­ Receptor (NLR),
and Toll-like receptors. Cytokines, particularly IL-1, TNF-α,
and IL-6, are crucial in driving the acute-phase response. Tables
23-1 through 23-8 provide descriptions of the function of cyto-
kines expressed within the acute inflammatory response.
be considered fluid states in which individual cells under
cytokine control transiently contribute to organized func-
tional subunits—such as the ectopic germinal center, syno-
vial lining layer, or renal interstitial nephritis—yet remain
competent to migrate thereafter under the influence of
chemotactic gradients on the extracellular matrix. Cyto-
kines also may promote cell death (apoptosis) either by
withdrawal (e.g., IL-2, IL-7, IL-15) or by binding cytokine
receptors containing death domains (e.g., TNF-R1). Cyto-
kines contribute at every stage of inflammatory lesion devel-
opment in a dynamic equilibrium, rather than in a static,
linear manner. Chronic inflammation in rheumatic disease
usually contains cytokine activities reminiscent of innate
and acquired immune responses. For convenience, cytokines
can be considered by their effect on cell subsets and cellular
interactions (Fig. 23-2 describes a notional positioning for
cytokine activity in a developing and chronic lesion). Inves-
tigation of cytokine-regulated pathways in several rheumatic
diseases has identified several common pathways.

CYTOKINES IN CHRONIC INFLAMMATION T Cell Effector Function in Chronic Inflammation

Cytokines critically modulate the cellular interactions that
characterize chronic inflammation. Studies using real-time
image analytic techniques, such as two-photon microscopy
and confocal scanning, suggest continuous cellular motility
during inflammation. Inflammatory lesions might properly
T cells depend on cytokine function at every develop­
m­ental stage from bone marrow stem cell maturation,
through thymic education, to functional determina-
tion and maturation after primary or secondary antigen
exposure. The last-mentioned is of prime importance
 372 Mcinnes  |  CYTOKINES

Table 23-4  Cytokines Described Initially with Primary Role in Regulation of T Cells*
Cytokine Size Receptors Major Cell Sources Key Functions
IL-12 IL-12/23p40 IL-12Rα Macrophages; DCs Th1 cell proliferation, maturation
IL-12p35 IL-12Rβ1 T cell cytotoxicity
IL-12Rβ2 B cell activation
IL-23 IL-12/23p40 IL-23R Macrophages; DCs Th17 cell expansion and activation; IL-17
release
IL-23p19 IL-12Rβ1
IL-15 15 kD IL-15Rα Monocytes; fibroblast; mast   T cell chemokinesis, activation, memory
cells; B cells; PMNs; DCs maintenance
IL-2/15Rβ γ-chain NK cell maturation, activation, cytotoxicity
Macrophage activation, suppression (dose
dependent)
PMN activation, adhesion molecule, oxida-
tive burst
Fibroblast activation
B cell differentiation and isotype switching
IL-21 15 kD IL-21R γ-chain Activated T cells; others (?) B cell activation
*Cytokines included in this table are now understood to exhibit considerable functional heterogeneity as shown. Other T cell regulatory cytokines have been
described, including IL-27, the functions of which are currently under investigation.
DC, dendritic cells; NK, natural killer; PMN, polymorphonuclear neutrophil.

Table 23-5  IL-10 Superfamily Cytokines*

Cytokine Receptors Cellular Sources Key Functions
IL-10 IL-10R1 Monocytes; T cells; B cells; DCs;   Macrophage cytokine release, iNOS, ROI ↓; soluble
epithelial cells; keratinocytes receptor ↑
IL-10R2 T cell cytokine release, MHC expression ↓; anergy
induction
Treg cell maturation; effector function (?)
DC activation, cytokine release ↓
Fibroblast MMP, collagen release ↓; no effect on TIMP
B cell isotype switching enhanced
IL-19 IL-20R1/IL-20R2 Monocytes; others (?) Monocyte cytokine and ROI release; monocyte apop-
tosis
IL-20 IL-22R/IL-20R2 Keratinocytes; others (?) Autocrine keratinocyte growth regulation
IL-20R1/IL-20R2
IL-22 IL-22R/IL-10R2 Th17 cells; CD8 T cells; γδ T cells;   Acute-phase response, keratinocyte activation prolif-
NK cells eration ↑
IL-24 IL-22R/IL-20R2 Monocytes; T cells Tumor apoptosis; Th1 cytokine release by PBMC
IL-20R1/IL-20R2
*Additional members include IL-26, IL-28, and IL-28A. Many functions of IL-10 superfamily are as yet poorly understood, but they likely reside beyond the
immune system.
DC, dendritic cell; iNOS, inducible nitric oxide synthase; MMP, matrix metalloproteinase; NK, natural killer; PBMC, peripheral blood mononuclear cells;
ROI, reactive oxygen intermediates; TIMP, tissue inhibitor of metalloproteinase.

because re-education of phenotypic T cell responses may
be achieved through alteration of the ambient cytokine
milieu. T cell receptor–peptide–major histocompatibil-
ity complex (MHC) interactions during T cell–dendritic
cell interaction rely on costimulatory molecule and local
cytokine expression to determine functional outcome
(see Tables 23-3 and 23-4). IL-12, in the presence of IL-
18, promotes type 1 phenotypic development, character-
ized ultimately by IFN-γ producing T helper type 1 (Th1)
effector cells.20 IFN-γ drives macrophage priming and
activation and adhesion molecule expression and pro-
motes granuloma formation and microbial killing. IFN-γ
has a complex role in tissue destruction, however, with
contradictory data obtained in inflammation models in
IFN-γ-deficient and IFN-γ receptor–deficient mice. IFN-γ
ultimately may retard tissue destruction, perhaps by sup-
pressing osteoclast activation.21
A novel T cell subset has been defined that secretes
IL-17A predominantly (Th17 effector cells), together
with IL-22. Th17 cells are generated in the presence of IL-
6 and transforming growth factor (TGF)-β, expanded by
IL-1β and IL-23, and antagonized by IL-25 (IL-17E) and
paradoxically by IFN-γ. IL-17A provides a direct and rapid
route to tissue damage via such means as osteoclast activa-
tion or FLS activation.22 The precise contribution of Th17
cells in human autoimmune disease is currently unclear.
There are, however, persuasive data from rodent models
indicating that Th17 cells may be of primary importance as
initiator and effector cells. IL-4 dominance during T cell–
dendritic cell interactions in the presence of IL-18 leads
to type 2 responses, which promote humoral ­ immunity
driven by Th2 cells synthesizing primarily IL-4, IL-5, IL-
10, and IL-13. Resulting pathogenesis more likely may be
B cell–mediated. Cytokines that predispose to regulatory
 Part 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION 373

Table 23-6  IL-6 Superfamily Cytokines*

Cytokine Size (kD) Receptors Major Cell Sources Key Functions
IL-6 21-28 IL-6R† gp130 Monocytes; fibroblasts; B cells; T cells B cell proliferation; immunoglobulin production
Hematopoiesis, thrombopoiesis
T cell proliferation, differentiation, cytotoxicity
Hepatic acute-phase response
Hypothalamic-pituitary-adrenal axis
Variable effects on cytokine release by monocytes
Oncostatin M 28 OMR gp130 Monocytes; activated T cells Megakaryocyte differentiation
Fibroblast, TIMP, and cytokine release
Acute-phase reactants, fibroblast protease inhibitors ↑
Monocyte TNF release ↓; IL-1 effector function ↓
Hypothalamic-pituitary axis ↑; corticosteroid release
Modulatory effect on osteoblast (?)
Proinflammatory effects in some models (?)
Leukemia   58 LIFR gp130 Fibroblasts; monocytes; lymphocytes; Acute-phase reactants ↑
inhibitory   mesangial cells; smooth muscle cells;
factor epithelial cells; mast cells
Hematopoiesis, thrombopoiesis
Role in neural development, neural effector function,
implantation
Bone metabolism; extracellular matrix regulation
Leukocyte adhesion molecule expression
Eosinophil priming
Mixed proinflammatory versus anti-inflammatory  
effects in models
*Additional members of potential importance include IL-11, cardiotropin-1, and ciliary neurotrophic factor. Note overlapping effects within family.
†Membrane or soluble form can dimerize gp130 to promote signaling, which promotes signal transduction.

LIFR, leukemia inhibitory factor receptor; OMR, oncostatin M receptor; TIMP, tissue inhibitor of metalloproteinase; TNF, tuimor necrosis factor.

T cell development are unclear, although high levels of IL-
10 or TGF-β have been suggested in this context.23 Effector
T cells can operate via secretion of cytokines to patterns
determined by their prior activatory conditions.
Cognate Cellular Interactions
In many inflammatory lesions, there is relative paucity
of inducer T cell–derived cytokines (especially IFN-γ),
despite abundant proinflammatory cytokine expression.
Cell-cell membrane interactions between leukocyte subsets
and between tissue cells and leukocytes have emerged as
a do­minant mechanism sustaining chronic inflammation.
Cytokines contribute to these interactions at several lev-
els (Fig. 23-3), including directly as membrane-expressed
ligands, indirectly via activating cells, and synergistically by
enhancing their subsequent cognate activities. The impor-
tance of cytokine-cell contact interactions is best studied in
synovial tissues, but applies to many inflammatory lesions.
Many data now show that cognate interactions between T
cells and adjacent macrophages constitute a major pathway
driving cytokine release, and that cytokines sustain this
pathway (see Fig. 23-3). Such interactions do not depend
on T cell receptor–mediated T cell activation and provide a
route to expansion of inflammation by T cells, but indepen-
dent of local autoantigen recognition.
Vey and colleagues24 first observed monocyte activa-
tion via cell contact with mitogen-stimulated T cells.
Freshly isolated synovial T cells activate macrophages by
this mechanism, confirming that contact-induced cellu-
lar activation is a fundamental property of inflammatory
T cells.25 Antigen-independent, cytokine-mediated bystander
activation confers this capacity on human CD4+ memory
T cells.26 Studies using synovial T cells from rheumatoid
arthritis and psoriatic arthritis tissues reveal that exposure of
memory T cells to synergistic combinations of cytokines are
most potent in this respect, particularly IL-15, TNF-α, and
IL-6.27,28 Cytokine activities also operate directly on macro-
phages to synergize with T cell contact. IFN-γ and IL-18 are
most potent in this respect, acting via increased adhesion
molecule expression.
Activated memory CD4+ and CD8+ T cells promote cyto-
kine release from macrophages via diverse membrane ligands,
including LFA-1/ICAM-1, CD69, and CD40/CD154.27,29 After
contact with T cells, macrophages release increased concentra-
tions of TNF-α and IL-1, but not IL-10, and they exhibit reduced
levels of IL-1Ra. Th1 cells promote relatively greater proinflam-
matory cytokine release than do Th2 cells after coculture. This
finding suggests that their functional phenotype extends beyond
cytokine secretion to include a differential membrane receptor
array.30 This su­ggestion is borne out further in the relative pheno-
typic distinctions between Th1 (CD40L, CCR5, IL-18Rα) and
Th2 (ST2, CXCR3) cells. The role of Th17 cells in this context is
unknown. Signaling pathways engaged in monocytes after such
T cell–membrane interactions are distinct from the pathways
activated by conventional c­ytokine-inducing agents. Distinct
use of phosphatidylinositol 3-kinase, NFκB, and p38 mitogen-
activated protein kinase pathways is observed.31 Similarly, dis-
crete macrophage signals follow contact with cytokine-activated
T cells (which resemble synovial T cells) compared with T cell
receptor–activated T cells.32 Such distinctions offer therapeutic
potential in targeting cytokine-activated, T cell–driven path-
ways, leaving antigen-driven responses relatively intact. The
activation state of memory T cells necessary for the previously
discussed interactions to proceed remains controversial. Purified
resting T cell subsets activate synovial fibroblasts to release IL-6,
IL-8, matrix metalloproteinase 3 (MMP3), and prostanoids, in
synergy with IL-17.33
 374 Mcinnes  |  CYTOKINES

Table 23-7  Growth Factors Relevant to Rheumatic Diseases

Cytokine Receptors Cellular Sources Key Functions
TGF-β* Type I TGFβR Broad—including fibroblasts,   Wound repair, matrix maintenance, and fibrosis
monocytes, T cells, platelets
Isoforms 1-3† Type II TGFβR Initial activation then suppression of inflammatory
responses
Others T cell (Treg and Th17) and NK cell proliferation and
effector function ↓
Early-phase leukocyte chemoattractant, gelatinase,
and integrin expression ↑
Early macrophage activation then suppression,
reduced iNOS expression
BMP family   BMPRI Varied (e.g., epithelial and   Regulate critical chemotaxis, mitosis, and differ-
(BMP2-15) mesenchymal embryonic tissues);   entiation processes during chondrogenesis and
bone-derived cell lineages osteogenesis, tissue morphogenesis (e.g., heart,
skin, eye)
BMPRII
PDGF PDGFRα Platelets; macrophages; endothelial   Local paracrine or autocrine growth factor for vari-
cells; fibroblasts; glial cells; astrocytes;   ety of lineages
myoblasts; smooth muscle cells
PDGFRβ Wound healing
FGF family FGFR (various) Widespread Growth and differentiation of mesenchymal,  
epithelial, and neuroectodermal cells
Basic FGF
Acidic FGF
*Members of TGF-β superfamily include BMP, growth and differentiation factor, inhibinA, inhibinB, müllerian inhibitory substance, glial-derived neurotrophic
factor, and macrophage inhibitory cytokine.
†Bound to latency-associated peptide to form small latency complex and to latent TGF-β binding protein to form large latent complex; activated by

­proteolytic and nonproteolytic pathways.

BMP, bone morphogenetic protein; FGF, fibroblast growth factor; iNOS, inducible nitric oxide synthase; NK, natural killer; PDGF, platelet-derived growth
factor; TGP, transforming growth factor.

It is likely that T cells may be activated by interac-
tions with diverse moieties, including extracellular matrix
c­omponents and potentially autoantigens. Nevertheless,
it is now clear that cytokines can promote chronicity by
a­ctivating T cells to promote inflammation regardless of
local (auto)antigen recognition, and that this has enormous
therapeutic potential (see Fig. 23-3).
Agonist/Antagonist Cytokine Activities in Chronic
Inflammation
Complex regulatory interactions exist to suppress ongoing
inflammatory responses. This is often achieved via parallel
secretion of antagonistic cytokines and soluble receptors to
regulate cytokine effector pathways. Th1 responses are sup-
pressed partly by cytokines of Th2 type (e.g., IL-4 or IL-10),
and consequently exaggerated Th1 responses arise in mod-
els in which the Th2 response is deficient.20 Th1 and Th2
cells similarly limit Th17 cell expansion.22 Similar regula-
tory loops operate for other leukocytes, exemplified by the
yin-yang effects of TNF-α and IL-10 on macrophage cyto-
kine release and effector function.34
Inhibitory cytokine activities usually are defined with
respect to a proinflammatory cytokine, and in other contexts
they may have quite distinct function, rendering prediction
of their net contribution to an inflammatory response dif-
ficult. IL-10 opposes many of the proinflammatory effects of
TNF-α and IL-1β (e.g., reduces adhesion molecule expres-
sion, MHC expression, and MMP release), but it potently
activates B cell activation and immunoglobulin secretion.34
Similarly, TNF-α, which is normally considered a proinflam-
matory moiety, may have an important role in ­ regulating
T cell function because T cells removed from sites of chronic
inflammation exhibit suppressed capacity to signal via their
T cell receptor that recovers on TNF-α neutralization.35
Such regulation is complicated further by the precise ratio
of cytokine to soluble receptor, such as TNF to sTNFR or
IL-10 to sIL-10R, within the local environment. Commen-
surate with this, administration of anti-inflammatory cyto-
kines, such as IL-4, IL-10, and IL-11, has generally proved
disappointing in the context of clinical inflammatory dis-
eases. An important caveat is the potential requirement of
combinations of cytokines to suppress inflammation opti-
mally (e.g., combinations including IL-4, IL-10, and IL-11).
Further functional antagonism is exemplified in the antago-
nistic activities of IL-1β and IL-1Ra and of IL-18 and IL-18
binding protein in regulating macrophage activation.
The role of cytokines in regulating cognate interac-
tions between leukocytes also has emerged more recently.
Although anti-inflammatory pathways are poorly induced
after cell contact, cytokine-activated T cells can induce
IL-10 release by monocytes.32 Rheumatoid arthritis synovial
membrane IL-10 release, which is partially T cell depen-
dent, feeds back to regulate TNF-α release. Cytokine pro-
duction from adjacent cell lineages within an inflammatory
lesion also may be suppressive. IFN-β reduces mitogen-
activated, T cell–induced macrophage release of TNF-
α and IL-1, whereas IL-1Ra release is enhanced.36 This
provides a mechanism whereby type I IFNs could modify
proinflammatory cytokine production. Regulation extends
beyond co­nventional cytokine activities. Prostaglandins
and lipoprotein moieties, particularly high-density lipopro-
tein, can suppress cytokine-mediated, T cell–macrophage
­interactions.37
 Part 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION 375

Table 23-8  Miscellaneous Cytokines with Potential Roles in Rheumatic Diseases

Cytokine Size (kD) Receptors Cellular Sources Key Functions
MIF 12 Unclear Macrophages; activated T cells;   Macrophage cytokine release, phagocyto-
fibroblasts (synoviocytes) sis, NO release ↑
T cell activation; DTH
Fibroblast proliferation; COX expression;
PLA2 expression
Intrinsic oxidoreductase activity  
(“cytozyme”)
HMGB1 30 RAGE, dsDNA Widespread expression; necrotic cells;   DNA-binding transcription factor
macrophages; pituicytes
Others (?) Necrosis-induced inflammation
Macrophage activation—delayed proin-
flammatory cytokine
Smooth muscle chemotaxis
Disrupts epithelial barrier function
Bactericidal (direct)
GM-CSF 14-35 GM-CSFRα T cells; macrophages; endothelial cells;   Granulocyte and monocyte maturation;
fibroblasts hemopoietic effects
GM-CSFRβ Leukocyte PG release; DC maturation
Pulmonary surfactant turnover
G-CSF 19 G-CSFR Monocytes; PMNs; endothelial cells;   Granulocyte maturation; promotes PMN
fibroblasts; various tumor cells;   function
stromal cells
M-CSF 28-44 M-CSFR Monocytes; fibroblasts; endothelial cells Monocyte activation, maturation
IL-32α-δ Unknown Unknown Monocytes; T cells; NK cells; epithelial cells Promotes proinflammatory cytokine
release from variety of cells
Type I   Various IFNαβR Widespread Antiviral response
interferons
IFNα/β  
family
Broad immunomodulatory effects (pro-
motes MHC expression)
Macrophage activation; lymphocyte
activation and survival
Antiproliferative, cytoskeletal alteration,
differentiation ↑
COX, Cyclooxygenase; DC, dendritic cell; DTH, delayed-type hypersensitivity; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage
colony-stimulating factor; HMGB, high mobility group box chromosomal protein; IFN, interferon; M-CSF, macrophage colony-stimulating factor; MIF, macro-
phage inhibitory factor; NO, nitric oxide; PG, prostaglandin; PLA, phospholipase A; PMN, polymorphonuclear neutrophil; RAGE, receptor for advanced glycation
end products.

Disease-Modifying Antirheumatic Drugs

Conventional disease-modifying antirheumatic drugs also can
act via modulation of cytokine production. Methotrexate mod-
ulates release of various cytokines in vitro, in part mediated via
adenosine–cyclic adenosine monophosphate pathway.38 The
active metabolite of the dihydroorotate dehydrogenase inhibi-
tor leflunomide, A77 11726, reduces TNF-α, IL-1β, IL-6, and
MMP-1, but it does not reduce IL-1Ra release by monocytes
after mitogen-activated T cell contact.39,40 Leflunomide may
mediate these activities through modulation of inhibitor of
NFκB (IκB)α phosphorylation and degradation and AP-1
and c-Jun N-terminal protein kinase activation.41 Finally, sul-
fasalazine is an inhibitor of proinflammatory cytokine-induced
NFκB. Biologic agents also potently modify cytokine expres-
sion in a variety of disease states.
Cellular Interactions across Diverse Tissues
Cytokines promote cognate cellular interactions across
a range of tissues. In contrast to T cell–macrophage and
T cell–dendritic cell interactions, in which adhesion ­molecule
and costimulatory pathways are often implicated, cell-cell
membrane communications apart from leu­kocyte-leukocyte
interactions are often mediated through membrane cytokine
expression (see Fig. 23-3). T cell contact–mediated activa-
tion of fibroblasts operates via membrane TNF-α and IFN-γ
to enhance fibroblast cytokine release and MMP—but not
tissue inhibitors of metalloproteinase—expression, favoring
tissue destruction.29 The source and activation status of the
fibroblast are vital; fibroblast-like synoviocytes, but not cuta-
neous fibroblasts, are potently activated by this route. Other
studies have shown T cell contact–mediated activation of
neutrophils, keratinocytes, mesangial cells (via a combina-
tion of membrane cytokine and CD40L expression), plate-
lets, and renal tubule epithelial cells. Cytokine-­activated
macrophages (via IFN-γ and sCD40L) may interact via
cell contact with mesangial cells to activate adhesion mol-
ecule and chemokine release by the latter. Cell-cell con-
tact between cells of the immune system and beyond likely
represents a ubiquitous mechanism whereby perpetuation
of chronic inflammation is potently influenced by local
production of cytokines.
 376 Mcinnes  |  CYTOKINES

Fibroblast
DC
IL-17 Neutrophil
? IL-22
IL-12, IL-23 C
Chemokines, ECM TNF-α
LTβ H
Co-stimulation R
IL-1
IL-15 Mast cell O
IL-18 N
IL-6 I
IL-20 C
± T1/T17 IL-32
IL-33 I
RANKL N
IL-17, IL-22 GM-CSF F
IFN-γ L
Cell contact, costimulation Tissue cell A
IL-10 M
IL-1Ra M
IL-18BP A
sIL-1R T
sTNFR Endothelial cell I
IL-27 O
Macrophage IL-35 N
TGF-β
TLR FcR
NLR BLyS
Peptidoglycan Immune complexes B cell
APRIL
Lipopolysaccharide Acute phase reactants
Heat shock proteins Complement

Figure 23-3  Cytokines regulate complex cellular networks in chronic inflammation. Cytokines regulate interactions between T1 cells and antigen
presenting cells (dendritic cells [DC]), and thereafter they promote cell-cell contact and soluble interactions between T cells, macrophages, neutrophils,
endothelial cells, fibroblasts, B cells, and target tissue cells (e.g., mesangial cells, renal tubular epithelial cells, keratinocytes). Tissue cells may make
­substantial contributions to organ dysfunction through inflammatory mediator release. Crucial to these pathways are the synergistic combinations of
cytokines that operate as “cassettes” (synergistic teams) or together with cell-cell, contact-dependent interactions. The latter are mediated via mem-
brane cytokine expression or through cell surface receptors, including integrin and immunoglobulin superfamily adhesion molecules and members of
the interleukin (IL)-1R and tumor necrosis factor (TNF)–receptor superfamilies. Chronicity is maintained on the basis of overproduction of proinflam-
matory moieties relative to the presence of anti-inflammatory mediators. Soluble cytokines also regulate activation of additional effector leukocytes
(see Tables 23-1 through 23-8), including mast cells and eosinophils, which are not shown here because they may not be characteristic of the T helper
type 1 (Th1) response shown. They may be relevant, however, in inflammatory arthritis. ECM, extracellular matrix; FcR, Fc receptor; IFN, interferon; TGF,
transforming growth factor; TLR, Toll-like receptor.

B Cells and Cytokine Release in Chronic Inflammation
Cytokines are crucial to B cell maturation, proliferation,
activation, isotype switching, and survival (see Chapter 10).
Cytokine-mediated B cell activation is important in immune
complex generation, B cell antigen presentation, B cell–T
cell interactions, and germinal center formation. Particular
importance has been placed on the TNF superfamily cyto-
kines, BLyS and APRIL. These cytokines are crucial for B
cell development, survival, and optimal activation. B cells
represent a potent source of cytokines such as IL-6 and IL-10.
B cells also have been considered important inducers of mac-
rophage-derived cytokine release. This process may operate
primarily via immune complex formation42 or through regu-
lation of T cell activation (with B cell help). Complex regula-
tory feedback loops involving cytokine expression and B cells
are likely important in a range of rheumatic diseases in which
B cells are of paramount pathophysiologic importance.
Innate Cell Lineages in Chronic Inflammation
Cytokines potently activate innate response cells that
c­ontribute to the chronic inflammatory lesion of a variety
of rheumatic diseases. Tables 23-1 through 23-8 document
relevant examples in which neutrophils, natural killer cells,
eosinophils, and mast cells may be recruited and activated
by the presence of appropriate cytokine combinations.
Growth Factors in Chronic Inflammation
Many data document the importance of growth factor fami-
lies in chronic inflammation. TGF-β superfamily members,
including TGF-β isoforms and bone morphogenetic pro-
tein family members, warrant particular reference. TGF-β
is critically involved in processes of cell proliferation, diff­
erentiation, inflammation, and wound healing.43 Bone mor-
phogenetic proteins, in addition to regulating inflammatory
responses, are paramount in determining cartilage and bone
tissue development and remodeling.44 As such, they are of
increasing interest in the pathogenesis of several rheumatic
diseases.
CYTOKINE EFFECTS BEYOND IMMUNE
REGULATION
A striking feature of the cytokine field concerns the broad
functional pleiotropy exemplified in the effects of cyto-
kines in normal physiologic and adaptive processes. Cyto-
kine activities are found in muscle, adipose tissue, central
nervous system, and liver, mediating normal regulation of
metabolic pathways and modulation imposed by altered tis-
sue conditions. Examples are found not only in the release
of adipokines that regulate adipose metabolic pathways, but
also in the release of conventional cytokines by fat pads in
inflammatory synovitis.
 Part 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
SUMMARY
Cytokines represent a diverse family of glycoproteins active
across a broad range of tissues. Their pleiotropic functions
and propensity for synergistic interactions and functional
redundancy render them intriguing therapeutic targets. So
far, single cytokine targeting has proved useful in several
rheumatic disease states. Further elucidation of the biology
and functional interactions within this expanding family of
bioactive moieties is likely to prove informative in resolving
pathogenesis and in generating novel therapeutic options.
REFERENCES
  1. Locksley RM, Killeen N, Lenardo MJ: The TNF and TNF recep-
tor superfamilies: Integrating mammalian biology. Cell 104:487,
2001.
  2. Marshak-Rothstein A: Toll-like receptors in systemic autoimmune
disease. Nat Rev Immunol 6:823, 2006.
  3. Bresnihan B, Baeten D, Firestein GS, et al: OMERACT 7 Special
Interest Group: Synovial tissue analysis in clinical trials. J Rheumatol
32:2481, 2005.
  4. Haringman JJ, Gerlag DM, Zwinderman AH, et al: Synovial tissue
macrophages: A sensitive biomarker for response to treatment in
patients with rheumatoid arthritis. Ann Rheum Dis 64:834, 2005.
  5. Gadina M, Hilton D, Johnston JA, et al: Signaling by type I and II
cytokine receptors: Ten years after. Curr Opin Immunol 13:363,
2001.
  6. Bravo J, Heath JK: Receptor recognition by gp130 cytokines.
EMBO J 19:2399, 2000.
  7. Dubois S, Mariner J, Waldmann TA, et al: IL-15Ralpha recycles and
presents IL-15 in trans to neighboring cells. Immunity 17:537, 2002.
  8. Francis K, Palsson BO: Effective intercellular communication dis-
tances are determined by the relative time constants for cyto/chemo-
kine secretion and diffusion. Proc Natl Acad Sci U S A 94:12258,
1997.
  9. Feldmann M, Andreakos E, Smith C, et al: Is NF-kappaB a useful thera-
peutic target in rheumatoid arthritis? Ann Rheum Dis 61(Suppl 2):13,
2002.
10. Hajeer AH, Hutchinson IV: TNF-alpha gene polymorphism: Clinical
and biological implications. Microsc Res Tech 50:216, 2000.
11. Hurme M, Lahdenpohja N, Santtila S: Gene polymorphisms of inter-
leukins 1 and 10 in infectious and autoimmune diseases. Ann Med
30:469, 1998.
12. Kontoyiannis D, Pasparakis M, Pizarro TT, et al: Impaired on/off regu-
lation of TNF biosynthesis in mice lacking TNF AU-rich elements:
Implications for joint and gut-associated immunopathologies. Immu-
nity 10:387, 1999.
13. Anderson P: Post-transcriptional regulation of tumour necrosis factor
alpha production. Ann Rheum Dis 59:3, 2000.
14. Gueydan C, Droogmans L, Chalon P, et al: Identification of TIAR as
a protein binding to the translational regulatory AU-rich element of
tumor necrosis factor alpha mRNA. J Biol Chem 274:2322, 1999.
15. Saito K, Chen S, Piecyk M, et al: TIA-1 regulates the production of
tumor necrosis factor in macrophages, but not in lymphocytes. Arthri-
tis Rheum 44:2879, 2001.
16. Budagian V, Bulanova E, Paus R, et al: IL-15/IL-15 receptor biology: A
guided tour through an expanding universe. Cytokine Growth Factor
Rev 17:259, 2006.
17. Ferrari D, Chiozzi P, Falzoni S, et al: Extracellular ATP triggers IL-1
beta release by activating the purinergic P2Z receptor of human mac-
rophages. J Immunol 159:1451, 1997.
18. Fantuzzi G, Dinarello CA: Interleukin-18 and interleukin-1 beta: Two
cytokine substrates for ICE (caspase-1). J Clin Immunol 19:1, 1999.
19. Wallach D, Varfolomeev EE, Malinin NL, et al: Tumor necrosis
factor receptor and Fas signaling mechanisms. Annu Rev Immunol
17:331, 1999.
20. Liew FY: T(H)1 and T(H)2 cells: A historical perspective. Nat Rev
Immunol 2:55, 2002.
21. Takayanagi H, Kim S, Taniguchi T: Signaling crosstalk between
RANKL and interferons in osteoclast differentiation. Arthritis Res
4(Suppl 3):S227, 2002.
377
22. Weaver CT, Harrington LE, Mangan PR, et al: Th17: An effector
CD4 T cell lineage with regulatory T cell ties. Immunity 24:677,
2006.
23. Shevach EM, DiPaolo RA, Andersson J, et al: The lifestyle of natu-
rally occurring CD4+ CD25+ Foxp3+ regulatory T cells. Immunol
Rev 212:60, 2006.
24. Vey E, Zhang JH, Dayer JM: IFN-gamma and 1,25(OH)2D3 induce
on THP-1 cells distinct patterns of cell surface antigen expression,
cytokine production, and responsiveness to contact with activated
T cells. J Immunol 149:2040, 1992.
25. McInnes IB, Leung BP, Sturrock RD, et al: Interleukin-15 medi-
ates T cell-dependent regulation of tumor necrosis factor-alpha
­production in rheumatoid arthritis. Nat Med 3:189, 1997.
26. Unutmaz D, Pileri P, Abrignani S: Antigen-independent activation
of naive and memory resting T cells by a cytokine combination. J Exp
Med 180:1159, 1994.
27. McInnes IB, Leung BP, Liew FY: Cell-cell interactions in synovitis:
Interactions between T lymphocytes and synovial cells. Arthritis Res
2:374, 2000.
28. Sebbag M, Parry SL, Brennan FM, et al: Cytokine stimulation of
T lymphocytes regulates their capacity to induce monocyte produc-
tion of tumor necrosis factor-alpha, but not interleukin-10: Possible
relevance to pathophysiology of rheumatoid arthritis. Eur J Immunol
27:624, 1997.
29. Dayer JM, Burger D: Cytokines and direct cell contact in synovitis:
Relevance to therapeutic intervention. Arthritis Res 1:17, 1999.
30. Ribbens C, Dayer JM, Chizzolini C: CD40-CD40 ligand (CD154)
engagement is required but may not be sufficient for human T helper
1 cell induction of interleukin-2- or interleukin-15-driven, contact-
dependent, interleukin-1beta production by monocytes. Immunology
99:279, 2000.
31. Hayes AL, Smith C, Foxwell BM, et al: CD45-induced tumor necro-
sis factor alpha production in monocytes is phosphatidylinositol
3-kinase-dependent and nuclear factor-kappaB-independent. J Biol
Chem 274:33455, 1999.
32. Foey A, Green P, Foxwell B, et al: Cytokine-stimulated T cells induce
macrophage IL-10 production dependent on phosphatidylinositol
3-kinase and p70S6K: Implications for rheumatoid arthritis. Arthritis
Res 4:64, 2002.
33. Yamamura Y, Gupta R, Morita Y, et al: Effector function of resting
T cells: Activation of synovial fibroblasts. J Immunol 166:2270, 2001.
34. Fickenscher H, Hor S, Kupers H, et al: The interleukin-10 family of
cytokines. Trends Immunol 23:89, 2002.
35. Cope AP: Studies of T-cell activation in chronic inflammation.
Arthritis Res 4(Suppl 3):S197, 2002.
36. Jungo F, Dayer JM, Modoux C, et al: IFN-beta inhibits the ability of
T lymphocytes to induce TNF-alpha and IL-1beta production in
monocytes upon direct cell-cell contact. Cytokine 14:272, 2001.
37. Hyka N, Dayer JM, Modoux C, et al: Apolipoprotein A-I inhibits
the production of interleukin-1beta and tumor necrosis factor-alpha
by blocking contact-mediated activation of monocytes by T lympho-
cytes. Blood 97:2381, 2001.
38. Chan ES, Cronstein BN: Molecular action of methotrexate in inflam-
matory diseases. Arthritis Res 4:266, 2002.
39. Breedveld FC, Dayer JM: Leflunomide: Mode of action in the treat-
ment of rheumatoid arthritis. Ann Rheum Dis 59:841, 2000.
40. Burger D, Begue-Pastor N, Benavent S, et al: The active metabolite of
leflunomide, A77 1726, inhibits the production of prostaglandin E(2),
matrix metalloproteinase 1 and interleukin 6 in human fibroblast-like
synoviocytes. Rheumatology (Oxf) 42:89, 2003.
41. Manna SK, Mukhopadhyay A, Aggarwal BB: Leflunomide suppresses
TNF-induced cellular responses: Effects on NF-kappa B, activator
protein-1, c-Jun N-terminal protein kinase, and apoptosis. J Immunol
165:5962, 2000.
42. Chantry D, Winearls CG, Maini RN, et al: Mechanism of immune
complex-mediated damage: Induction of interleukin 1 by immune
complexes and synergy with interferon-gamma and tumor necrosis
factor-alpha. Eur J Immunol 19:189, 1989.
43. Chen W, Wahl SM: TGF-beta: Receptors, signaling pathways and
autoimmunity. Curr Dir Autoimmun 5:62, 2002.
44. Abe E: Function of BMPs and BMP antagonists in adult bone. Ann N
Y Acad Sci 1068:41, 2006.
 24
KEY POINTS
The three types of cell death are apoptosis, autophagy, and
necrosis.
Apoptosis proceeds through defined biochemical pathways
that are initiated through death receptors on the cell surface
or by intracellular signals emanating from damaged organ-
elles.
Phagocytes recognize alterations on the surface of dead and
dying cells signaling them to engulf the intact apoptotic cell
or necrotic cell debris.
Defects in apoptosis and defective clearance of dead and
dying cells lead to immune responses to self (autoimmunity).
Many anti-inflammatory, immunomodulatory, and newer
biologics affect cell survival pathways and offer new opportu-
nities for therapeutic intervention.
HISTORY AND CONCEPTS
APOPTOSIS
Illustrations of cells undergoing apoptosis were made almost
as soon as stains were used to examine the appearance of cells
in different tissues. Drawings of ovarian follicles undergoing
cell death made more than 100 years ago show cell shrink-
age and nuclear condensation. Subsequent descriptions of
the appearance of subcellular particles during cell death,
referred to as “pyknosis,” chromatin margination, and other
terms, included many features now recognized as apoptosis.
The history of this subject is reviewed elsewhere.1
The modern understanding of apoptosis began with the
electron microscopic descriptions of morphologic changes
characterized by shrinkage of hepatocytes (i.e., shrinkage
necrosis) after ischemic or toxic injury to the liver. The
term apoptosis was coined by Kerr and colleagues in 19722
to describe the form of death that was “consistent with an
active, inherently controlled phenomenon” characterized
by cell shrinkage, nuclear condensation, and cell blebbing
(Fig. 24-1).2 This term also conveyed the concept of cell
death that was similar to leaves falling from a tree (apo
means “from” and ptosis means “a fall” in Greek), implying
a regulated “mechanism of cell deletion, which is comple-
mentary to mitosis.”2
Further developments in apoptosis paralleled advances
in molecular biology, genetics, and biochemistry. The
detection of a nucleosomal ladder3 was important because
it defined a biochemical event (i.e., nucleosomal cleav-
age) and provided a simple electrophoretic test for detec-
tion of apoptotic cell death that remains a standard in
Cell Survival and Death
in Rheumatic Diseases
Keith B. Elkon
the field (see Methods of Detection later in this chapter).
Another landmark was the discovery in the 1980s that the
death of cells during the development of the nematode
Caenorhabditis elegans was under strict genetic control. The
death of these cells could be perturbed by mutation of a few
genes called ced (for cell death abnormal) genes.4 Ellis and
Horvitz4 determined that two ced genes, ced3 and ced4,
encoded death effectors, whereas ced9 was an antiapoptotic
gene. Most of the remaining ced genes were responsible for
engulfment and removal of the “corpses.” This simple model
in which CED-3 is the main death protease that is activated by
CED-4 and inhibited by CED-9 has served as a paradigm for
defining apoptotic pathways in mammalian cells (Fig. 24-2).
In 2002, Horvitz was awarded the Nobel Prize for his dis-
coveries in regard to genetic regulation of programmed cell
death.
Mammalian cells are more complex and, as discussed later
in detail, have multiple defined pathways that follow the
basic C. elegans model. The molecules within these path-
ways, the downstream effectors of apoptosis, the caspases
(cysteine aspartate proteases), and the proteins implicated
in the clearance of apoptotic cells are discussed in detail
later. Regulation of cell death is important in many diseases,
including cancers, autoimmune diseases, and degenerative
disorders.5,6 The relevance of apoptosis to rheumatic disor-
ders is summarized at the end of this chapter.
PROGRAMMED CELL DEATH
Although the term apoptosis originally referred to the
appearance of dying cells in certain contexts as explained
previously, the concepts of atrophy, cellular or tissue involu-
tion or regression, and degeneration also had been appre-
ciated for hundreds of years, yet the two phenomena were
not associated until more recently. Perhaps the most precise
descriptions of cells that died in an orderly and apparently
programmed fashion were documented in developmental
biology. Examples included the involution of cells between
digits, metamorphosis of insect larvae, and the death of spe-
cific cells during the development of C. elegans.
NECROSIS
Necrosis (from the Greek nekros meaning “corpse”) is dis-
tinguished from apoptosis predominantly by morphologic
appearances.7 Necrotic cells are swollen, and electron
microscopy reveals disorderly fragmentation of chromatin
and severe damage to the mitochondria (see Fig. 24-1). The
cellular membrane loses integrity and becomes permeable to
vital dyes such as trypan blue and propidium iodide (Fig. 24-3).
The distinction between apoptosis and necrosis remains
379
 380 ELKON  |  Cell Survival and Death in Rheumatic Diseases

M C

V P

A B C
Figure 24-1  Electron microscopic morphology of apoptosis. A, Cytotoxic T cell (lower left) conjugated to its target, P815 (a murine mast cell), before
the initiation of cell death. B, Induction of apoptotic changes in P815. Note the reduction in target cell size, nuclear condensation, and vacuoles with
relative preservation of organelles. C, Osmotic lysis and necrosis in P815 induced by antibody and complement. Note the increased size of the nucleus
and apparently random fragmentation of the chromatin. Organelles are severely disrupted. C, dense chromatin; M, mitochondria; P, nuclear pore; 
V, vacuoles. (Adapted from Russell JH, Masakowski V, Rucinsky T, et al: Mechanisms of immune lysis, III: Characterization of the nature and kinetics of the cyto-
toxic T lymphocyte induced nuclear lesion in the target. J Immunol 128:2087, 1982.)

important from numerous perspectives. In contrast to the

genetic and biochemical programs that regulate apoptosis,
necrotic cells usually result from death “by accident”—
from thermal or drug injury, infection, or infarction of an
organ. Because of the uncontrolled release of lysosomal and
C. elegans Mammalian granular contents, necrotic cells induce a proinflammatory
immune response, whereas apoptotic cells usually elicit an
Egl-1 ? Bax anti-inflammatory response.
Regulation The same inducers (e.g., ischemia, hydrogen peroxide)
may produce apoptosis or necrosis, depending on the sever-
CED-9 Bcl-2
ity of the injury and the rapidity of cell death. The cell’s fate
CED-4 Apaf-1 is determined partly by cellular energy reserves, especially
Execution
CED-3 Caspase 9 adenosine triphosphate (ATP).8 ATP is generated by oxida-
tive phosphorylation in mitochondria and by glycolysis in
CED-2 crkII
the cytosol. Some inducers may cause apoptosis initially fol-
lowed by necrosis (postapoptotic necrosis). This is likely to
CED-12 ELMO
1. occur when removal of the apoptotic cells is delayed.
CED-5 DOCK 180
Engulfment CED-10 Rac
AUTOPHAGY
CED-1 CD91
2. Autophagy is an alternative, nonapoptotic form of pro-
CED-6 GULP
grammed cell death that has gained attention more recently.
CED-7 ABC-1
The term autophagy means to eat oneself, and in this pro-
Figure 24-2  Caenorhabditis elegans paradigm of apoptosis. Genes in- cess, cells switch to a catabolic program in which cellular
volved in the regulation, execution, and clearance of apoptotic cells dur-
ing the development of C. elegans and their mammalian homologues are constituents are degraded for energy production as a sur-
shown. In this figure, only the most closely related homologues are indi- vival mechanism during periods of nutrient stress. In cells
cated, but, as described in the text and shown in subsequent figures, the undergoing autophagy, a double membrane vesicle forms
complexity in mammalian cells is much greater. At least half of the CED and encapsulates whole organelles leading to degradation
proteins are involved in engulfment and removal of apoptotic corpses.
There are two distinct, but partially overlapping pathways of engulfment
after fusion with lysosomes. The ATG gene family, includ-
in C. elegans: CED-2, CED-12, CED-5, and CED-10, which most likely regulate ing ATG7 and beclin1, a protein that is highly conserved
cytoskeletal changes, and CED-1 and CED-6, which may be involved in rec- from yeast through humans, is involved in autophagic cell
ognition (CED-1) and upstream signal transduction (CED-6). CED-7 is ho- death.9 The protein beclin1 is monoallelically deleted in a
mologous to the mammalian ATP-binding cassette transporter-1 (ABC-1),  variety of human cancers and may function in control of
which likely affects membrane dynamics. The lower right panel shows a
schematic of the two proposed pathways for apoptotic cell ingestion in cell growth and tumor suppression.10 When metabolic sub-
mammalian cells. In pathway (1), an unknown receptor triggers activation strates are depleted, autophagic death shares many morpho-
of the GTPase, RhoG, which leads to transport of the scaffolding protein, logic features with necrotic cell death11 and may be seen in
ELMO, to the cell membrane. There, Dock 180, a guanine exchange fac- neuronal cell death associated with polyglutamine repeats.
tor, promotes Rac activation leading to activation of the cytoskeleton and
phagocytosis. In pathway (2), activation of the CED1 homologue (most
Autophagy may play a role in the survival of lymphocytes
likely CD91) interacts with the adapter protein, GULP, and downstream after growth factor withdrawal.11 Additional details of the
activation of the cytoskeleton. autophagic process are reviewed elsewhere.12
 104

103 200
160
120
102 80

Counts

PI
40 M1
0
100 101 102 103 104 0 4 6 hr
101

100
100 101 102 103 104
A Annexin V B TMRM C D
PART 3 
| 

G0/G1
200

M1 M1

Counts
0
E F 0 200 400 0 200 400 G H
Figure 24-3  Methods for detection of apoptotic cells. A variety of methods are available for the identification and quantification of dying cells; a sampling is shown here. Methods A-C depend on changes
to the cell surface membrane, mitochondria, and caspase activation, whereas methods D-H detect changes in the nucleus. A, Apoptotic thymocytes were incubated with fluorescein isothiocyanate– 
conjugated annexin V in the presence of the dye propidium iodide (PI), which permeates cells with severely damaged cell membranes. Cells in the bottom left quadrant (not stained for annexin or PI) are alive;
cells in the lower right quadrant are early apoptotic; cells in the upper right quadrant are late apoptotic (stain with annexin and admit PI). For cells in suspension, such as lymphocytes, annexin V binding to
phosphatidylserine is the most commonly used method to detect early apoptotic cells. B, Human embryonic kidney cells were incubated in medium alone (upper panel) or medium containing valinomycin, an
ionophore that increases ionic permeability of the inner mitochondrial membrane (lower panel). The cells were incubated with tetramethylrhodamine methyl ester (TMRM), a cell-permeable dye that binds to
the outer mitochondrial membrane proportional to its membrane potential (Δψ), and analyzed by flow cytometry. The fluorescence intensity for the apoptotic cells is lower as a result of loss of mitochondrial
membrane potential. Other probes used in similar assays for mitochondrial potential are rhodamine 123 and the carbocyanine dye DiOC6. C, Cells were induced to undergo apoptosis by anti-Fas antibodies.
Cell extracts taken at 0, 4, and 6 hours postinduction were analyzed for poly-ADP ribose polymerase (PARP) cleavage by Western blot analysis. The 4-hour and 6-hour samples show partial cleavage of PARP
(arrow). Western blot for detection of activated caspase 3 also is used. D, Mouse peritoneal macrophages were incubated with apoptotic thymocytes, cytocentrifuged onto glass slides, and stained with Diff-
quik (Dade Behring, AG, Deefield, IL). Arrows indicate ingested apoptotic cells with condensed nuclei. E, Jurkat T cells were induced to undergo apoptosis, stained with the dye bisBENZIMIDE (Hoechst No.
33342; Sigma, St. Louis, Mo) and viewed by immunofluorescence microscopy. Arrows indicate condensed nuclei. F, Normal (left panel) or apoptotic (right panel) cells were permeabilized and incubated with
EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION

RNase, and their DNA was stained with PI according to the method of Nicoletti and colleagues.81 The cells were analyzed by flow cytometry, and staining in the subdiploid peak (smaller than the G0/G1 peak
and labeled M1 in the histogram) reflects the extent of apoptosis. G, Normal and apoptotic cells were lysed, and the nuclei were removed by centrifugation. Cytosolic extracts were applied to agarose gels, and
components were resolved by electrophoresis. Ethidium bromide staining of the extract made from live cells reveals high-molecular-weight DNA that remains close to the application well (left lane), whereas
the extract from apoptotic cells shows loss of high-molecular-weight DNA and the appearance of the typical ladder of nucleosomes. H, Six-micrometer sections were made from a normal mouse thymus. The
381

cells were permeabilized and incubated with biotinylated deoxyuridine triphosphate in the presence of terminal deoxynucleotidyl transferase. Nicked DNA incorporated the labeled deoxynucleotide and was
detected by staining with peroxidase-labeled streptavidin and substrate (dark area).
 382 ELKON  |  Cell Survival and Death in Rheumatic Diseases
Table 24-1  Modular Components of Proteins   ­Receptors in the TNF family (TNFR) include at least six
Involved in Apoptosis and Inflammation*
Module* Component of Function
DD Death receptors, adapters,   Protein-protein interaction
kinases
DED Adapters, caspases Protein-protein interaction
CARD Caspases, adapters, NODs Protein-protein interaction
BH (1-4) Bcl-2 family Protein-protein interaction
Pyrin Pyrin family Protein-protein interaction
LRR Pyrin family, NODs Protein-protein interaction
NBS/NOD Pyrin family, NODs Nucleotide binding,  
oligomerization
*See references 50 and 161 for further discussion.
BH, Bcl-2 homology; CARD, caspase recruitment domain; DD, death domain;
DED, death effector domain; LRR, leucine-rich repeats; NBS, nucleotide binding
site; NOD, nucleotide oligomerization domain.
BIOCHEMISTRY OF APOPTOSIS
A schematic diagram of the cell death program is shown
in Figure 24-2. A brief outline of each major functional
component within the program, from the signals for death
to removal of the apoptotic cells, is discussed here, but
space limitations preclude a detailed analysis of the lay-
ers of regulation at each step of the pathway. Post-trans-
lational protein modifications, such as phosphorylation,
nitrosylation, and oxidation, provide additional complex-
ities that are under intense study. Apoptosis and its Rel-
evance to Autoimmunity13 offers more detailed reviews of
the biochemistry and relationship of apoptotic pathways
to immune function.
The specialized proteins involved in apoptosis and its
regulation contain numerous modules or domains that
are predominantly involved in promoting protein-protein
interactions (Table 24-1). These domains may be found
in receptors, adapters, effectors, or inhibitors. As discussed
subsequently, these domains occur in proteins involved
in apoptosis and inflammation. It has been suggested that
death domain (DD), death effector domain (DED), cas-
pase recruitment domain (CARD), and Pyrin domains all
evolved from the prototypic DD fold corresponding to an
antiparallel six-helix bundle.14 In general, similar domains
bind so as to facilitate homotypic interactions leading to
oligomerization of the same protein or binding to different
proteins in a signaling pathway. These changes usually lead
to conformational alterations, which lead to further protein
recruitment. Other domains, such as nucleotide binding site
(NBS), specify nucleotide binding.
DEATH LIGANDS, RECEPTORS,
AND SIGNALS
Death of a cell may result from intracellular stress activating
an intrinsic death program or may be forced on the cell by
the interaction of a death ligand with a death receptor (see
Fig. 24-2). Death receptors belong to the tumor necrosis fac-
tor (TNF) receptor superfamily of proteins, which comprises
approximately 25 members.15,16 This family of receptors is
responsible for diverse biologic responses, such as inflam-
mation, proliferation, antiviral activity, and cell death.
receptors capable of transmitting apoptosis (see later) and
receptors such as CD40, CD30, BlyS/BAFF/TALL, and
TACI15 that trigger survival or proliferation or both partly
through activation of nuclear factor kB (NFkB). Although
most receptors of the TNFR family exert their effects pri-
marily within the immune system, some members (e.g.,
p75NGFR, TNFR I and II) seem to serve important func-
tions in the nervous system and other organs. Some TNFR-
like proteins, such as PV-T2, PV-A53R, and CAR1, are
encoded by viruses and may contribute to their virulence.15
DEATH RECEPTORS
The death receptors identified, including Fas, TNFR I, DR-3
(TRAMP/wsl/APO-3), DR-4 (TRAIL), DR-5, and DR-6,
share homology in their intracellular domains over a
70-amino-acid region termed the death domain.17 Three
decoy receptors have been identified, two (DcR1 and DcR2)
that bind and inhibit their ligand, TRAIL, and one (DcR3)
that binds Fas ligand. These decoy receptors presumably
modulate cytotoxic function of the ligands, but the biologic
contexts remain to be fully defined. Alternative splice forms
and shedding of the receptors and ligands also down-modulate
their function.
TNFR family members are characterized by two to six cys-
teine-rich domains (CRDs) in their extracellular regions.15
The co-crystal structure of TNFR I and lymphotoxin-α
indicates that the CRDs project from the cell surface in a
linear array, making distinct contacts with ligands at sub-
unit interfaces. The first CRD also may be responsible for
preassembly of the receptor as trimers that undergo further
conformational alterations on ligand engagement.
The three-dimensional structure of the DD has been solved
by nuclear magnetic resonance spectroscopy and has been
shown to consist of six amphipathic α-helices that create a
unique fold.18 Functionally, the DD seems to be a novel pro-
tein-protein association motif that facilitates homotypic inter-
actions. The DD of Fas and TNFR I self-associate, recruiting
adapter proteins that also contain DD and that directly or indi-
rectly mediate receptor signal transduction (see Fig. 24-2).
DEATH RECEPTOR SIGNAL TRANSDUCTION
This section focuses predominantly on signaling from Fas and
TNFR because these are the best-characterized members of
the death receptor subfamily, and it is likely that other death
receptors signal through similar pathways. As illustrated in
Figure 24-4, Fas and TNFR1 share a common death pathway.
Binding of Fas ligand to Fas causes conformational changes
in the receptor cluster leading to recruitment of intracellular
adapter molecules. Initially, aggregation of Fas induces uptake
of the adapter protein, FADD, to the DD of Fas. FADD has
two structural domains—a C-terminal DD, which mediates
Fas binding, and an N-terminal DED. The FADD DED allows
recruitment of procaspase 819,20 and procaspase 1021 through
DED-DED interactions. Procaspases 8 and 10 have a bipar-
tite structure comprising a DED and an enzymatic caspase
domain, the latter linking Fas aggregation with the execution
phase of apoptosis. The apposition of procaspases 8 and 10 to
the activated Fas complex leads to autocatalytic cleavage and
conversion of the proenzymes to activated proteases, which
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION 383

Induction Immune homeostasis Genotoxic damage, p53 Abnormal protein processing-

Immune privilege Drugs, loss of growth factor folding, glycosylation
Increased Ca2+

Death receptors Mitochondrial stress ER stress

Fas Bax
Pore
TNFR Bak
DD Bip
Cyt c
TRADD FADD
TRAF
PERK
RIP IRE
DED Bid
Caspase 9
TRAF Caspase 12, (7)
NFκB Apoptosome

Caspase 8,10
Survival

Execution
Attenuation JNK,
of protein synthesis ATF6
Caspase 3, (6)
Dissolution
CAD, Acinus, AIF, DNase II

Potential binding of Cleavage of

β2 glycoprotein, structural (fodrin, lamins),
annexin V, functional (DNA-PK,Ö)
PS
clotting factors proteins
Chromatin cleavage nucleosomes
Figure 24-4  Mammalian apoptotic pathways. Cell death can be initiated by multiple pathways, including an extrinsic ligand-induced pathway (left
panel), an intrinsic pathway mediated by the mitochondria (middle panel), and an intrinsic pathway mediated through the endoplasmic reticulum (ER)
(right panel). Examples of stimuli that can induce each of these pathways are shown and discussed in further detail in the text. These pathways differ
in the upstream caspases activated, but converge to cleave the effector caspases, such as caspase 3, during execution of apoptosis. Tumor necrosis
factor receptor (TNF-R) and other “death receptors” also can signal cell survival by activation of NFкB. During ER stress, unfolded proteins release the ER
chaperone protein, Bip, from binding to the stress sensor proteins, IRE, PERK, and ATF6. PERK attenuates protein synthesis, whereas ATF6 and JNK are
transcription factors that upregulate the expression of proapoptotic proteins that contain unfolded protein response elements such as CHOP (GADD
153) and caspases. In mice, caspase 12 is the initiator caspase, whereas in human cells, caspase 4 and caspase 7 have been implicated. The alterations
that occur during dissolution of the cell are too numerous to mention, but a few are highlighted in view of their potential relevance to autoimmunity
(see text). Exposure of phosphatidylserine (PS) on the cell surface (left lower panel) provides a simple means for detection of apoptotic cells through
binding annexin V and may be relevant to the generation of antiphospholipid autoantibodies and coagulation disorders in vivo. Cleavage products
of chromatin (lower middle panel) and proteins, such as lamins and DNA-PK, may be antigenic. AIF, apoptosis-inducing factor; CAD, caspase-activated

are released and able to initiate a proteolytic cascade lead-
ing to programmed cell death. In some cell lines, caspase 8
cleavage also results in cleavage of the proapoptotic molecule
Bid, which activates the mitochondrial amplification cascade
(type II pathway) (see Fig. 24-4).22
Although the six DD-containing receptors initiate cell
death in certain contexts, all may signal cell survival and
proliferation in different cell types and in different contexts.
The ability to signal an opposite cell fate depends on the
recruitment of proteins such as the TNFR-associated factors
that activate NFкB, promoting cell survival (see later).
DEATH LIGANDS
Fas ligand (FasL) (CD178) is a 40-kD type II transmembrane
protein that shares 15% to 35% amino acid identity with the
TNF superfamily of ligands. FasL is expressed constitutively
in the anterior chamber of the eye and in the testis, but is
induced when CD8+ cells, T helper type 1 CD4+ T cells,
and some natural killer cell populations become activated.23
In lymphocytes, expression of ligand is tightly regulated,
and activity on the cell surface is short-lived because metal-
loproteases cleave the extracellular portion of the ligand
into soluble, functional molecules. The zinc metalloprote-
ase, TNF-α converting enzyme (TACE), is a membrane-
anchored member of the disintegrin family of proteases that
cleaves active TNF-α from the cell surface.24,25
FUNCTION IN IMMUNE REGULATION
Although the role of Fas and FasL interactions in the thy-
mus is controversial,26 this pathway is involved in the main-
tenance of immune privilege in the eye and the testis, in the
pathogenesis of graft-versus-host disease, and in immune
 384 ELKON  |  Cell Survival and Death in Rheumatic Diseases
evasion by tumors.23 The major physiologic function of
Fas and FasL in the immune system is the preservation of
peripheral tolerance. This preservation is achieved by the
phenomenon of activation-induced cell death, whereby
CD8+ cells, T helper type 1 CD4+ T cells, and possibly natu-
ral killer cells induce apoptosis of activated T cells, B cells,
and macrophages. The deletion of activated immune cells
removes the source of proinflammatory molecules, prevents
the continued presentation of self-peptides by primed (high
levels of costimulatory molecules) antigen presenting cells,
and eliminates B cells that have mutated to self-specificity in
the germinal centers.27 These topics are discussed in greater
detail elsewhere in this textbook, and the consequences of
Fas deficiency are described later in this chapter.
Although TRAIL signals apoptosis through DR4 and
DR5 predominantly in tumor cells, more recent evidence
suggests that TRAIL plays a role in negative selection of
thymocytes.28 Similarly, DR3 (the receptor for the ligand
TWEAK) also has been implicated in negative selection.29
Finally, DR6 plays a role in immunologic homeostasis as
evidenced by enhanced T cell and B cell proliferation in
DR6-deficient mice.30
INTRINSIC CELL DEATH PATHWAYS:
INITIATION AND EXECUTION OF
APOPTOSIS
Cells need constant sources of nutrition and depend on a
variety of signals for active maintenance of survival. Loss of
signals from neighboring cells31 or withdrawal of growth fac-
tors or cytokines results in initiation of a cell death program.
Damage or stress to intracellular organelles may be induced
from outside or within the cell. This section discussses
injury or stress to DNA, mitochondria, and the endoplas-
mic reticulum.
GENOTOXIC INJURY
Mutations occur frequently in mammalian DNA and usu-
ally are promptly repaired. If repair fails, or DNA is severely
damaged by radiation or drugs, the transcription factor p53
(“guardian of the genome”) is upregulated and phosphory-
lated by DNA damage sensors, such as ATR and ATM.
Activated p53 induces a cell cycle arrest through induction
of the cyclin-dependent kinase inhibitor p21. If the DNA
damage is repaired, cell cycle arrest is abrogated, whereas if
the injury cannot be repaired, the cell undergoes apoptosis.
The importance of p53 as a tumor suppressor is illustrated
by the high frequency of p53 mutations in cancers.32 p53
induces apoptosis partly by transcription of death effectors,
such as Bax, which cause mitochondrial stress. In addition,
activation of the transcription factor Foxo-1 upregulates the
expression of Bim, FasL, and TRAIL.33
MITOCHONDRIAL STRESS
Mitochondria are cytoplasmic organelles that contain their
own 16-kb genome encased by inner and outer membranes
with numerous proteins, including cytochrome c, situated
between these membranes (see Fig. 24-4). Mitochondria
help to maintain redox potential and constitute the energy
powerhouse of the cell through the generation of ATP by
oxidative phosphorylation. These biochemical pathways
create an electrochemical gradient (Δψ) that is positive
and acidic on the outside and alkaline on the inside of the
mitochondrial membrane. Spanning the inner membrane
is the adenine nuclear translocator, which mediates ATP
transport (with the voltage-dependent anion channel
[VDAC]—see later) to the cytosol. The VDAC, which is
permeable to solutes of approximately 5000 kD, is situated
on the outer mitochondrial membrane.34
Genotoxic injury, reduced supply of nutritional or growth
factors, increased intracellular calcium, reactive oxygen
intermediates, and exposure to certain chemicals, such as
staurosporine, cause mitochondrial stress. These initiating
factors lead to selective mitochondrial membrane permeabi-
lization with the resulting dissipation of the proton gradient
responsible for the Δψ (permeability transition), permeabi-
lization of the outer membrane, and loss of ATP production.
Mitochondria themselves are the major producers of reac-
tive oxygen intermediates, which in excess damage nucleic
acids, proteins, and membrane lipids.
When mitochondrial membrane permeabilization is ini-
tiated, cytochrome c is released from the intermitochondrial
space into the cytosol (see Fig. 24-4). In the cytosol, cyto-
chrome c and the cofactors Apaf-1 and ATP or dATP assem-
ble with caspase 9 to form a molecular aggregate called the
apoptosome that promotes the cleavage of procaspase 9 into
its active form.35 Caspase 9 acts on effector caspases, such
as caspase 3, resulting in the caspase cascade that leads to
the cleavage and inactivation of a wide variety of substrates
within the cell (see Fig. 24-4). A caspase-independent,
apoptosis-inducing factor (AIF) also is released from the
mitochondria and induces nuclear changes and cell death
by less well-defined pathways.36
ENDOPLASMIC RETICULUM STRESS
The main functions of the endoplasmic reticulum are to
regulate intracellular calcium flux and to promote proper
folding of nascent proteins. In the contiguous conduit, the
Golgi apparatus, post-translation modifications, such as
glycosylation and isoprenylation, are executed. Elaborate
mechanisms are in place to ensure that errors in protein
folding do not occur, but if they do, an “unfolded protein
response” is initiated (see Fig. 24-4). The endoplasmic
reticulum/Golgi apparatus initiates apoptosis if calcium flux
is excessive, if unfolded proteins persist, or if post-transla-
tional protein modification is abnormal.37 Release of the
cleaved IRE protein leads to degradation of 28S RNA and
termination of protein synthesis. In contrast to the death
receptor or mitochondrial pathways, apoptosis is executed
through caspase 12 in mice and possibly caspase 4 in mam-
mals.38 Many of the same molecules that regulate apoptosis
in the mitochondria, the Bcl-2 family in particular, also
affect endoplasmic reticulum–mediated apoptosis, how-
ever, possibly through control of intraluminal calcium.
ANTIAPOPTOTIC PROTEINS: FLIP, Bcl-2, IAPs,
and Akt
Cellular homeostasis within each bodily system is care-
fully regulated. Excessive cell growth or premature cell
death translates into diseases, as discussed in the last part
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
of this chapter. The death and survival pathways are finely
balanced, and each cell death program may be attenuated,
often at multiple levels. Before discussion about how the
death program is executed, the way in which cell death is
regulated by inhibitors of apoptosis is discussed.
Inhibition of Death Receptors
In most resting cell types that express Fas on their cell
surface (e.g., lymphocytes), the receptor is nonfunctional.
Resistance to death is explained by low levels of expres-
sion of the receptor and by active inhibition by a protein
called Fas long inhibitory protein (FLIP). FLIP resembles
the structure of caspase 8 and competes with caspase 8 for
recruitment to FADD. This prevents FADD from initiat-
ing apoptosis. When lymphocytes become activated, FLIP is
usually degraded, allowing Fas signal transduction to occur
unimpeded. Similarly, a protein called SODD (silencer of
death domain), attenuates TNFR1 signal transduction.
Bcl-2 Family of Cell Death Regulators
The Bcl family comprises more than 18 members.39 Bcl-2
is the prototype antiapoptotic protein that was first discov-
ered to be overexpressed in certain B cell lymphomas (Bcl).
Of particular significance, Bcl-2 overexpression did not
enhance cell proliferation (most cells were in the G0/G1
phase of the cell cycle), but rendered the cells more resistant
to death. The antiapoptotic members (Bcl-2, Bcl-XL, Bcl-w,
Mcl-1, and A1) contain three to four of the characteristic
Bcl-2 homology (BH) domain motifs, and most possess the
N-terminal BH4 domain and the hydrophobic C-terminal
membrane anchor, accounting for their attachment to mito-
chondrial, endoplasmic reticulum, and nuclear membranes.
Virus-encoded proteins (BHRF1, LMW5-HL, ORF16,
KS-Bcl-2, and E1B-19K) have similar antiapoptotic func-
tional properties to Bcl-2. The proapoptotic members of this
family can be subdivided into two groups—the Bax/Bak-like
proteins (Bax, Bak, Bok, and BCl-Xs), which contain two to
three BH3 domains, and the BH3-only subset (Bad, Bik, Bid,
Hrk, Bim, Noxa, Puma, and Bmf), which contain only the
single domain. Bcls are regulated at the transcriptional and
the post-transcriptional levels by a multitude of stimuli.
How do Bcls regulate apoptosis? One level of regulation
is conferred by binding interactions (homodimerization or
heterodimerization) between members via their BH1, BH2,
and BH3 domains.40 Although the outcomes vary for each
specific pair, homodimerization of Bcl-2 or Bax potentiates
their antiapoptotic or proapoptotic function respectively,
whereas heterodimers may potentiate or abrogate function
of one member of the pair. Bax and Bak have been shown to
be pivotal downstream effectors of intrinsic apoptotic path-
ways. A possible model is that Bcl-2 (or homologues) usu-
ally heterodimerizes with Bax and Bak preventing apoptosis.
Increased expression of a BH3 proapoptotic protein binds to
Bcl-2, releasing Bax and Bak to induce apoptosis. Bcls such
as Bcl-2 and Bcl-XL also may bind to Apaf-1 and prevent
it from activating caspase 9, analogous to the regulation of
CED-4 by CED-9 in C. elegans (see Fig. 24-1).
Bcl regulation of cell death is closely connected to mito-
chondrial function. The physical association of Bcl-2 fam-
ily proteins with the outer mitochondrial membrane and
385
the close structural similarity between the BH1 and BH2
domains and bacterial pore-forming proteins such as coli-
cin41 allow them to regulate ion fluxes or the transfer of
small molecules from the membrane. In vitro models suggest
that Bax and Bak promote opening of the VDAC, allowing
the release of cytochrome c into the cytosol, whereas Bcl-2
binds directly to the VDAC and closes it.34
Intracellular Inhibitors of Apoptosis
Intracellular inhibitors of apoptosis (IAPs) are a separate
family of antiapoptotic proteins that are highly conserved
through evolution. The neuronal apoptosis inhibitory pro-
tein (NAIP) was discovered through the association of
NAIP mutations in patients with the severe form of spinal
muscular atrophy. Seven additional members of the family
(c-IAP-1, c-IAP-2, X-IAP, survivin, ILP2, ML-IAP, and
Bruce) that share a baculovirus IAP repeat domain have
subsequently been identified, and most contain a RING
domain that functions as an E3 ligase presumably target-
ing interacting proteins for proteasomal degradation. IAPs
such as X-IAP directly inhibit effector caspases.42 IAPs
block apoptosis induced by a variety of stimuli, including
Fas, TNF-α, ultraviolet irradiation, and serum withdrawal,
and survivin is overexpressed in certain cancers and in the
rheumatoid arthritis (RA) synovium.43 In some cells, the
antiapoptotic effect of IAPs is eliminated by the release of a
protein called Smac/Diablo from the mitochondria.
Akt
Akt is a cytosolic protein kinase (protein kinase B) that plays
a special role in prevention of apoptosis because it links cell
activation through PI-3 kinase with multiple transcription
factors. Phosphorylation of Akt is antagonized by the phos-
phatase PTEN. When phosphorylated, Akt promotes cell
survival by altering the function of the intrinsic (mitochon-
drial) and the extrinsic (death receptor) pathways of apop-
tosis. Specifically, this includes inactivation of proapoptotic
molecules, such as caspase 9 and Bad, and activation of sur-
vival pathways that include NFкB and forkhead transcrip-
tion factors that inhibit FasL.44 The antiapoptotic molecules
also may be cell type specific or context specific.
CASPASES
Caspases are cysteine-containing proteases that have an
unusual substrate specificity for peptidyl sequences with a P1
aspartate residue.45,46 These proteases are 30 to 50 kD in size
and comprise an aminoterminal prodomain, a large subunit
domain, and a small subunit domain. The active site cyste-
ine residue is contained within the conserved pentapeptide,
QACxG, on the large subunit of the enzyme, whereas most
of the substrate specificity is determined by the small sub-
unit. The upstream caspases 8, 9, 10, 2, and 4 have large
prodomains that interact with regulatory proteins such as
FADD for caspases 8 and 10 and Apaf-1 for caspase 9 (see
Fig. 24-4). Presumably, clustering of these complexes allows
autocatalytic cleavage of the large and small subdomains to
form the active tetramer. Effector caspases such as caspases
3, 6, and 7 have small prodomains and are thought to be
cleaved into their active forms by the upstream caspases.
 386 ELKON  |  Cell Survival and Death in Rheumatic Diseases
Members of the caspase family can be divided into three
functional subgroups based on their substrate specificities.47
Group I members (caspases 1, 4, and 5) are potently inhib-
ited by the serpin CrmA; group II members (caspases 2, 3,
and 7) are specific for DExD; and group III members (cas-
pases 6, 8, 9, and 10) are specific for I/V/LExD, a sequence
that also is contained at the junctions of the caspase sub-
units themselves. Granzyme B produced by cytotoxic T cells
has a substrate specificity similar to that of group III caspases
and is capable of inducing apoptosis through this pathway.
Identification of the substrate specificity of caspases has led
to numerous practical applications, including the ability to
quantify activity using fluorogenic tetrapeptide substrates
and blockade of proteolytic activity with noncleavable cell-
permeable tetrapeptide analogues.
The effector caspases are necessary for the execution
of apoptosis. They cleave specific substrates, such as the
structural proteins fodrin, gelsolin, and lamins, key intra-
cellular enzymes involved in DNA repair (e.g., poly-ADP
ribose polymerase, DNA-PK) (see Fig. 24-4). These changes
facilitate inactivation of synthetic functions of the cell, dis-
solution of the nuclear membrane, and packaging of cellular
proteins into apoptotic blebs on the cell surface. Caspases
also cleave regulatory proteins such as Bcl family mem-
bers and the inhibitor of caspase-activated DNase (CAD).
Cleavage of CAD inhibitor leads to the release of active
CAD, which enters the nucleus and cleaves nucleosomes at
the linker region, yielding the characteristic “DNA ladder”
(see Figs. 24-3 and 24-4).48,49
Not all caspases are involved in the execution of apop-
tosis. Human caspases 1, 4, 5, and 12 and mouse caspases
1, 4, 11, and 12 are most likely involved in inflammation.
Caspase 1 originally was defined as the enzyme that cleaves
interleukin (IL)-1β (IL-1-converting enzyme) into its active
form. It has been shown more recently that caspase 1 and
caspase 5 interact and form a multiprotein complex that has
been called the inflammasome (analogous to the apopto-
some).50 Caspase 1 and caspase 5 bind to the adapter pro-
teins ASC (PYCARD) (caspase 1) and NALP1 (DECAP)
(caspase 5) by their CARD domains. ASC and NALP1
are mutidomain proteins that contain many of the protein
interaction domains listed in Table 24-1. The N-terminus
of the protein, pyrin, which is mutated in familial Mediter-
ranean fever, binds to ASC. More recent studies indicate,
however, that the C-terminus of pyrin, which is mutated in
familial Mediterranean fever, binds directly to IL-1β, sug-
gesting that pyrin normally directly exerts an inhibitory
effect on IL-1β.51
Caspases are tightly regulated by their own prodomains
and by Bcl and IAP family members. In addition, viral pro-
teins, such as the serpin CrmA, produced by cowpox, and
p35, produced by baculovirus, are potent inhibitors of cas-
pases.
REMOVAL AND DEGRADATION
OF APOPTOTIC AND NECROTIC CELLS
Of the 14 C. elegans death genes (ced1 through ced14), at
least half encode proteins that are required for engulfment
of apoptotic cells (see Fig. 24-2).52 CED-7 is present in the
membrane of the apoptotic cell and the phagocyte, whereas
the remaining proteins function in the phagocyte to execute
two partially overlapping pathways of engulfment. CED-1 is
a receptor that recognizes changes in the apoptotic cell and
signals through CED-6 to activate the phagocyte. CED-2,
CED-5, CED-10, and CED-12 most likely form a functional
complex that promotes the cytoskeletal changes required to
engulf the apoptotic prey.53 The mammalian counterparts
are described in Figure 24-2, and their signaling pathways
are discussed in detail elsewhere.54
Within the immune system alone, more than 109 apop-
totic cells are removed from the body each day. These
apoptotic cells are generated in vast numbers in the central
lymphoid organs, such as the thymus and bone marrow, by
out-of-frame rearrangements of antigen receptors, negative
selection, or simple “neglect.” A significant load of apoptotic
cells is produced in the peripheral immune system because
of the relatively short life span of lymphocytes and myeloid
cells and secondary selection of high-affinity B cells in ger-
minal centers. The specialized sites of selection (i.e., thy-
mus, bone marrow, and lymphoid follicles) have remarkably
efficient phagocytes that rapidly remove the dying cells.
An early event that occurs in apoptotic cells is the appear-
ance of phosphatidylserine on the cell surface membrane
(see Fig. 24-4). This membrane asymmetry (phosphatidyl-
serine is usually located on the inner surface of the mem-
brane) is caused by the reduced function of a translocase
and possibly by activation of a lipid scramblase.55 Phospha-
tidylserine probably does not act as a ligand itself, although
oxidation of phosphatidylserine or phosphatidylcholine
may engage the scavenger receptor, CD36.56 Phosphati-
dylserine is recognized by numerous serum opsonins, such
as annexin I, Gas6, β2 glycoprotein 1, and milk fat globule
epidermal growth factor 8 (MFG-E8). This heterogeneity
allows the protein bridge to interact with different recep-
tors (Fig. 24-5). MFG-E8 facilitates apoptotic cell clear-
ance in germinal centers,57 whereas C1q deficiency leads to
apoptotic cell accumulation in the kidney.58 Complement
components, such as C1q and iC3b, also opsonize apoptotic
cells for recognition and efficient clearance by macrophages
as discussed later. Natural IgM antibodies and acute-phase
proteins such as C-reactive protein amplify serum comple-
ment deposition.59,60 Other serum opsonins or bridging mol-
ecules include thrombospondin, which bridges the αvβ3 and
CD36 receptors,61 and collectins (mannose binding protein,
C1q and surfactant proteins). Collectin binding receptors
are controversial (see Stuart and colleagues62 for discussion).
The endoplasmic reticulum protein calreticulin is unique in
that it is translocated from the endoplasmic reticulum to the
cell surface of apoptotic cells, but also can be detected at low
concentrations on live cells.63
Despite the detection of only limited chemical altera-
tions on the apoptotic cell membrane, blockade of a large
and diverse number of receptors on phagocytes can impair
the uptake of apoptotic cells (see Fig. 24-5). This diversity
may be explained partly by the different cells and conditions
used for phagocytic assays, but it likely also reflects the over-
lapping and partially redundant function of each individual
receptor. All of the receptors identified have other functions,
perhaps reflecting an evolution from receptors designed to
remove apoptotic cells during development to pattern rec-
ognition receptors useful for host defense.64 Many of the
receptors are integrins comprising the vitronectin receptor,
αvβ3,65 αvβ5,66 complement receptors 3 (CD11b/CD18)
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
Apoptotic cell Phagocyte
Annexin I
CD91
CRT
Gas6
PS MER
MFG-E8
αv β3/5
Ox
CD36
? in lysosomes and degrades ingested DNA, whereas multiple
TSP
LFA-3
CD14
C3bi
CR3, CR4
LyPtC
lgM/
CRP
Mannose
? CD91
Collectins
Figure 24-5  Receptors, ligands, and opsonins (bridging proteins) im-
plicated in recognition or phagocytosis of apoptotic cells (see text for
details). CRP, C-reactive protein; CRT, calreticulin; LyPtC, lysophospha-
tidylcholine; Ox, oxidized form; PS, phosphatidylserine; PSR, phosphati­
dylserine receptor; TSP, thrombospondin.
and 4 (CD11c/CD18),67 and class A and B scavenger recep-
tors. Nonintegrin receptors include the ATP-binding cas-
sette transporter (ABC1)68; CD1469; and the closely related
Tyro 3 family receptor tyrosine kinases, c-Mer, TYRO, and
Axl.70 CD91 (low-density lipoprotein receptor–related pro-
tein), a multifunctional receptor, recognizes 30 different
ligands, calreticulin being one.63 According to the “tether
and tickle” model,71 some receptors, such as CD14 or CR3,
serve as recognition structures and contribute to adhesion;
others, such as CD91, convey signals for engulfment; and
others, such as SIRP-α, prevent uptake.
The ingestion of apoptotic cells has significant effects
on the phagocyte and potentially on the T cell response to
ingested antigens. In vitro72,73 and some in vivo74 studies sug-
gest that uptake of apoptotic cells by macrophages induces
the expression of immunosuppressive cytokines, such as trans-
forming growth factor (TGF)-β1, prostaglandin E2, and pos-
sibly IL-10, by macrophages. These cytokines tend to dampen
an immune response to self-antigens. Ligands for c-Mer, such
as Gas6 and protein S on apoptotic cells, suppress production
of IL-12 or TNF-α by macrophages. Apoptotic cells activate a
specific transcriptional repressor of IL-12, GC-BP,75 which also
suppresses adaptive immune responses. Because some peptides
derived from apoptotic cells can be presented to lymphocytes
by dendritic cells and possibly by macrophages through cross-
priming,66,76 a question of paramount importance to studies
of autoimmunity is whether self-peptides are presented after
phagocytosis of apoptotic cells, and under what conditions
they induce tolerance or immunity.
387
Engulfment of apoptotic cells or necrotic cell debris is the
first step of the “clean-up” process, but swift degradation of
cellular contents within the phagocyte and extracellularly
is equally important. It has been shown that self-nucleic
acids may be potent inducers of type 1 interferons and other
inflammatory cytokines through activation of Toll-like
receptors and Toll-like receptor–independent pathways.77
Extracellularly, opsonins, such as C-reactive protein, func-
tion as a scavenger for nucleoproteins,78 and serum contains
a potent DNase, DNase 1, and abundant RNases. Within
the cell, a specific acid-activated DNase, DNase II, resides
different RNases have been described. Failure to degrade
these molecules likely explains why the debris from necrotic
cells induces type 1 interferon, TNF-α, and other proinflam-
matory cytokines.72,79
DETECTION OF APOPTOSIS
Numerous methods have been devised to detect cells under-
going apoptosis. These methods depend on the ­biochemical
changes in the cell described earlier and are depicted in
Figure 24-3. Electron microscopic examination (see Fig. 24-1)
is still regarded as the “gold standard.”
CELL MEMBRANE ALTERATIONS
Annexin V binds to negatively charged phospholipids in a
calcium-dependent manner and can readily detect the flip
of phosphatidylserine to the outer surface of the cell mem-
brane (see Fig. 24-3A). When annexin V is conjugated
to fluorescein isothiocyanate, biotin, or other markers, it
provides a convenient tag for detecting apoptotic cells by
flow cytometry.80 Flow cytometry detection with annexin
V is simple, is sensitive, and detects cells at an early stage
of apoptosis. Annexin V also binds to necrotic cell mem-
branes before complete rupture of the cell. Entry of trypan
blue, as seen by light microscopy, or propidium iodide, as
quantified by flow cytometry, into the cell indicates pro-
found damage to the cell membrane indicative of necrosis.
LOSS OF MITOCHONDRIAL MEMBRANE
POTENTIAL
As discussed earlier, multiple stimuli lead to apoptosis
through the intrinsic mitochondrial pathway resulting in
a loss of membrane potential. Several dyes, including rho-
damine 123, tetramethylrhodamine methyl ester (see Fig.
24-3B), and DiOC6, bind selectively to mitochondria and
provide a fairly sensitive measure of membrane potential.
CASPASE ACTIVATION
As discussed previously, caspases are normally present in an
inactive state, but when activated, caspases recognize­ spe-
cific tetrapetide sequences. Caspase activity can be quantified
directly in intact cells by flow cytometry­ analysis with cell-
permeable fluorochrome tetrapeptide conjugates or in cell
extracts by enzyme-linked immunosorbent assay that detects
release of colorimetric dyes conjugated to the ­ tetrapeptides.
Caspase activation also can be quantified indirectly, by West-
ern blot analysis with antibodies specific for cleaved (activated)
 388 ELKON  |  Cell Survival and Death in Rheumatic Diseases
caspase 3 or by cleavage of specific caspase substrates. Cleav-
age of the nuclear protein, poly-ADP ribose polymerase, also
is used to evaluate activation of caspase 3 (see Fig. 24-3C).
CHROMATIN CONDENSATION AND DNA
FRAGMENTATION
Condensation of chromatin can be seen by light micros-
copy after nuclear staining (see Fig. 24-3D). This can be
a sensitive screening method depending on the experience
of the viewer, but confirmation with a more specific assay
usually is required for verification. Chromatin condensa-
tion is more easily seen by staining with vital dyes, such
as Hoechst No. 33342 bisBENZIMIDE (2′-(4-ethoxyphe-
nyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole)
or DAPI (4′,6-diamidino-2-phenylindole), and inspection
under fluorescence microscopy (see Fig. 24-3E). For precise
quantification of nuclear condensation, DNA staining with
propidium iodide and flow cytometry analysis of condensed
(subdiploid) DNA is widely used (see Fig. 24-3F).81
As mentioned previously, DNA is cleaved by multiple
DNases leading to the cleavage of nucleosomes at the linker
region between histone binding, yielding the characteristic
180-bp DNA ladder (see Fig. 24-3G). DNA fragmentation
results in free 3′-OH groups, which can be detected within
the nuclei in tissue sections using biotinylated dNTPs
(TUNEL assay) (see Fig. 24-3H).82 Although formation of
the ladder is specific to apoptosis, generation of free ends
of DNA is not and may be detected in DNA damaged by
necrosis. It has been reported that TUNEL and in situ DNA
incorporation methods may yield positive results in cells
undergoing extensive DNA repair or rapid proliferation.83,84
APOPTOSIS IN RELATION TO RHEUMATIC
DISORDERS
The regulation of apoptosis is highly relevant to the patho-
genesis and treatment of rheumatic disorders. Pertinent
examples are discussed in the following sections.
DEFECTIVE APOPTOSIS OF IMMUNE CELLS
Mice with mutations of Fas or FasL develop a syndrome
characterized by lymphoproliferation (lpr) and generalized
lymphadenopathy (gld) together with systemic autoimmu-
nity.85 As might be expected from the key role described for
Fas in activation-induced cell death, lymphadenopathy and
splenomegaly are the consequence of failure of activated
lymphocytes to die, resulting in an absolute increase in the
numbers of T and B lymphocytes and by the accumulation
of an unusual subset of T cells that do not express CD4+ or
CD8+ coreceptors (i.e., double-negative T cells). The nature
and extent of systemic autoimmunity vary according to the
strain into which the Fas or FasL mutation has been bred.85
A syndrome of massive lymphadenopathy with systemic
autoimmunity in children was reported by Canale and
Smith in 1967. Subsequently, these and other lpr patients
were found to have mutations in Fas.86-88 The syndrome
(called Canale-Smith syndrome or autoimmune lympho­
proliferative syndrome) is characterized by lymphadenopathy
or splenomegaly, autoimmune cytopenias (most commonly
affecting platelets and red blood cells), and an increase
(>5%) in ­circulating double-negative T cells. These patients
had defective lymphocyte apoptosis in response to anti-Fas
antibodies or FasL when tested in vitro, and most had het-
erozygous mutations in Fas affecting DD. These mutations
impair Fas-mediated apoptosis through a dominant negative
effect.89,90 Although Fas/FasL mutations are an exception-
ally rare contributory factor to systemic lupus erythematosus
(SLE), the Canale-Smith syndrome is informative because
it suggests that defective apoptosis of cells of the immune
system can cause systemic autoimmune diseases, such as
idiopathic thrombocytopenic purpura, autoimmune hemo-
lytic anemia, and Guillain-Barré syndrome. It illustrates (as
do the mouse models) that even when a single gene has a
powerful effect on predisposition to systemic autoimmunity,
the clinical expression of disease depends on the precise
nature of the mutation89,90 and the interaction with modify-
ing genes.
There are several other examples of genetic alterations
in death or survival genes that lead to lupus-like diseases
in mice.91 Of particular interest is the overexpression of the
ligand for the BlyS/BAFF/TALL/zTNF receptor that pro-
motes the survival of B lymphocytes92 because increased
expression of this ligand has been reported in SLE, Sjögren’s
syndrome, and RA.93 Mutations in the p55/TNFR1/CD120a
receptor in humans results in a periodic autoinflammatory
syndrome called TNFR-associated periodic syndrome.
Mutations predominantly occur in the first two CRDs of the
receptor, resulting in reduced shedding of the extracellular
domain of the receptor and reduced neutralization of cir-
culating TNF-α.94 Increased lymphocyte survival resulting
from overexpression of Bcl-2, knockout of Bim, reduction in
PTEN activity,95 and increased survival of dendritic cells96,97
causes lupus-like autoimmunity in mice.
Defective apoptosis of B cells or T cells may lead to inap-
propriate survival of self-reactive cells in the central (thymus
or bone marrow) or peripheral immune system. Enhanced
survival of dendritic cells may promote the activation and
expansion of low-affinity self-reactive T cells.
DEFECTIVE UPTAKE AND PROCESSING
OF APOPTOTIC CELLS
Autoantibodies were discovered in the 1940s and 1950s, and
their molecular and functional identities were characterized
in the 1980s and 1990s. Why the immune system targets
a select subset of self-antigens (mainly nucleoproteins) in
each disease has never been satisfactorily explained. The
facts that autoantibodies target nucleosomes in SLE,98,99 and
certain anticardiolipin antibodies cross-react with phos-
phatidylserine, which translocates to the cell surface during
apoptosis,100 support the idea that autoantibodies target the
products of apoptotic cells. Additional inferential evidence
for this hypothesis comes from detection of lupus antigens
in apoptotic blebs,101 modification of antigens by cleavage,
and phosphorylation during apoptosis.102,103
Because apoptosis occurs on a vast scale in the central
lymphoid organs and should render the host tolerant, under
what conditions would apoptotic cells immunize? If apop-
tosis occurs in the presence of an adjuvant (virus, bacteria,
or chemicals), tolerance may be lost, at least transiently.
Abnormalities leading to the accelerated apoptosis of cells
in the periphery or reduced uptake of dying cells allow
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
cells to undergo postapoptotic necrosis which provokes a
proinflammatory cytokine response from phagocytes72 as
explained earlier. An increase in the rate of apoptosis of
SLE peripheral blood mononuclear cells has been observed
in vitro,104,105 suggesting that accelerated apoptosis occurs
in vivo. Although SLE macrophage survival in vitro also
is compromised, reduced macrophage phagocytosis of apop-
totic cells has been reported in SLE.106
Experiments in mice and in human cells in culture indi-
cate that phagocytosis of apoptotic cells is impaired in the
absence of early complement components,58,67 and it is well
known that deficiencies of early complement components
predispose to human SLE.107 Knockout of members of the
Tyro 3 receptor tyrosine kinases is associated with defective
clearance of apoptotic cells and the expression of a lupus-
like disease.70,108 It also is likely that the lupus-like diseases
that occur in mice deficient in the acute-phase protein SAP
result from reduced clearance of apoptotic cell debris.109,110
As mentioned previously, mammalian nucleic acids can
potently stimulate Toll-like receptor–dependent and Toll-
like receptor–independent pathways to generate inflamma-
tory cytokines. A deficiency of DNase 1 led to lupus, and a
conditional deficiency of DNase II caused RA-like disease
in mice.110,111 In addition, nucleic acids contained within
immune complexes have been shown to stimulate inter-
feron-α by plasmacytoid dendritic cells, providing a plau-
sible explanation for increased interferon-α observed in SLE
and amplification of disease activity.112
PROLONGED EXPOSURE TO GROWTH FACTORS
Histologically, RA is characterized by an accumulation of
inflammatory cells in the synovium, leading to pannus for-
mation and destruction of cartilage and bone. Although Fas
and FasL can be detected in RA synovium, and evidence
of apoptosis has been detected in RA synoviocytes,113,114 the
extent of synoviocyte apoptosis is inadequate to counteract
ongoing proliferation. This imbalance is explained by many
factors. First, FasL is expressed at relatively low levels on
synovial T cells,115 and soluble Fas or FasL competitors may
impede Fas-induced apoptosis. Cytokines, such as TNF-α,
IL-1β, and TGF-β1, which are overexpressed in the joints
of patients with RA, favor synoviocyte proliferation and
inhibit susceptibility to apoptosis.116-118 These and other sig-
nals reduce apoptosis of synoviocytes through activation of
NFkB and increased expression of antiapoptotic proteins,
including X-IAP and Akt.119-121 Growth of the pannus is
compounded by inflammatory changes such as oxidation,
which result in upregulation and mutations of the growth
suppressor protein p53122 and IL-23, which promote or sta-
bilize IL-17-producing T helper cells (Fig. 24-6). IL-23 has
been shown to promote cell survival.123
The observations regarding apoptosis regulators in RA
are significant because they provide an opportunity for thera-
peutic manipulation. Local administration of anti-Fas mono-
clonal antibodies to human T cell lymphotropic virus-1 tax
transgenic mice or Fas ligand to collagen arthritis (mouse
models of RA) led to an improvement in arthritis.124,125 Sev-
eral strategies to modulate NFkB attenuate the growth of
synovial cells. Administration of TRAIL also attenuated
experimental arthritis, although this was not thought to be
due to apoptosis,126 and an antibody to TRAIL-R2 (DR5)
389
Growth cytokines, TNF-α, IL-1-β, TGF-β
NFkB
Bcls
Lymphocytes
Fas ligand flow
Bcls p53 NFκB IAPs Bcls
O*, NO
Figure 24-6  Antiapoptotic phenotype of rheumatoid synovial fibro-
blasts. Cytokines and growth factors produced by macrophages and T
cells lead to activation of NFкB and overexpression of antiapoptotic pro-
teins, such as B cell lymphoma-2 (Bcl-2) family (Bcls). Inflammatory stim-
uli, release of nitric oxide (NO), and reactive oxygen intermediates (O*)
upregulate and induce mutations of p53. Infiltrating lymphocytes have a
Fas ligand “low” phenotype. IAPs, intracellular inhibitors of apoptosis; IL,
interleukin; TGF, transforming growth factor; TNF, tumor necrosis factor.
induced apoptosis of RA synovial fibroblasts.127 Evidence
is accumulating that fibroblasts in scleroderma also may be
more resistant to apoptosis, and that TGF-β may promote
this phenotype.128
TISSUE INJURY IN ORGAN-SPECIFIC
AUTOIMMUNITY
In contrast to systemic autoimmune diseases that are char-
acterized by B lymphocyte stimulation leading to antibody-
mediated and immune complex–mediated tissue injury,
many organ-specific autoimmune diseases are caused by a
cell-mediated attack leading to the death of specific cell
types within the organ. Cell targets are beta cells of the islets
of Langerhans of the pancreas in insulin-dependent diabetes
mellitus, oligodendrocytes in the brain in multiple sclerosis,
the salivary and lacrimal glands in Sjögren’s syndrome, and
myocytes in polymyositis.129 Programmed pathways of cell
death (apoptosis) can be implicated in pathogenesis of some
of these diseases, as illustrated by the resistance of Fas-defi-
cient (lpr) mice to diseases such as diabetes and experimen-
tal encephalomyelitis. In many of these diseases, cell death
at the site of injury also can be directly shown by DNA frag-
mentation (TUNEL staining) in situ. Apoptosis usually is
considered noninflammatory, but, as discussed earlier, the
context of cell death influences the immune response. Cyto-
toxic lymphocytes that induce cell death predominantly by
perforin-mediated cell lysis (necrosis, although apoptotic
changes also are observed through caspase 3 activation) or
macrophage defects leading to delayed clearance of dying
cells promote the release of proinflammatory cytokines,
such as TNF-α.
 390 ELKON  |  Cell Survival and Death in Rheumatic Diseases
In most organ-specific autoimmune diseases, especially
diseases for which adoptive transfers have been performed
in animal models, CD4+ T cells have been shown to be
critically involved in disease pathogenesis. Disease-promot-
ing CD4+ T cells are restricted by major histocompatibility
complex class II molecules and are unlikely to exert a direct
cytotoxic action on the class I–bearing target cell (although
CD4+ T cells can upregulate FasL). CD4+ cells may arm
other effectors through the production of cytokines (inter-
feron-γ); they may induce tissue injury through a “bystander
pathway” involving macrophages or induce receptors for
cell death on the target cell “assisted suicide pathway.” In
Sjögren’s syndrome in humans, there is controversy about
whether Fas or FasL is constitutively expressed in normal
salivary glands, but the coexpression of both molecules in
patients with Sjögren’s syndrome presumably causes cell
death of acinar and ductal cells.130
Inflammatory myopathies, such as polymyositis and
dermatomyositis, are autoimmune diseases that result in
destruction of skeletal muscle fibers. Although Fas is upreg-
ulated on the myocytes in these diseases, expression also is
increased in nonautoimmune muscle disorders, such as in
metabolic myopathies, denervating disorders, and muscu-
lar dystrophies, but not in normal human muscle tissue.131
Detection of FasL on mononuclear cells invading the
muscles in polymyositis and dermatomyositis patients with
apoptosis of muscle cells implicates Fas/FasL in tissue injury
in myositis.132 Increased expression of the T cell cytotoxic
mediator perforin in some polymyositis and dermatomyo-
sitis patients133 indicates that granzyme-mediated myocyte
injury also is involved. The tRNA synthetase antigens or
their cleavage products may perpetuate inflammation by
exerting chemotactic recruitment of immune cells through
chemokine receptors.134
ACCELERATED APOPTOSIS IN DEGENERATIVE
RHEUMATIC DISORDERS
Apoptosis of chondrocytes occurs during normal develop-
ment of joints, and accelerated cell death may be important
in diseases such as osteoarthritis. The main mechanism
underlying primary or secondary osteoarthritis is degrada-
tion of cartilage. Degradation is mediated by enzymatic
and nitric oxide–induced breakdown of the extracellular
matrix and insufficient new matrix synthesis. Normal and
osteoarthritis-derived chondrocytes in the superficial and
middle cartilage zones, the major areas involved in early
cartilage degeneration, express Fas and are sensitive to Fas-
mediated death.135 Chondrocytes obtained from patients
with osteoarthritis have enhanced spontaneous apopto-
sis in these zones compared with normal controls.136,137
Although nitric oxide also is capable of inducing apop-
tosis in chondrocytes, it does not seem to act through the
Fas pathway.135 In an experimental model of osteoarthritis,
transgenic mice lacking type II collagen, the main con-
stituent of the extracellular matrix in cartilage, had high
levels of apoptosis in their chondrocytes.138 Together, these
findings suggest that apoptosis of chondrocytes plays a role
in osteoarthritis, and that inhibitors of nitric oxide synthe-
sis may be valuable in treating this disease. The therapeutic
use of intra-articular Fas agonists in RA may be deleterious
to chondrocytes.
Osteoporosis is a common disorder resulting from
increased bone resorption, decreased bone synthesis, or a
combination of the two. Several reports support the concept
that estrogen exerts its beneficial effect in preventing osteo-
porosis by the induction of apoptosis in the bone-resorbing
osteoclasts,139,140 and glucocorticoid-induced osteoporo-
sis may be explained by an increased rate of apoptosis of
osteoblasts and osteocytes.141 In the presence of macrophage
colony-stimulating factor, osteoclasts differentiate from a
myeloid precursor common to macrophages and dendritic
cells. In addition to the numerous factors influencing bone
turnover,142 a soluble member of the TNFR family, osteo-
protegerin or osteoclastogenesis-inhibitory factor, inhib-
its osteoclast activity after binding to its cognate ligand,
RANK ligand (osteoprotegerin ligand/TRANCE).143,144
RANK ligand is expressed on osteoblasts and on activated
T cells. Engagement of the membrane form of the receptor
induces activation of NFкB, enhancing the formation, sur-
vival, and resorptive activity of osteoclasts. A monoclonal
antibody that blocks RANK ligand is currently in clinical
trial for treatment of osteoporosis.
DRUGS THAT AFFECT APOPTOTIC PATHWAYS
Until more recently, therapy for inflammatory rheumatic
disorders has been largely empiric. The types of drugs used
include anti-inflammatory agents, such as corticosteroids and
nonsteroidal anti-inflammatory drugs (NSAIDs); immuno-
modulatory drugs, such as cyclosporine; and cytotoxic drugs,
such as cyclophosphamide and azathioprine. Because most
of these drugs impinge on crucial biochemical events within
the cell, they have effects on pathways of apoptosis.
Anti-inflammatory Drugs
Glucocorticoids at high doses induce the death of lymphoid
cells through transcriptional regulation by the glucocorticoid
receptor. Corticosteroids modulate expression of numer-
ous molecules that affect apoptotic programs—cytokines,
cell cycle control proteins, c-myc, and Bcl-2—and inhibit
NFкB activation, but the precise pathways that may operate
in a cell-specific fashion145 and that are relevant to clinical
efficacy remain to be defined. Patients on long-term steroid
therapy also are susceptible to osteoporosis and osteonecro-
sis, which may be explained by bone loss caused by apoptosis
of osteoblasts and osteocytes.141
The major mechanism of action of NSAIDs is inhibi-
tion of cyclooxygenases, which reduce the production of
proinflammatory cytokines and prostaglandins (see Chap-
ter 54). NSAIDs also are effective in the chemoprevention
of colorectal tumors in genetically susceptible individuals.
Their antineoplastic properties may be explained by an
increase of the prostaglandin precursor arachidonic acid and
by conversion of sphingomyelin to ceramide, a proapoptotic
lipid.146,147
Immunomodulatory Drugs
Cyclosporine and a closely related macrolide antibiotic,
FK506, have potent immunosuppressive properties and are
used to prevent allograft rejection. Both drugs modulate T
cell and B cell immune responses by interfering with IL-2
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
gene transcription mediated by nuclear factor of activated
T cells, nitric oxide synthase activation, cell degranulation,
and apoptosis.148 The reduced cytotoxic T lymphocyte activ-
ity is explained partly by impaired FasL induction secondary
to the effect on nuclear factor of activated T cells,149,150 but
effects on mitochondrial function also have been shown.
Certain cell types, such as renal proximal tubules and syn-
oviocytes or endothelial cells in RA, may be more suscep-
tible to the proapoptotic effects of cyclosporine.151
Cytotoxic Drugs
Many cytotoxic or immunosuppressive drugs that induce
the suicide of lymphocytes, mostly through the p53 path-
way (see Fig. 24-4), exert some anti-inflammatory effect.
Although methotrexate has effects on adenosine recep-
tors, even low-dose methotrexate induces apoptosis of
activated lymphocytes in vitro and in RA patients, prob-
ably in a Fas-independent manner.152 Cyclophosphamide is
an alkylating agent commonly used to treat many human
cancers and severe autoimmune disease. Its efficacy has
been attributed partly to apoptosis of tumor cells and per-
haps of mesangial cells in glomerulonephritis.153 Induction
of apoptosis also may account for certain adverse effects,
such as oligospermia or azoospermia and pancreatic beta
cell destruction.
Bisphosphonates are the most potent antiresorptive
drugs available and are widely used to treat various meta-
bolic bone diseases, such as Paget’s disease, bone tumors,
ectopic calcification, and osteoporosis. Although individual
members of this family differ in their effects, their general
mechanisms of action include direct and indirect effects on
osteoclast recruitment, function, and survival.154
BIOLOGICS
The remarkable success of anti-TNF-α therapy for the treat-
ment of RA, other arthritides, and Crohn’s disease is gen-
erally attributed to blockade of TNF-α stimulation of the
proinflammatory NFkB pathway (see Fig 24-4).155 In Crohn’s
disease, it also has been reported, however, that anti-TNF-α
monoclonal antibodies ameliorate disease through bind-
ing to cell-associated TNF-α and induction of apoptosis of
macrophages and T cells.156,157 Etanercept and infliximab
induced apoptosis of monocyte/macrophages in RA syno-
vial tissue.158
B cell depletion therapy with anti-CD20 (rituximab) and
other B cell antigens is becoming an increasingly used thera-
peutic modality. The mechanism of action has been studied
most intensively in chronic lymphocytic leukemia B cells.
Rituximab depletes B cells by induction of apoptosis associ-
ated with down-modulation of Bcl-2 and XIAP, activation
of complement, and antibody-dependent, cell-mediated
cytotoxicity.159 See also Chapters 58 and 59.
THERAPEUTIC INTERVENTION
Understanding the biochemical pathways that regulate
apoptosis offers new opportunities for therapeutic inter-
vention, some of which are already in practice (Fig. 24-7).
In antibody-mediated diseases, induction of apoptosis of B
cells is effective. In cell-mediated diseases, such as Sjögren’s
391
syndrome and polymyositis, it may be beneficial to induce
apoptosis of the cytotoxic effector cell. In diseases charac-
terized by macrophage activation and inflammatory tissue
growth, induction of macrophage apoptosis by anti-TNF-α
reagents already has been shown to be effective in RA and
Crohn’s disease (see Fig. 24-7, A1).
If a death receptor is selectively expressed on the cell to
be killed, a death ligand could be administered. Examples
of such a strategy are the use of Fas agonists or anti-TRAIL
receptor antibodies in arthritis. Proapoptotic pathways
also could be initiated from within the cell by gene ther-
apy approaches (see Fig. 24-7, A2). Examples include
blockade of NFkB or overexpression of Bax. Similarly,
as discussed earlier, a cytokine or unwanted growth/dif-
ferentiation promoter, such as TNF-α in RA or osteopro-
tegerin/RANK ligand in osteoporosis, can be blocked by
a monoclonal antibody or soluble receptor fusion ­protein
(see Fig. 24-7, A3).
In diseases in which apoptotic cell death leads to loss
of organ function, the death ligand could be blocked by a
A Effector or target cell
2
Key:
1 3 Activator
Inhibitor
B Target cell
Death receptor
Growth receptor
1
2
Caspases
3
Figure 24-7  Avenues for therapeutic manipulation of apoptosis— 
biologics in practice or clinical trials. Anti–tumor necrosis factor (TNF) 
reagents work in part by engaging membrane TNF (mTNF) on activated
macrophages and inducing apoptosis. Anti-CD20 antibodies eliminate
B cells in part by inducing apoptosis. Anti-BAFF/BLyS reagents reduce B
cell and possibly macrophage survival. Antibodies to RANK ligand reduce
differentiation or survival of osteoclasts. Estrogens promote osteoclast
apoptosis and bisphosphonates prevent it. Other approaches to modula-
tion of apoptotic pathways are discussed in the text.
 392 ELKON  |  Cell Survival and Death in Rheumatic Diseases
­ onoclonal antibody or soluble receptor fusion protein (see
m REFERENCES
Fig. 24-7, B1). Even when the death pathway has not been fully  
­determined, attempts can be made to interfere with
upstream components of apoptosis well before the “point of  
no return.” Antiapoptotic genes with limited (e.g., protein
FLIP blocks Fas pathway) or broad (e.g., Bcl-2 family) speci-  
ficity can be introduced into the cell (see Fig. 24-7, B2).
Further downstream, cell-permeable caspase inhibitors can
block the execution phase of apoptosis in vivo, as illustrated  
experimentally (see Fig. 24-7, B3).160 All of these approaches
 
are feasible, but they are limited by their potential adverse
effects. Therapy must be relatively specific for the target cell  
because widespread prevention of cell death for sustained
periods is likely to predispose to neoplasia.  
 
SUMMARY
Appreciation that death and survival of cells are highly  
regulated, and dissection of the biochemical pathways
that are activated by different modes of intracellular stress  
have made an enormous contribution to understand-
ing of the pathophysiology of human disease. Inherited
mutations of apoptosis-regulatory molecules may cause  
systemic autoimmunity, dysregulation, or misdirection of
other cell death and survival molecules, contributing to  
a whole range of musculoskeletal disorders. Many of the
drugs used to treat musculoskeletal disorders exert potent
effects on apoptotic programs. New biologic therapies that  
induce apoptosis of selected targets are already in use in
rheumatic diseases, and many more, perhaps with greater  
selectivity, are likely to be developed. All of these find-
ings suggest that further understanding of the regulation  
of apoptosis would have widespread implications for the
pathogenesis and therapeutic manipulation of rheumatic  
disorders.
 
ADDENDUM  
Two additional receptors for the uptake the apoptotic cells
have recently been described. Tim (T-cell immunoglobulin-  
and mucin-domain-containing molecule) 4 is a member of
the Tim family of type I membrane proteins that contain  
an immunoglobulin variable region-like domain, a mucin-
like domain, a transmembrane region, and a variable intra-
cellular domain. Tim4 is expressed on macrophages and  
dendritic cells and is reported to bind to PS on apoptotic
cells and exosomes.162 The second receptor is brain specific
angiogenesis inhibitor 1 (BAI1).163 BAI1 is a 7 transmem-  
brane protein that belongs to the adhesion-type-G-protein
coupled receptor (GPCR) family. The large extracellular  
 
portion of the molecule contains an integrin binding motif
and thrombospondin type 1 repeats that mediate binding  
to PS. BAI1 is highly expressed on astrocytes in the brain
but is also expressed on peripheral macrophages. Of note,
BAI1 binds to ELMO (see Fig. 24-2) and is therefore con-  
sidered the putative receptor for the type 1 pathway.  
Acknowledgments  
Help and discussion from current and past laboratory members is gratefully
acknowledged.
1. Majno G, Joris I: Apoptosis, oncosis, and necrosis: An overview of
cell death. Am J Pathol 146:3-19, 1995.
2. Kerr J, Wyllie A, Currie A: Apoptosis: A basic biological ­phenomenon
with wide-ranging implications in tissue kinetics. Br J Cancer 26:
239-257, 1972.
3. Wyllie AH, Morris RG, Smith AL, et al: Chromatin cleavage in
apoptosis: Association with condensed chromatin morphology and
dependence on macromolecular synthesis. J Pathol 142:67-77, 1984.
4. Ellis H, Horvitz H: Genetic control of programmed cell death in
the nematode C. elegans. Cell 44:817-829, 1986.
5. Thompson CB: Apoptosis in the pathogenesis and treatment of
­disease. Science 267:1456-1462, 1995.
6. Vaishnaw AK, McNally JD, Elkon KB: Apoptosis in the rheumatic
diseases. Arthritis Rheum 40:1917-1927, 1997.
7. Duvall E, Wyllie AH: Death and the cell. Immunol Today 7:115,
1986.
8. Leist M, Single B, Castoldi AF, et al: Intracellular adenosine
­triphosphate (ATP) concentration: A switch in the decision between
apoptosis and necrosis. J Exp Med 185:1481-1486, 1997.
9. Yu L, Alva A, Su H, et al: Regulation of an ATG7-beclin 1 ­program
of autophagic cell death by caspase-8. Science 304:1500-1502,
2004.
10. Qu X, Yu J, Bhagat G, et al: Promotion of tumorigenesis by
­heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest
112:1809-1820, 2003.
11. Lum JJ, Bauer DE, Kong M, et al: Growth factor regulation of
­autophagy and cell survival in the absence of apoptosis. Cell 120:
237-248, 2005.
12. Levine B, Klionsky DJ: Development by self-digestion: ­ Molecular
mechanisms and biological functions of autophagy. Dev Cell 6:
463-477, 2004.
13. Theofilopoulos AN: Current directions in autoimmunity. In Elkon
KB (ed): Apoptosis and its Relevance to Autoimmunity, Vol 9. Basel,
Karger, 2006, pp 1-204.
14. Aravind L, Dixit VM, Koonin EV: The domains of death: Evolution
of the apoptosis machinery. Trends Biochem Sci 24:47-53, 1999.
15. Smith C, Farrah T, Goodwin R: The TNF receptor superfamily of
cellular and viral proteins: Activation, costimulation, and death.
Cell 76:959-962, 1994.
16. Locksley RM, Killeen N, Lenardo MJ: The TNF and TNF ­ receptor
superfamilies: Integrating mammalian biology. Cell 104:487-501, 2001.
17. Ashkenazi A, Dixit VM: Apoptosis control by death and decoy
receptors. Curr Opin Cell Biol 11:255-260, 1999.
18. Huang B, Eberstadt M, Olejniczak E, et al: NMR structure and muta-
genesis of the Fas (APO-1/CD95) death domain. Nature 384:638-
641, 1996.
19. Muzio M, Chinnaiyan A, Kischkel F, et al: FLICE, a novel FADD-
homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/
APO-1) death-inducing signaling complex. Cell 85:817-827, 1996.
20. Boldin M, Goncharov T, Goltsev Y, et al: Involvement of MACH, a
novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF
receptor-induced cell death. Cell 85:803-815, 1996.
21. Vincenz C, Dixit V: Fas-associated death domain protein interleu-
kin-1β-converting enzyme 2 (FLICE2), an ICE/Ced-3 homologue,
is proximally involved in CD95- and p55-mediated death signaling.
J Biol Chem 272:6578-6583, 1997.
22. Scaffidi C, Fulda S, Srinivasan A, et al: Two CD95 (APO-1/Fas) sig-
naling pathways. EMBO J 17:1675-1687, 1998.
23. Nagata S: Apoptosis by death factor. Cell 88:355-365, 1997.
24. Black R, Rauch C, Kozlosky C, et al: A metalloproteinase disintegrin that
releases tumor necrosis factor-α from cells. Nature 385:729-733, 1997.
25. Moss M, Jin S-LC, Milla M, et al: Cloning of a disintegrin
­metalloproteinase that processes precursor tumor necrosis factor-α.
Nature 385:733-736, 1997.
26. Kishimoto H, Sprent J: Negative selection in the thymus includes
semimature T cells. J Exp Med 185:263-271, 1997.
27. Elkon KB, Marshak-Rothstein A: B cells in systemic autoimmune
disease: Recent insights from Fas-deficient mice and men. Curr Opin
Immunol 8:852-859, 1996.
28. Lamhamedi-Cherradi SE, Zheng S, Tisch RM, et al: Critical roles
of tumor necrosis factor-related apoptosis-inducing ligand in type 1
diabetes. Diabetes 52:2274-2278, 2003.
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
  29. Wang EC, Thern A, Denzel A, et al: DR3 regulates negative selection  
during thymocyte development. Mol Cell Biol 21:3451-3461, 2001.
  30. Schmidt CS, Liu J, Zhang T, et al: Enhanced B cell expansion,
­survival, and humoral responses by targeting death receptor 6. J Exp  
Med 197:51-62, 2003.
  31. Raff M: Social controls on cell survival and cell death. Nature 356:
397-400, 1992.
  32. Levine AJ: P53, the cellular gatekeeper for growth and division. Cell
88:323-331, 1997.  
  33. Huang H, Regan KM, Lou Z, et al: CDK2-dependent ­phosphorylation
of FOXO1 as an apoptotic response to DNA damage. Science
314:294-297, 2006.
  34. Kroemer G, Reed JC: Mitochondrial control of cell death. Nat Med  
6:513-519, 2000.
  35. Zou H, Henzel W, Liu Z, et al: Apaf-1, a human protein homologous
to C. elegans CED-4, participates in cytochrome c-dependent activa-  
tion of caspase 3. Cell 90:405-413, 1997.
  36. Susin SA, Lorenzo HK, Zamzami N, et al: Molecular characterization of
mitochondrial apoptosis-inducing factor. Nature 397:441-446, 1999.  
  37. Kaufman RJ: Orchestrating the unfolded protein response in health
and disease. J Clin Invest 110:1389-1398, 2002.
  38. Boyce M, Yuan J: Cellular response to endoplasmic reticulum stress:  
A matter of life or death. Cell Death Differ 13:363-373, 2006.
  39. Adams JM, Cory S: The Bcl-s protein family: Arbiters of cell ­survival.
Science 281:1322-1326, 1998.  
  40. Oltvai ZN, Korsmeyer SJ: Checkpoints of dueling dimers foil
death wishes. Cell 79:189-192, 1994.  
  41. Muchmore SW, Sattler M, Liang H, et al: X-ray and NMR ­structure
of human Bcl-xL, an inhibitor of programmed cell death. Nature
381:335-341, 1996.
  42. Roy N, Deveraux QL, Takahashi R, et al: The c-IAP-1 and c-IAP-2  
proteins are direct inhibitors of specific caspases. EMBO J 16:
6914-6925, 1997.
  43. Deveraux QL, Reed JC: IAP family proteins—suppressors of  
­apoptosis. Genes Dev 13:239-252, 1999.
  44. Downward J: PI 3-kinase, Akt and cell survival. Semin Cell Dev Biol
15:177-182, 2004.  
  45. Thornberry NA, Lazebnik Y: Caspases: Enemies within. Science
281:1312-1316, 1998.
  46. Los M, Wesselborg S: Schulze-Osthoff K: The role of caspases in  
development, immunity, and apoptotic signal transduction: Lessons
from knockout mice. Immunity 10:629-639, 1999.
  47. Garcia-Calvo M, Peterson EP, Leiting B, et al: Inhibition of human  
caspases by peptide-based and macrophage inhibitors. J Biol Chem
273:32608-32613, 1998.  
  48. Enari M, Sakahira H, Yokoyama H, et al: A caspase-activated DNase
that degrades DNA during apoptosis, and its inhibitor ICAD. Nature
391:43-50, 1998.
  49. Sakahira H, Enari M, Nagata S: Cleavage of CAD inhibitor in CAD  
activation and DNA degradation during apoptosis. Nature 391:
96-99, 1998.  
  50. Mariathasan S, Monack DM: Inflammasome adaptors and sensors:
Intracellular regulators of infection and inflammation. Nat Rev
Immunol 7:31-40, 2007.  
  51. Chae JJ, Wood G, Masters SL, et al: The B30.2 domain of pyrin, the
familial Mediterranean fever protein, interacts directly with caspase-1
to modulate IL-1beta production. Proc Natl Acad Sci U S A 103:  
9982-9987, 2006.
  52. Ellis RE, Jacobson DM, Horvitz R: Genes required for the ­engulfment  
of cell corpses during programmed cell death in Caenorhabditis ­elegans.
Genetics 129:79-94, 1991.  
  53. Hengartner MO: Apoptosis: Corralling the corpses. Cell 104:325-
328, 2001.
  54. deBakker CD, Haney LB, Kinchen JM, et al: Phagocytosis of apoptotic  
cells is regulated by a UNC-73/TRIO-MIG-2/RhoG signaling module
and armadillo repeats of CED-12/ELMO. Curr Biol 14:2208-2216, 2004.  
  55. Verhoven B, Schlegel RA, Williamson P: Mechanisms of phospha-
tidylserine exposure, a phagocyte recognition signal, on apoptotic
T lymphocytes. J Exp Med 182:1597-1601, 1995.
  56. Greenberg ME, Sun M, Zhang R, et al: Oxidized phosphatidylserine-  
CD36interactions play an essential role in macrophage-dependent
phagocytosis of apoptotic cells. J Exp Med 203:2613-2625, 2006.
  57. Hanayama R, Tanaka M, Miyasaka K, et al: Autoimmune disease and  
impaired uptake of apoptotic cells in MFG-E8-deficient mice. Sci-
ence 304:1147-1150, 2004.
393
58. Botto M, Dell’Agnola C, Bygrave AE, et al: Homozygous C1q
deficiency causes glomerulonephritis associated with multiple
apoptotic bodies. Nat Genet 19:56-59, 1998.
59. Gershov D, Kim S, Brot N, et al: C-reactive protein binds to apoptotic
cells, protects the cells from assembly of the terminal complement com-
ponents, and sustains an antiinflammatory innate immune response:
Implications for systemic autoimmunity. J Exp Med 192:1353-1364,
2000.
60. Kim G, Jun JB, Elkon KB: Necessary role of phosphatidylinositol
3-kinase in transforming growth factor beta-mediated activation of
Akt in normal and rheumatoid arthritis synovial fibroblasts. Arthritis
Rheum 46:1504-1511, 2002.
61. Ren Y, Silverstein RL, Allen J, et al: CD36 gene transfer confers
capacity for phagocytosis of cells undergoing apoptosis. J Exp Med
181:1857-1862, 1995.
62. Stuart LM, Henson PM, Vandivier RW: Collectins: Opsonins for
apoptotic cells and regulators of inflammation. Curr Dir Autoimmun
9:143-161, 2006.
63. Gardai SJ, McPhillips KA, Frasch SC, et al: Cell-surface calreticulin
initiates clearance of viable or apoptotic cells through trans-activa-
tion of LRP on the phagocyte. Cell 123:321-334, 2005.
64. Franc NC, Dimarcq J-L, Lagueux M, et al: Croquemort, a novel
­drosophila hemocyte/macrophage receptor that recognizes apoptotic
cells. Immunity 4:431-443, 1996.
65. Savill J, Dransfield Hogg N, Haslett C: Vitronectin receptor-mediated
phagocytosis of cells undergoing apoptosis. Nature 343:170-173, 1990.
66. Albert ML, Pearce SFA, Francisco LM, et al: Immature dendritic
cells phagocytose apoptotic cells via alpha-v-beta-5 and CD36, and
cross-present antigens to cytotoxic T lymphocytes. J Exp Med 188:
1359-1368, 1998.
67. Mevorach D, Mascarenhas J, Gershov DA, et al: Complement-
dependent clearance of apoptotic cells by human macrophages. J Exp
Med 188:2313-2320, 1998.
68. Luciani M-F, Chimini G: The ATP binding cassette transporter,
ABC1, is required for the engulfment of corpses generated by apop-
totic cell death. Eur Mol Biol Organ J 15:226-235, 1996.
69. Devitt A, Moffatt OD, Raykundalia C, et al: Human CD14 mediates
recognition and phagocytosis of apoptotic cells. Nature 392:505-509,
1998.
70. Lu Q, Lemke G: Homeostatic regulation of the immune system by
receptor tyrosine kinases of the Tyro 3 family. Science 293:306-
311, 2001.
71. Somersan S, Bhardwaj N: Tethering and tickling: A new role for the
phosphatidylserine receptor. J Cell Biol 155:501-504, 2001.
72. Fadok VA, Bratton DL, Konowal A, et al: Macrophages that have
ingested apoptotic cells in vitro inhibit proinflammatory cytokine
production through autocrine/paracrine mechanisms involving
TGF-beta, PGE2, and PAF. J Clin Invest 101:890-898, 1998.
73. Voll RE, Herrmann M, Roth EA, et al: Immunosuppressive effects of
apoptotic cells. Nature 390:350-351, 1997.
74. Huynh ML, Fadok VA, Henson PM: Phosphatidylserine-dependent
ingestion of apoptotic cells promotes TGF-beta1 secretion and the
resolution of inflammation. J Clin Invest 109:41-50, 2002.
75. Kim SJ, Elkon KB, Ma X: Transcriptional suppression of interleukin-12
gene expression following phagocytosis of apoptotic cells. ­ Immunity
21:643-653, 2004.
76. Bellone M, Iezzi G, Rovere P, et al: Processing of engulfed apoptotic
bodies yields T cell epitopes. J Immunol 159:5391-5399, 1997.
77. Ishii KJ, Akira S: Innate immune recognition of, and regulation by,
DNA. Trends Immunol 27:525-532, 2006.
78. Du Clos TW: The interaction of C-reactive protein and serum
­amyloid P component with nuclear antigens. Molec Biol Rep 23:
253-260, 1996.
79. Gallucci S, Lolkema M, Matzinger P: Natural adjuvants: Endogenous
activators of dendritic cells. Nat Med 5:1249-1255, 1999.
80. Vermes I, Haanen C, Steffens-Nakken H, et al: A novel assay for apop-
tosis: Flow cytometric detection of phosphatidylserine ­expression on
early apoptotic cells using fluorescein-labeled Annexin V. J Immunol
Methods 184:39-51, 1995.
81. Nicoletti I, Migliorati G, Pagliacci MC, et al: A rapid and simple
method for measuring thymocytes apoptosis by propidium iodide
staining and flow cytometry. J Immunol Meth 139:1173-1181, 1991.
82. Gavrieli Y, Sherman Y, Ben-Sasson SA: Identification of programmed 
cell death in situ via specific labeling of nuclear DNA fragmentation.
J Cell Biol 119:493-501, 1992.
 394 ELKON  |  Cell Survival and Death in Rheumatic Diseases
  83. Kanoh M, Takemura G, Misao J, et al: Significance of myocytes with
positive DNA in situ nick end-labeling (TUNEL) in hearts with
dilated cardiomyopathy: Not apoptosis but DNA repair. Circulation
99:2757-2764, 1999.
  84. Wrone-Smith T, Mitra RS, Thompson CB, et al: Keratinocytes
derived from psoriatic plaques are resistant to apoptosis compared
with normal skin. Am J Pathol 151:1321-1329, 1997.
  85. Cohen PL, Eisenberg RA: lpr and gld: Single gene models of systemic
autoimmunity and lymphoproliferative disease. Annu Rev Immunol
9:243-269, 1991.
  86. Fisher GH, Rosenberg FJ, Straus SE, et al: Dominant interfering Fas
gene mutations impair apoptosis in a human lymphoproliferative
syndrome. Cell 81:935-946, 1995.
  87. Rieux-Laucat F, Le Deist F, Hivroz C, et al: Mutations in Fas ­associated
with human lymphoproliferative syndrome and ­ autoimmunity.
­Science 268:1347-1349, 1995.
  88. Drappa J, Vaishnaw AK, Sullivan KE, et al: The Canale-Smith
­syndrome: An inherited autoimmune disorder associated with
­defective lymphocyte apoptosis and mutations in the Fas gene.
N Engl J Med 335:1643-1649, 1996.
  89. Vaishnaw AK, Orlinick JR, Chu JL, et al: Molecular basis for the
apoptotic defects in patients with CD95 (Fas/Apo-1) mutations.
J Clin Invest 103:355-363, 1999.
  90. Martin DA, Zheng L, Siegel RM, et al: Defective CD95/APO-1/Fas signal
complex formation in the human autoimmune lymphoproliferative
syndrome, type Ia. Proc Natl Acad Sci U S A 96:4552-4557, 1999.
  91. Kim SJ, Gershov D, Ma X, et al: Opsonization of apoptotic cells and
its effect on macrophage and T cell immune responses. Ann N Y
Acad Sci 987:68-78, 2003.
  92. Khare SD, Sarosi I, Xia XZ, et al: Severe B cell hyperplasia and auto-
immune disease in TALL-1 transgenic mice. Proc Natl Acad Sci U S
A 97:3370-3375, 2000.
  93. Baker KP: BLyS—an essential survival factor for B cells: Basic biol-
ogy, links to pathology and therapeutic target. Autoimmun Rev
3:368-375, 2004.
  94. Hull KM, Shoham N, Chae JJ, et al: The expanding spectrum of
systemic autoinflammatory disorders and their rheumatic manifesta-
tions. Curr Opin Rheumatol 15:61-69, 2003.
  95. Di Cristofano A, Kotsi P, Peng YP, et al: Impaired Fas response and
autoimmunity in Pten (+/−) mice. Science 285:2122-2125, 1999.
  96. Zhu J, Liu X, Xie C, et al: T cell hyperactivity in lupus as a ­consequence
of hyperstimulatory antigen-presenting cells. J Clin Invest 115:
1869-1878, 2005.
  97. Chen M, Wang YH, Wang Y, et al: Dendritic cell apoptosis in the
maintenance of immune tolerance. Science 311:1160-1164, 2006.
  98. Burlingame RW, Rubin RL, Balderas RS, et al: Genesis and evolu-
tion of anti-chromatin autoantibodies in murine lupus implicates
T-dependent immunization and self antigen. J Clin Invest 91:
1687-1696, 1993.
  99. Amoura Z, Chabre H, Koutouzov S, et al: Nucleosome restricted
antibodies are detected before anti-ds DNA and/or anti-histone anti-
bodies in serum of MRL-Mp lpr/lpr and +/+ mice, adn are ­ present
in kidney eluates of lupus mice with proteinuria. Arthritis Rheum
37:1684-1688, 1994.
100. Price BE, Rauch J, Shia MA, et al: Antiphospholipid autoantibodies
bind to apoptotic, but not viable, thymocytes in a beta2-glycoprotein
I-dependent manner. J Immunol 157:2201-2208, 1996.
101. Casciola-Rosen LA, Anhalt G, Rosen A: Autoantigens targeted
in systemic lupus erythematosus are clustered in two populations
of surface structures on apoptotic keratinocytes. J Exp Med 179:
1317-1330, 1994.
102. Casciola-Rosen LA, Anhalt GJ, Rosen A: DNA-dependent protein
kinase is one of a subset of autoantigens specifically cleaved early
during apoptosis. J Exp Med 182:1625-1634, 1995.
103. Utz PJ, Hottelet M, Schur PH, et al: Proteins phosphorylated during
stress-induced apoptosis are common targets for autoantibody pro-
duction in patients with systemic lupus erythematosus. J Exp Med
185:843-854, 1997.
104. Emlen W, Niebur J-A, Kadera R: Accelerated in vitro apoptosis
of lymphocytes from patients with systemic lupus erythematosus.
J Immunol 152:3685-3692, 1994.
105. Perniok A, Wedekind F, Herrmann M, et al: High levels of ­circulating
early apoptotic peripheral blood mononuclear cells in systemic lupus
erythematosus. Lupus 7:113-118, 1998.
106. Herrmann M, Voll RE, Zoller OM, et al: Impaired phagocyto-
sis of apoptotic cell material by monocyte-derived macrophages
from patients with systemic lupus erythematosus. Arthritis Rheum
41:1241-1250, 1998.
107. Morgan BP, Walport MJ: Complement deficiency and disease.
­Immunol Today 12:301-306, 1991.
108. Scott RS, McMahon EJ, Pop SM, et al: Phagocytosis and clear-
ance of apoptotic cells is mediated by MER. Nature 411:
207-211, 2001.
109. Bickerstaff MCM, Botto M, Hutchinson WL, et al: Serum amyloid P
component controls chromatin degradation and prevents antinuclear
autoimmunity. Nat Med 5:694-697, 1999.
110. Napirei M, Karsunky H, Zevnik B, et al: Features of systemic lupus
erythematosus in Dnase1-deficient mice. Nat Genet 25:177-181,
2000.
111. Kawane K, Ohtani M, Miwa K, et al: Chronic polyarthritis caused
by mammalian DNA that escapes from degradation in macrophages.
Nature 443:998-1002, 2006.
112. Ronnblom L, Eloranta ML, Alm GV: The type I interferon
­system in systemic lupus erythematosus. Arthritis Rheum 54:
408-420, 2006.
113. Nakajima T, Aono H, Hasunuma T, et al: Apoptosis and functional
Fas antigen in rheumatoid arthritis synoviocytes. Arthritis Rheum
38:485-491, 1995.
114. Firestein GS, Yeo M, Zvaifler NJ: Apoptosis in rheumatoid arthri-
tis synovium. J Clin Invest 96:1631-1638, 1995.
115. Cantwell MJ, Hua T, Zvaifler NJ, et al: Deficient Fas ligand ­expression
by synovial lymphocytes from patients with rheumatoid arthritis.
Arthritis Rheum 40:1644-1652, 1997.
116. Kawakami A, Eguchi K, Matsuoka N, et al: Thyroid-stimulating
­hormone inhibits Fas antigen-mediated apoptosis of human thyro-
cytes in vitro. Endocrinology 137:3163-3169, 1996.
117. Tsuboi M, Eguchi K, Kawakami A, et al: Fas antigen expression on
synovial cells was downregulated by interleukin 1. Biochem Biophys
Res Commun 218:280-285, 1996.
118. Salmon M, Scheel-Toellner D, Huissoon AP, et al: Inhibition of T
cell apoptosis in the rheumatoid synovium. J Clin Invest 99:439-446,
1997.
119. Fujisawa K, Aono H, Hasunuma T, et al: Activation of transcription
factor NF-κB in human synovial cells in response to tumor necrosis
factor. Arthritis Rheum 39:197-203, 1996.
120. Marok R, Winyard PG, Coumbe A, et al: Activation of the tran-
scription factor nuclear factor κ-B in human inflamed synovial tissue.
Arthritis Rheum 39:583-591, 1996.
121. Sugiyama M, Tsukazaki T, Yonekura A, et al: Localization of apoptosis
and expression of apoptosis-related proteins in the synovium of patients
with rheumatoid arthritis. Ann Rheum Dis 55:442-449, 1996.
122. Tak PP, Smeets TJ, Boyle DL, et al: P53 overexpression in synovial
tissue from patients with early and longstanding rheumatoid arthri-
tis compared with patients with reactive arthritis and osteoarthritis.
Arthritis Rheum 42:948-953, 1999.
123. Langowski JL, Zhang X, Wu L, et al: IL-23 promotes tumour inci-
dence and growth. Nature 442:461-465, 2006.
124. Fujisawa K, Asahara H, Okamoto K, et al: Therapeutic effect of the
anti-Fas antibody on arthritis in HTLV-1 tax transgenic mice. J Clin
Invest 98:271-278, 1996.
125. Zhang H, Yang Y, Horton JL, et al: Amelioration of collagen-
induced arthritis by CD95 (Apo-1/Fas)-ligand gene transfer.
J Clin Invest 100:1951-1957, 1997.
126. Song K, Chen Y, Goke R, et al: Tumor necrosis factor-related ­apoptosis-
inducing ligand (TRAIL) is an inhibitor of autoimmune inflammation
and cell cycle progression. J Exp Med 191:1095-1104, 2000.
127. Ichikawa K, Liu W, Fleck M, et al: TRAIL-R2 (DR5) mediates
apoptosis of synovial fibroblasts in rheumatoid arthritis. J Immunol
171:1061-1069, 2003.
128. Jelaska A, Korn JH: Role of apoptosis and transforming growth ­factor
beta1 in fibroblast selection and activation in systemic sclerosis.
Arthritis Rheum 43:2230-2239, 2000.
129. Ohsako S, Elkon KB: Apoptosis in the effector phase of autoimmune
diabetes, multiple sclerosis and thyroiditis. Cell Death Diff 6:13-21,
1999.
130. Kong L, Ogawa N, Masago R, et al: Bcl-2 family in salivary gland
from Sjogren’s syndrome: Bax may be involved in the destruction of
SS salivary glandular epithelium. Arthritis Rheum 39:S289, 1996.
 PART 3  |  EFFECTOR MECHANISMS IN AUTOIMMUNITY AND INFLAMMATION
131. Behrens L, Bender A, Johnson MA, et al: Cytotoxic mechanisms in
inflammatory myopathies: Co-expression of Fas and protective Bcl-2
in muscle fibres and inflammatory cells. Brain 120:929-938, 1997.
132. Sugiura T, Murakawa Y, Nagai A, et al: Fas and Fas ligand ­interaction
induces apoptosis in inflammatory myopathies: CD4+ T cells injury
in polymyositis. Arthritis Rheum 42:291-298, 1999.
133. Goebels N, Michaelis D, Engelhardt M, et al: Differential expression
of perforin in muscle-infiltrating T cells in myositis and dermatomyo-
sitis. J Clin Invest 97:2905-2910, 1996.
134. Howard OM, Dong HF, Yang D, et al: Histidyl-tRNA synthetase and
asparaginyl-tRNA synthetase, autoantigens in myositis, activate che-
mokine receptors on T lymphocytes and immature dendritic cells.
J Exp Med 196:781-791, 2002.
135. Hashimoto S, Setareh M, Ochs RL, et al: Fas/Fas ligand ­expression
and induction of apoptosis in chondrocytes. Arthritis Rheum
40:1749-1755, 1997.
136. Hashimoto S, Ochs RL, Komiya S, et al: Linkage of chondrocyte
apoptosis and cartilage degradation in human osteoarthritis. ­Arthritis
Rheum 41:1632-1638, 1998.
137. Blanco FJ, Guitian R, Vazquez ME, et al: Osteoarthritis chondrocytes
die by apoptosis: A possible pathway for osteoarthritis pathology.
Arthritis Rheum 41:284-289, 1998.
138. Yang C, Li SW, Helminen HJ, et al: Apoptosis of ­ chondrocytes
in transgenic mice lacking collagen II. Exp Cell Res 235:
370-373, 1997.
139. Kameda T, Mano H, Yuasa T, et al: Estrogen inhibits bone ­resorption
by directly inducing apoptosis of the bone-resorbing osteoclasts.
J Exp Med 186:489-495, 1997.
140. Okahashi N, Koide M, Jimi E, et al: Caspases (interleukin-1beta-
converting enzyme family proteases) are involved in the regulation
of the survival of osteoclasts. Bone 23:33-41, 1998.
141. Weinstein RS, Jilka RL, Parfitt AM, et al: Inhibition of osteoblasto-
genesis and promotion of apoptosis of osteoblasts and osteocytes by
glucocorticoids: Potential mechanisms of their deleterious effects on
bone. J Clin Invest 102:274-282, 1998.
142. Jilka RL, Weinstein RS, Bellido T, et al: Osteoblast programmed
cell death (apoptosis): Modulation by growth factors and cytokines.
J Bone Miner Res 13:793-802, 1998.
143. Yasuda H, Shima N, Nakagawa N, et al: Osteoclast differentiation
factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory
factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci
U S A 95:3597-3602, 1998.
144. Kong YY, Yoshida H, Sarosi I, et al: OPGL is a key regulator of
­osteoclastogenesis, lymphocyte development and lymph-node
­organogenesis. Nature 397:315-323, 1999.
145. Wang D, Muller N, McPherson KG, et al: Glucocorti-
coids engage different signal transduction pathways to induce
­apoptosis in thymocytes and mature T cells. J Immunol 176:
1695-1702, 2006.
146. Chan TA, Morin PJ, Vogelstein B, et al: Mechanisms underlying
nonsteroidal antiinflammatory drug-mediated apoptosis. Proc Natl
Acad Sci U S A 95:681-686, 1998.
147. Schwenger P, Bellosta P, Vietor I, et al: Sodium salicylate induces
apoptosis via p38 mitogen-activated protein kinase but inhibits
tumor necrosis factor-induced c-Jun N-terminal kinase/stress-acti-
vated protein kinase activation. Proc Natl Acad Sci U S A 94:
2869-2873, 1997.
395
148. Thomson AW, Bonham CA, Zeevi A: Mode of action of tacrolimus
(FK506): Molecular and cellular mechanisms. Therap Drug Monitor
17:584-591, 1995.
149. Anel A, Buferne M, Boyer C, et al: T cell receptor-induced Fas
ligand expression in cytotoxic T lymphocyte clones is blocked by
protein tyrosine kinase inhibitors and cyclosporin A. Eur J Immunol
24:2469-2476, 1994.
150. Migita K, Eguchi K, Kawabe Y, et al: FK506 augments activation-
induced programmed cell death of T lymphocytes in vivo. J Clin
Invest 96:727-732, 1995.
151. Cutolo M, Barone A, Accardo S, et al: Effect of cyclosporin on
­apoptosis in human cultured monocytic THP-1 cells and synovial
macrophages. Clin Exp Rheumatol 16:417-422, 1998.
152. Genestier L, Paillot R, Fournel S, et al: Immunosuppressive ­properties
of methotrexate: Apoptosis and clonal deletion of ­activated ­peripheral
T cells. J Clin Invest 102:322-328, 1998.
153. Cha DR, Feld SM, Nast C, et al: Apoptosis in mesangial cells induced
by ionizing radiation and cytotoxic drugs. Kidney Int 50:1565-1571,
1996.
154. Hughes DE, Wright KR, Uy HL, et al: Bisphosphonates ­ promote
apoptosis in murine osteoclasts in vitro and in vivo. J Bone Miner
Res 10:1478-1487, 1995.
155. Tak PP, Taylor PC, Breedveld FC, et al: Decrease in cellularity and
expression of adhesion molecules by anti-tumor necrosis ­ factor
α monoclonal antibody treatments in patients with rheumatoid
­arthritis. Arthritis Rheum 39:1077-1081, 1996.
156. Lugering A, Schmidt M, Lugering N, et al: Infliximab induces
­apoptosis in monocytes from patients with chronic active Crohn’s
disease by using a caspase-dependent pathway. Gastroenterology
121:1145-1157, 2001.
157. Van den Brande JM, Braat H, van den Brink GR, et al: Infliximab but
not etanercept induces apoptosis in lamina propria T-lymphocytes
from patients with Crohn’s disease. Gastroenterology 124:
1774-1785, 2003.
158. Catrina AI, Trollmo C, af Klint E, et al: Evidence that anti-tumor
necrosis factor therapy with both etanercept and infliximab induces
apoptosis in macrophages, but not lymphocytes, in rheumatoid
arthritis joints: Extended report. Arthritis Rheum 52:61-72, 2005.
159. Clark EA, Ledbetter JA: How does B cell depletion ­ therapy
work, and how can it be improved? Ann Rheum Dis 64(Suppl
4):iv-77-iv-80, 2005.
160. Rodriguez I, Matsuura K, Ody C, et al: Systemic injection of a
tripeptide inhibits the intracellular activation of CPP32-like
proteases in vivo and fully protects mice against Fas-mediated
fulminant liver destruction and death. J Exp Med 184:
2067-2072, 1996.
161. Martinon F, Tschopp J: NLRs join TLRs as innate sensors of
­pathogens. Trends Immunol 26:447-454, 2005.
162. Miyanishi M, Tada K, Koike M et al. Identification of Tim4 as a phos-
phatidylserine receptor. Nature 450:435-439, 2007.
163. Park D, Tosello-Trampont AC, Elliott MR, et al. BAI1 is an engulf-
ment receptor for apoptotic cells upstream of the ELMO/Dock180/
Rac module. Nature 450:430-434, 2007.
 25
KEY POINTS
Animal models of arthritis do not fully resemble human rheu-
matoid arthritis.
Animal models are tools to mimic various aspects of rheuma-
toid arthritis.
Arthritis can be induced in animals by immunization with
cartilage components, nonspecific immune stimuli, bacterial
or viral components, or transgenic manipulation.
Models have a defined onset and are useful for the kinetic
evaluation of arthritis, cell and mediator involvement, and
detailed analysis of joint erosion.
Animal models provide direction for novel approaches to
treatment, such as cytokine inhibition.
Experimental animal models of arthritis have contributed
to the understanding of basic mechanisms of joint disease.
There is marked diversity among the numerous models, and
arthritis has been induced by various stimuli, including the
generation of autoimmunity to cartilage components, non-
specific skewing of autoimmunity with adjuvants, and trig-
gering with exogenous agents such as bacteria and viruses.
More recently, focused transgenic manipulation has added
novel variants.
No single animal model of arthritis truly represents
the human disease. The wide variety of agents that can
induce experimental arthritis with clinical and histopatho-
logic features close to those of human arthritides indicates
that disparate etiologic pathways may exist in rheumatoid
arthritis (RA). Aspects peculiar to individual models are of
value but must be interpreted with caution. Much can be
learned from the general validity of mediator involvement
and other common concepts. In particular, models provide
valuable preclinical data for the development of novel
treatments, both pharmacologic and biologic, and insights
into relevant mechanisms common to both experimental
arthritis and RA.
Characteristic histopathologic features include immune
complexes (ICs) in the articular cartilage layers and vari-
able amounts of macrophages, T cells, and plasma cells in
the synovium, often accompanied by fibrosis and synovial
hyperplasia. Formation of autoantibodies, including rheu-
matoid factor and anticitrulline antibodies, is prominent,
making B cell activation and IC-mediated cellular inflam-
mation likely contributors to pathogenesis. Indeed, percep-
tions have changed over the years, and it is now accepted
that IC arthritis models have their value, although it must be
emphasized that erosive arthritis is achieved only with high
Animal Models of
Inflammatory Arthritis
Wim B. Van Den Berg
amounts of defined antibody cocktails and that chronicity
and joint erosions are markedly amplified by the presence of
a T cell component. Apart from insights into immune regu-
lation, the further identification of downstream mediators
of synovial macrophage and fibroblast activation and their
involvement in joint erosion remains a prime focus. The
process of cartilage erosion is hard to evaluate in patients.
Synovial biopsies are now performed in many early arthritis
clinics, but samples of damaged bone and cartilage become
available only late in the process, after joint replacement.
Models therefore provide the optimal tools for the detailed
evaluation of mechanisms and kinetics of cartilage and bone
destruction.
MODELS OF ARTHRITIS
INDUCED MODELS
The models most widely used in a historical context for
the investigation of RA were adjuvant arthritis (AA), col-
lagen-induced arthritis (CIA), antigen-induced arthritis
(AIA), and streptococcal cell wall (SCW) arthritis (Table
25-1).1 These models are classic examples of three driving
elements: nonspecific immune deviation, targeted cartilage
autoimmunity, and abundant exogenous or infectious trig-
gers. T cells play a dominant part in all these models, and
this feature is a major principle of chronic erosive arthritis.
Although T cell–directed therapy in RA was once ques-
tionable, new insight into T cell subclasses and more subtle
targeting of CTLA4 on T cell subsets show promise in this
regard. The recent discovery of T helper type 17 (Th17)
cells as a distinct, pathogenic T cell subset will boost the
interest in T cell–driven arthritis models.
A second common principle in the classic arthritis mod-
els is the presence of a chronic stimulus, either a persistent
antigen or autoantigen akin to joint structures. Examples of
persisting exogenous antigens are nondegradable bacterial
cell walls in the synovial tissue or antigen trapped in col-
lagenous reservoirs such as ligaments and articular cartilage.
Both conditions reflect escape from proper clearance by the
phagocytic system. By nature, autoantigens from the articu-
lar cartilage, such as collagen type II and proteoglycans, are
persistent stimuli. These potential triggers become true arth-
ritogens when tolerance is lost. Regulation of tolerance and
underlying mechanisms of sensitivity of various mouse and
rat strains has been a major research topic in animal models.
It remains to be identified whether tolerance against endog-
enous autoantigens or so-called exogenous but commensal
bacterial or viral antigens is regulated differently.
Intriguingly, progression in human RA tends to decline
when cartilage is fully destroyed. Moreover, total joint
397
 398 van den Berg  | Animal Models of Inflammatory Arthritis

Table 25-1  Models of Arthritis

Model Abbreviation Species* Feature IC† T Cell Referencess
Trigger-Induced Models, Nonspecific Immune Stimuli
Adjuvant arthritis AA Lewis rat AI – + 2, 5
Oil-induced arthritis OIA DA rat AI – + 3
Pristane-induced arthritis PIA DA rat AI – + 4, 6
Cartilage-Directed Autoimmunity
Collagen-induced arthritis CIA DBA mouse CII-AI + + 10, 11
Proteoglycan-­induced PG-A BALB/c mouse PG-AI + + 12, 13
arthritis
Infectious Agents, Exogenous Triggers
Streptococcal cell wall arthritis SCW-A Lewis rat Persistent – + 15, 16
bacteria–AI
Flare SCW-F Mouse Th17 – + 18, 19
Antigen-induced arthritis AIA Rabbit, mouse Persistent antigen + + 23, 24
Flare AIA-F Mouse Th17 – + 26
Transgenic Spontaneous Models
HTLV-induced arthritis HTLV Mouse Viral Tax antigen – + 28
KRN arthritis KRN K/BxN mouse GPI-AI + + 29, 30
SKG arthritis SKG Mouse ZAP-70 T cell defect – + 31, 32
GP130 arthritis GP130 Mouse STAT3, T cell defect – + 33, 34
TNF transgenic arthritis TNFtg Mouse TNF overexpression – – 35, 36
IL-1ra transgenic arthritis IL-1ra –/– BALB/c mouse Autoimmune T cells ± + 40, 41
IL-1 transgenic arthritis IL-1tg Mouse IL-1 overexpression – – 39
Immune Complex Models
Collagen type II P-CA DBA mouse Mouse CII antibody + –
KRN serum P-GPI BALB/c mouse Mouse GPI antibody + – 30, 47
PLL-lysozyme PLL-L DBA mouse Cationic antigen + – 44
*Used most commonly.
†Immune complex (IC) as an early feature.

AI, autoimmune; CII, collagen type II; GP, glycoprotein; GPI, glucose-6-phosphate isomerase; HTLV, human T cell leukemia virus; IL, interleukin; PLL, poly-L-
lysine; Th17, T helper type 17; TNF, tumor necrosis factor.

replacement often results in complete remission of arthri-
tis in that joint, without the need for synovectomy. These
are arguments for a direct role of cartilage antigens in the
pathogenesis of arthritis or for an indirect role of cartilage
components in the maintenance of arthritis. Structural
mimicry does exist between bacterial peptidoglycans and
cartilage proteoglycans, and bacterial triggers may influ-
ence autoimmune responses through their nonspecific
adjuvant properties or antigenic mimicry. Bacterial flora
has a major impact on numerous arthritis models, and the
recent discovery of Toll-like receptors (TLRs) mediat-
ing bacterial signaling has boosted interest in its role in
­arthritis.
TRANSGENIC MODELS
Transgenic variants have been developed in recent years.
They provide attractive new models by virtue of distinct
engineering yet follow many of the aforementioned prin-
ciples. Although these models can identify potential dis-
turbances underlying autoimmune arthritis and the ensuing
cytokines or autoantibodies that drive it, transgenic mod-
els are not necessarily more useful for the development of
advanced targeted therapy in RA. Several examples are
worthy of mention here and are discussed in more detail
later.
Transgenic overexpression of a T cell viral antigen results
in chronic arthritis, suggesting that a viral infection may be
involved in RA. The production of autoantibodies and the
accelerated onset of human T cell leukemia virus (HTLV)
arthritis after collagen type II immunization implies that
viral skewing of T cell responses to cartilage antigens may
be one of the underlying mechanisms.
In the KRN arthritis model, overexpression of a self-
reactive T cell receptor leads to skewed regulation of T
cell tolerance, with an ensuing excessive production of
anti–glucose-6-phosphate isomerase (GPI) antibodies. This
model resembles both CIA and AIA, where IC formation
at the cartilage surface is a salient feature. GPI is a cationic
endogenous protein with affinity for the articular cartilage,
and autoantibodies accumulate at that site, explaining the
predominance of joint disease in this general autoimmune
model.
Intriguingly, when regulatory control of interleukin (IL)-
1 is lost, T cell–dependent arthritogenic autoimmunity can
develop. This was found in BALB/c mice depleted of IL-1ra,
and IL-1, tumor necrosis factor (TNF), and IL-17 appeared
to be critical players. It is likely that excessive cytokine pro-
duction, including IL-1, is also an underlying mechanism in
the classic adjuvant or oil-induced models.
Other examples of manipulated T cell function leading
to autoimmune arthritis are the SKG and GP130­ arthritis
 PART 3  |  effector mechanisms in autoimmunity and inflammation
models. In SKG arthritis, aberrant T cell receptor func-
tion allows the positive selection of autoimmune T cells; in
the GP130 model, a mutation in the IL-6 receptor induces
enhanced signaling, illustrating that excessive IL-6 signal-
ing can drive T cell–dependent autoimmune arthritis.
Apart from these examples of T cell–related arthritis,
transgenic models have taught us that cytokine overexpres-
sion is a driving arthritic principle, without the need for T
or B cells. TNF transgenic mice develop arthritis that can be
transferred with fibroblasts. Likewise, IL-1–­overproducing
transgenic mice develop nonimmune arthritis. Human TNF
transgenic mice have been instrumental in the ­development
of the successful anti-TNF therapy with monoclonal
­antibodies and soluble receptors in RA patients.
IMMUNE COMPLEX MODELS
In line with the growing belief that autoantibodies are of
pathogenic importance in RA, models based on the passive
administration of autoantibodies have gained popularity.
This has been enhanced by the successful introduction of
anti–B cell therapy (rituximab) in RA and the identifica-
tion of anticitrulline antibodies as early markers of RA
development. Standard models use a cocktail of mono-
clonal anti–type II collagen (CII) antibodies or serum
containing anti-GPI antibodies obtained from arthritic
K/BxN mice. A fully passive system can be generated by
the transfer of antilysozyme antibodies to mice bearing
planted cationic lysozyme in the joint. The IC models are
best suited for the identification of pathways of inflamma-
tion and tissue destruction downstream of IC activation.
Such models are not suited for investigating B cell target-
ing. In addition, it should be stressed that excessive IC for-
mation is needed for tissue destruction and that the models
miss the unequivocal accelerating and exaggerating poten-
tial of combined IC and T cell triggering, which is evident
in CIA and AIA.
MECHANISMS OF ARTHRITIS IN SPECIFIC
MODELS
RESPONSE TO NONSPECIFIC IMMUNOLOGIC
STIMULI
Injection of nonspecific agents with adjuvant activity—that
is, the capacity to elicit an indirect immunologic response—
can provoke experimental arthritis in certain species. The
classic example is adjuvant arthritis (AA) in the Lewis rat.2
The precise contributions of the oil and mycobacterial com-
ponents of Freund’s complete adjuvant in the pathologic
pathway of this experimental disease remain unclear, but
both may contribute.
Adjuvant and Pristane Arthritis
A bacterium-specific pathogenesis seems likely in AA,
because conventionally bred rats are generally resistant to
AA, whereas germ-free Fischer or Wistar rats are suscep-
tible. Germ-free rats lack early contact with bacteria and
therefore are not tolerized. This is in sharp contrast to
­pristane (mineral oil)–induced arthritis, which is suppressed
in germ-free mice.
399
Incomplete Freund’s adjuvant (lacking mycobacteria)
and pristane can also induce arthritis in susceptible rats
and mice, indicating that oil can override natural toler-
ance.3,4 Ultimately, arthritogenic consequences appear to
depend on a reaction against either exogenous or autolo-
gous antigens. Heat shock proteins may be regulators in
AA.5 In susceptible strains of animals, macrophages phago-
cytose nondegradable oil components, become intensely
activated, produce large concentrations of proinflamma-
tory cytokines (IL-1, IL-6, TNF), and subsequently may
activate T and B cells. The results of polyclonal activa-
tion are apparent in pristane-induced arthritis, in which
lymphadenopathy and hypergammaglobulinemia precede
the onset of disease. Naturally occurring T cells that rec-
ognize T cell receptor peptides may regulate susceptibility,
because these cells become activated before the develop-
ment of disease.
Regulatory network reactions, which, by definition,
are autoimmune in nature, can skew responses toward
pathogenic consequences. Expansion of autoreactive T
and B cell populations can occur as a consequence of the
adjuvant activation process, resulting in the migration of
cells to the joint and subsequent immune-mediated joint
damage and the production of a spectrum of autoantibod-
ies against cartilage antigens. Production of rheumatoid
factors in pristane-induced arthritis may amplify the auto-
reactive process. Disease onset is rapid in AA (2 weeks),
often followed by spontaneous remission. Pristane arthritis
has a slower onset (months) and exhibits remissions and
relapses, with no indication of B cell involvement at the
onset. The pristane model is highly suited to study genes
controlling onset, severity, and chronicity.6 The sponta-
neous remission and lack of susceptibility to reinduction
makes AA a suitable model for studies on the regulation of
T cell tolerance.
Histopathology of AA reveals major involvement of
the bone marrow, marked bone erosion, and bone appo-
sition and minor direct cartilage damage at early stages
(Table 25-2). Indirect cartilage damage occurs later, mainly
as a consequence of the loss of underlying bone. The latter
may explain the cartilage protective effect of treatment with
osteoprotegerin, which is a selective inhibitor of the osteo-
clast activator RANKL.7,8 Combination blocking of TNF
and IL-1 results in optimal control of arthritis and joint
destruction.9
AUTOIMMUNITY TO CARTILAGE COMPONENTS
Articular cartilage is an intriguing tissue. It is the target of
the disease, but it may also function as the trigger by releasing
potential autoantigens and trapping exogenous antigens in
its avascular matrix. Destructive forms of RA tend to decline
when cartilage is fully destroyed, and arthritis also wanes in
joints undergoing replacement surgery, both of which argue
for cartilage’s crucial role in the arthritic process.
Collagen and Proteoglycan Arthritis
Classic arthritis models based on cartilage autoimmunity
can be induced by immunization with either of the two
major components of hyaline cartilage: CII and aggrecan
proteoglycans. More recently, similar models have ­identified
 400 van den Berg  | Animal Models of Inflammatory Arthritis

Table 25-2 Features of Common Arthritis Models

Feature AA SCW-A CIA AIA P-GPI TNFtg IL-1ra –/– SKG
Impact of flora* + + – – ? ? + +
Induction Systemic FCA Systemic bacterial Systemic CII, Local antigen Systemic Transgenic Transgenic Trans-
fragment FCA injection serum genic
Arthritic stimulus Bacteria, oil Persistent bacteria CII Planted antigen GPI TNF IL-1, bacteria Yeast
Mechanism T cell T cell T cell, IC T cell, IC IC TNF IL-1–IL-17 IL-1–
IL-17
Self-limited arthritis + – ± – – – – –
Flares Refractory Spontaneous Inducible Inducible Inducible – – –
Chronic ± ++ + ++ + ++ ++ ++
synovitis
Bone marrow ++ + ± ± – + + +
inflammation†
Main site of Ankle Ankle Peripheral Various‡ Paw Paw Ankle Ankle
expression
Bone erosion ++ + ++ + ++ ++ + +
Bone +++ + ++ + + – + +
apposition
Cartilage ± ± ++ ++ ++ ± + +
erosion
Salient feature Periostitis Fibrosis Fast erosion Local Fast erosion Role of IL-1 Role of IL-17 Role of
hyperreactivity in erosion IL-17
*Impact of microflora on susceptibility of strains.
†As an early feature.
‡Chosen by intra-articular injection.

AA, adjuvant arthritis; AIA, antigen-induced arthritis; CIA, collagen-induced arthritis; CII, collagen type II; FCA, Freund’s complete adjuvant; GPI, glucose-6-
­phosphate isomerase; IC, ­ immune complex; IL, interleukin; P-GPI, passive GPI arthritis; SCW-A, streptococcal cell wall arthritis; TNFtg, tumor necrosis factor
transgenic­.

other, less abundant cartilage components as potential auto-
antigens in arthritis. In principle, any cartilage matrix com-
ponent can be a potential arthritogen, provided it is released
in substantial amounts and natural tolerance is lost. CIA can
be elicited in mice, rats, and primates,10,11 whereas proteo-
glycan arthritis has been established only in distinct BALB/
c mice.12,13 Both models require the induction of a vigorous
immune response directed initially against the immunizing
(heterologous) cartilage antigen, which subsequently reacts
with autologous cartilage antigens. Susceptible strains of
animals immunized with either CII or high-density aggrecan
proteoglycans in adjuvant recognize specific epitopes. Auto-
reactive CD4+ T cells respond against either cross-reactive
epitopes (antigens common to both the heterologous and
autologous cartilage components) or cryptic epitopes (privi-
leged antigens normally concealed from immune surveil-
lance), and they elicit connective tissue antigen-specific T
helper cells and autoantibodies. Adoptive and passive trans-
fer experiments suggest that both immunologic elements are
required for the generation of chronic arthritis, although
transient but destructive forms can be elicited with cocktails
of anti-CII autoantibodies only. The localization of anti-
body and reactive cells in synovial joints causes immune-
mediated cartilage damage, and the immune response may
be perpetuated by the release of cartilage antigens.
The pathologies of CIA and proteoglycan arthritis appear
to be similar and include synovial hypertrophy and hyper-
plasia, giving rise to pannus formation, and severe cartilage
erosion (Fig. 25-1). Although both models are considered
T helper 1 (Th1) driven, interferon-γ (IFN-γ) deficiency
makes BALB/c mice more susceptible to CIA, whereas it
prevents proteoglycan arthritis. Diversity in the antibody
subclasses involved and skewed participation of the classic
Th1 IFN-γ–producing and Th17 IL-17–producing cells may
be responsible for this difference. Neutralization of TNF is
efficacious in early CIA, whereas IL-1 blockade suppresses
both early and fully established arthritis.14
INFECTIOUS AGENTS AND EXOGENOUS
TRIGGERS
The development of arthritis as a consequence of infection
is apparent in both natural and induced animal models.
Infectious agents may be trophic for the joint as a result
of expression of adhesins or other molecules that promote
sequestration within synovial tissues. Bacterial cell wall
and mycoplasma membrane components may also provide
nonspecific immune activation through mitogenic activity.
Viral infection of synovial cells may elicit novel cell sur-
face antigens or abnormal expression of activation antigens
and the overproduction of autoantigens. Viral infection may
also disrupt immune regulation, increasing proinflammatory
cytokines and immune activity against normally immuno-
logically privileged components of the joint.
Streptococcal Cell Wall Arthritis
Persistence of antigens from microorganisms within the
joint is critical for the induction of arthritis. SCW experi-
mental arthritis15 is induced in Lewis rats by the systemic
injection of cell wall fragments of group A streptococci,
which are highly resistant to biodegradation. A similar
 PART 3  |  effector mechanisms in autoimmunity and inflammation
d­ isease can be induced with cell wall fragments from other
bacteria, such as Lactobacillus casei or Eubacterium aerofa-
ciens. The underlying principle is the poor degradability
of the fragments, thereby creating a persistent stimulus.
The Lactobacillus and Eubacterium models are of particu-
lar interest for human disease, because these bacteria are
part of the normal gastrointestinal flora.16 Extrapolation
of this model to humans suggests that an enormous load of
potential arthritogenic stimuli is continuously present in
the normal gastrointestinal tract; from there, it may spread
to other tissues and therefore requires tight immunoregu-
lation. Within 24 hours of the administration of cell wall
fragments in rats, acute inflammation develops in periph-
eral joints, coincident with dissemination of cell wall frag-
ments in blood vessels of the synovium and subchondral
bone marrow. Acute, complement-dependent inflamma-
tion subsides over the next week and is followed within 2
weeks by a chronic, T cell–dependent erosive polyarthritis
involving mainly peripheral joints. In contrast with the
acute-phase lesion, chronic joint inflammation develops
only in susceptible strains that lose tolerance and display
SCW-specific T cell responses, with the highest incidence
in Lewis rats. It is tempting to speculate that similar loss of
tolerance may occur in RA patients. Mouse strains studied
so far are not susceptible to the single-injection model.
In addition to SCW-specific T cell reactions, cross-reac-
tive autoimmunity to cartilage proteoglycans may contribute
to chronicity. However, it is unlikely that this cross-reactiv-
ity is a major factor at the onset, because the loss of proteo-
glycans from articular cartilage is limited at that stage. Only
later are marked pannus formation and severe erosions of
underlying cartilage and bone frequently observed. In line
with the tumor-like behavior of synovial cells in patients
with RA, synovial cells from arthritic rats show continued
proliferation ex vivo, with apparent paracrine and autocrine
regulation by growth factors.17 This observation delineates
that sustained macrophage-fibroblast activation by retained
bacterial components may be a perpetuating principle, but
401
Figure 25-1  Macroscopic signs of peripheral inflam-
mation in murine collagen arthritis. Histology shows ex-
amples of mild cartilage proteoglycan loss in the super-
ficial cartilage layer at an early stage, advancing to full
surface loss at later stages.
in vivo, the T cell is still a critical, driving factor. Studies on
the involvement of cytokines in SCW arthritis show a com-
bined role of TNF and IL-1, as is found in AA.9,18 In mice,
a chronic relapsing SCW model can be induced by repeated
weekly injection of SCW fragments directly into the knee
joint, displaying a gradually increasing role of T cell–derived
IL-17 and synovial IL-17 receptor–bearing cells with every
flare.19 TLR2 is the driving receptor of SCW recognition,
and arthritis is markedly suppressed in TLR2 –/– mice and
absent in MyD88 –/– mice.20
As an extension of the involvement of bacteria in
­arthritis, bacterial DNA can induce arthritis. In particular,
the CpG motifs in bacterial DNA are arthritogenic, and
substantial amounts can be found in joint tissues.21 Mac-
rophages play a major role in this arthritis through the
­production of TNF. However, in comparison to cell wall
fragments, the ­cytokine-inducing capacity is weak. Normal
joints of individuals contain both bacterial fragments and
bacterial cpG (CP61) motifs, and it is conceivable that both
factors contribute to arthritis.
ANTIGEN-INDUCED ARTHRITIS
AIA provides an antigen-defined model, reflecting a sus-
tained immune reaction to a persistent trigger in joint
tissues—in this case, an exogenous protein antigen. It
is elicited by local injection of a high dose of antigen in
the knee joint of an animal previously hyperimmunized
with that antigen in Freund’s complete adjuvant. Such
a model was first developed by Dumonde and Glynn22
in rabbits. In principle, it can be induced in any species,
provided that proper immunity to a particular antigen
can be mounted. Applications have been developed in
mice, rats, and guinea pigs. In contrast to the polyarthri-
tis models described so far, this type of arthritis remains
confined to the injected joint. Commonly used antigens
are ovalbumin, bovine serum albumin (BSA), fibrin, or
cationic forms such as methylated BSA. Preimmunization
 402 van den Berg  | Animal Models of Inflammatory Arthritis
with antigen in complete Freund’s adjuvant induces strong
humoral as well as cell-mediated immunity. Arthritis is
usually induced 3 weeks later by local injection of a large
amount of antigen in the knee joint. Initially, an IC-type
reaction dominates, followed by T cell–mediated chronic
inflammation. In the rabbit, chronicity may last for years.
Histopathology shows a granulocyte-rich exudate in the
joint space, thickening of the synovial lining layer, and,
at later stages, a predominantly mononuclear infiltrate in
the synovium, which later includes numerous T cells and
clusters of plasma cells. Interestingly, a large proportion
(50%) of these plasma cells are making antibodies to the
inciting antigen, suggesting that retained antigen is a driv-
ing force in chronic arthritis. Intense IC formation is seen
in superficial layers of the articular cartilage, which may
contribute to localized cartilage destruction. Early loss of
proteoglycans, followed by pannus formation and cartilage
and bone erosion, is a common finding.
Two important principles emerge from this model: first,
chronicity is found only in the presence of sufficient anti-
gen retention in joint tissues, in combination with proper
T cell–mediated delayed hypersensitivity; second, joints
contain numerous non- or avascular collagenous tissues
such as cartilage, ligaments, and tendons, which allows for
prolonged antigen retention by antibody-mediated trap-
ping and charge-mediated binding.23,24 Importantly, anti-
gen injected in the skin produces transient inflammation,
whereas a similar dose in the joints causes chronic inflam-
mation. Chronicity is due to the generation of local hyper-
reactivity. Antigen that is initially trapped in collagenous
tissues is slowly released, sustaining low-grade chronic
arthritis. As a consequence, the local T cell infiltrate gains
specificity, because the retention of specific T cells is shaped
by homologous antigen. Small amounts of antigen are suf-
ficient to sustain arthritis, whereas relatively large amounts
are needed for induction. This condition forms the basis for
exacerbations (flares) of arthritis with low doses of antigen
(described later).
In rabbits, antibody responses are generally high and
allow for sufficient IC-mediated trapping of antigen in the
joint. Cationic antigens are effective arthritogens in the
murine model, owing to their ability to stick to the nega-
tively charged collagenous structures of the joint and to
accumulate IC formation at the surface.24 Of interest, this
principle may extend to cationic bacterial or viral compo-
nents and appears to be important in the more recently
developed transgenic KRN model of arthritis, where anti-
GPI antibodies stick to GPI antigen trapped at cartilage
surfaces.
In AIA in the rabbit and the mouse, elimination of
TNF-α and IL-1 was poorly effective in suppressing joint
inflammation, pointing to substantial “overkill” by other
mediators in this severe-onset arthritis. However, elimina-
tion of IL-1 yielded impressive protection against cartilage
destruction.25 The model of AIA is most suited to studies of
the mechanism of cartilage destruction as induced by a mix
of ICs and T cell reactivity. It is facilitated by knowledge of
the exact time of onset, accessibility of the knee joint (as
compared with the ankle), and the presence of a contralat-
eral control joint. Moreover, the model can be used to eval-
uate the regulation of local T cell hyperreactivity against a
retained foreign antigen.
FLARES OF ARTHRITIS
In comparison to the chronicity of human RA, most
­animal models show a relatively short duration of severe,
­rapidly destructive inflammation. In that respect, models of
repeated flares of arthritis, with the slower development of
lesions, provide a valuable extension.
An arthritic joint bearing retained antigen and a chronic
antigen-specific T cell infiltrate displays a state of local
hyperreactivity. This situation is not restricted to retained
antigen but also applies to new antigen entering the sensi-
tized joint from the circulation. Flares of smoldering arthri-
tis can be induced with as little as 10 ng of antigen and are
highly dependent on T cell–derived IL-17.26 Flares can be
induced by local, intravenous, or even oral rechallenge. An
interesting situation is exacerbation induced by cytokines.
Joints bearing a macrophage infiltrate are more sensitive
to IL-1 and IL-17 and easily allow for marked cytokine-
­mediated destruction.
In addition to flare models based on protein antigen,
similar models have been developed in rats and mice using
bacterial cell wall constituents. In contrast to small protein
antigens, which are inflammatory only in the context of an
immune response, bacterial fragments may function as an
antigen but directly trigger TLRs as well; the ensuing reac-
tions are a mixture of T cell– and macrophage-fibroblast–
driven processes. The generation of local hyperreactivity
requires large, persistent bacterial peptidoglycan-polysac-
charide components, but a recurrence may be induced with
a variety of components, ranging from cell wall fragments,
lipopolysaccharide, CpG motifs, and cytokines such as IL-1.
The strongest flares occur in the presence of T cell immunity,
and a correlation is found between the fragments’ potential
to induce an exacerbation and to elicit cell wall–specific
T cell proliferation. In the mouse system, flares are a mix-
ture of macrophage and T cell reactivity. Separate roles of
TNF and IL-1 are found in swelling and erosion.18 A TNF-
dependent swelling response is seen in each flare, but IL-1 is
dominant in the chronic erosive process. Erosion is absent
in IL-1–deficient mice but does occur in TNF-deficient mice
(Fig. 25-2). The model is more severe and erosive in DBA
mice than in C57Bl mice; erosion is absent in T or B cell–
deficient RAG mice, and both IL-12 and IL-18 promote an
erosive phenotype.
Of note, considerable cross-reactivity occurs between
cell walls from different bacterial origins, and flares may
result from homologous as well as heterologous frag-
ments.27 This may extend to cross-reactive autoantigens
from cartilage, which underlines the fact that arthritis
may start against a particular antigen but spread to other
antigens, including autoantigens. Recently, TLRs have
been identified as pattern-recognition molecules for bac-
teria; TLR4 in particular cross-reacts with numerous frag-
ments of damaged connective tissue components. Their
presence on dendritic cells and their role in regulating
autoimmune responses are intriguing. These principles
open up a wide range of putative stimuli involved in
exacerbations, simultaneously complicating the search
for the driving “antigen” in humans. Figure 25-3 illus-
trates the erosive character of flares, which can be effi-
ciently blocked with a combination of antibodies to TNF,
IL-1, and IL-17.19,26 The potential efficacy of TLR block-
ers remains to be elucidated.
 PART 3  |  effector mechanisms in autoimmunity and inflammation
TRANSGENIC MODELS
Transgenic animals, defined as novel strains generated
by the manipulation of particular genes, have resulted in
several new models that prove the importance of certain
principles in arthritis and are useful for distinct screening.
They do not necessarily provide better translational models
for drug targeting in RA. Some recent examples of skewed
T cell responses are discussed below.
HTLV Arthritis
Mice transgenic for the env-pX region of the HTLV type 1
genome develop a spontaneous chronic arthritis as a result of
expression of the tax gene.28 Onset occurs at 2 to 3 months
of age in female mice but is delayed several months in male
animals. Ankle joints are most frequently affected, and
pannus formation leading to severe erosions of cartilage and
bone is observed in transgenic mice after several months of
disease. The mice produce autoantibodies, and collagen
immunization can provoke the onset or exacerbation of
arthritis. It is tempting to speculate that a retrovirus could be
involved in the pathogenesis of RA, possibly by influencing
T cell responses to cartilage antigens.
KRN Arthritis
An intriguing, novel arthritis model emerged from experi-
ments in transgenic mice overexpressing a self-reactive T
cell receptor (TCR). The cross of K/BxN mice developed
arthritis.29 In principle, many insults or adjuvants that skew
the regulation of T cell tolerance have the potential to create
autoimmune pathology, including joint inflammation. The
403
Figure 25-2  Safranin O staining of
a joint subjected to repeated flares
of streptococcal cell wall arthritis
(day 28) in wild-type mice (left) and
TNF –/– mice (right). Note the full
progression of synovitis and joint
damage in the TNF –/– condition.
major breakthrough of the KRN model is the elucidation of
the driving antigen and the finding that passive transfer with
antibodies induces a protracted arthritis. The TCR recognizes
the ubiquitous self-antigen GPI and provokes, through B cell
differentiation and proliferation, high levels of anti-GPI anti-
bodies. These antibodies are directly pathogenic upon trans-
fer and appear to recognize endogenous GPI, which seems
to associate preferentially with the cartilage surface.30 The
latter finding may underlie the dominance of joint pathology
in these mice, although GPI is also abundant at other body
sites. Immunoglobulin (Ig) G1 antibodies are the major sub-
class and cause a sustained, erosive arthritis after continued
transfer, with BALB/c mice showing high sensitivity. The
pathology brings the model close to passive CIA or IC arthri-
tis, with IC formation at the cartilage surface (see the section
on passive IC arthritis for details). Differences between the
models relate to the IgG subclasses involved.
SKG and GP130 Arthritis
Another recent example of a transgenic T cell model is pro-
vided by the occurrence of chronic autoimmune arthritis in
mice with a point mutation of the gene encoding ZAP-70, a
key signal transduction molecule in T cells.31 The aberrant
TCR function leads to positive selection of otherwise nega-
tively selected autoimmmune T cells. Of great interest is that
these mice fail to develop disease under a ­microbially clean
condition, despite the active production of arthritogenic
autoimmune cells. A single injection of zymosan provokes
arthritis in a Dectin-1–dependent but TLR–­independent
manner.32 The latter is in sharp contrast with the arthritis
Figure 25-3  Impact of IL-17
blocking in flares of antigen-induced
arthritis. Note the marked synovitis
and cathepsin K–positive osteoclast-
mediated bone attack in the control
flare (left) and suppression of this
reaction in the anti–IL-17 treated
joint (right).
 404 van den Berg  | Animal Models of Inflammatory Arthritis
in IL-1ra –/– mice, which is similarly dependent on flora but
clearly TLR4 dependent as well. Th17 cells play a crucial
role in this model and show that environmental factors such
as yeast may drive or accelerate Th17 arthritis pathology.
Mice with a homozygous mutation in the GP130 IL-6
receptor subunit show enhanced signal transduction and
STAT3 activation and develop a lymphocyte-mediated
RA-like joint disease. This is another example of skewed
T cell function resulting in arthritis. Increased proliferation
of CD4+ T cells occurs due to the elevated production of T
cell–activating IL-7 by nonhematopoietic cells.33,34
Tumor Necrosis Factor Transgenic Arthritis
An elegant series of experiments provided insight into the
possible role of TNF in arthritis induction. By introducing
into mice a modified human TNF transgene (lacking a TNF
3'UTR region, involved in the translational repression of
TNF), it was shown that pronounced TNF overexpression
results in chronic polyarthritis with a 100% incidence.35
Hyperplasia of the synovium, inflammatory infiltrates in
the joint space, pannus formation, and cartilage and bone
destruction were observed. Intriguingly, a similar form of
arthritis also developed in targeted mutant mice lacking the
3'AU-rich elements, confirming the role of these elements
in maintaining a physiologic TNF response in the joint.36 A
proposed mechanism is the inability of natural anti-inflam-
matory signals, such as IL-10, to suppress TNF production
under these conditions. These exciting findings stimulated
a major search for functional mutations around TNF pro-
duction in RA patients; however, no clear indications have
been found so far.
The model is of great interest to identify pathways of
TNF-induced arthritis and to screen the efficacy of various
TNF-directed therapies. It is not surprising that anti-TNF
treatment blocks the pathology, but it is a remarkable obser-
vation that antibodies to the IL-1 receptor also prevent
arthritis, suggesting that much of this arthritis occurs by the
induction of IL-1.37 The model does not need T or B cells,
because arthritis occurs in TNF transgenics backcrossed
to RAG mice, and the pathology can be transferred with
selected TNF-producing fibroblasts. Further investigation of
TNF receptor involvement showed a crucial role for the p55
type I receptor in mediating TNF pathology and a suppres-
sive role for the p75 type II receptor.
Apparently, the type II receptor does not have a clear sup-
pressive role in inflammatory bowel disease, another pathol-
ogy found in TNF transgenic mice. The latter is a T or B
cell–dependent disease. It is known that cytotoxic anti-TNF
and TNF and lymphotoxin scavenging TNF-soluble receptor
treatments have different efficacies in human RA compared
with Crohn’s disease, but the reason is not fully understood.
Recent studies substantiate a dual proinflammatory and
immunosuppressive role for TNF and the heterogeneity of
TNF receptor usage in autoimmune suppression versus inflam-
matory tissue damage.38 These observations provide a ratio-
nale for the future treatment of RA with selective anti–TNF
receptor instead of anti-TNF. However, full understanding
of this system is complicated by the finding of cooperative
activity of p55 and p75 TNF receptors in arthritis induced
with membrane-bound TNF, in line with the identification
of preferential binding of transmembrane TNF to the p75
receptor. It remains to be elucidated to what extent human
RA is driven by soluble or membrane TNF. Of note, soluble
TNF is hard to detect in RA synovial fluid, and models with
dominant overexpression of soluble TNF hamper the proper
identification of the role of p75 TNF receptor.
Interleukin-1 Transgenic Mice
Transgenic IL-1α overexpression has been shown to induce
chronic, destructive arthritis.39 Transgenic mice expressing
human IL-1α had high serum levels of IL-1 and developed
a severe polyarthritis by 4 weeks of age. Hyperplasia of the
synovial lining, pannus formation, and, ultimately, carti-
lage destruction were evident. T and B cells were scant, but
active granulocytes were abundant.
The opposite approach—elimination of IL-1 control by
gene targeting of the endogenous IL-1 receptor antagonist
(IL-1ra)—yielded a T cell–driven model of arthritis. IL-1ra
deficiency in a BALB/c background resulted in pronounced
arthritis at age 8 weeks.40 Marked synovial and periarticu-
lar inflammation was noted, with invasion of granulation
tissue and articular erosion (Fig. 25-4). Moreover, elevated
levels of antibodies against immunoglobulins, CII, and
double-stranded DNA were found, suggesting an autoim-
mune response. Intriguingly, IL-1ra deficiency in a C57Bl/6j
background did not yield arthritis; arteritis was produced
instead. This genetic variation, though not well understood,
underscores an immunologic pathogenic pathway. Overex-
pression of a range of cytokines, including IL-1b, TNF, and
IL-6, was observed in the joints before the onset of arthritis.
Interestingly, autoantibody levels did not correlate with dis-
ease severity, which may imply a reaction to damaged joint
tissue.
In sharp contrast to the TNF transgenic model, the arthri-
tis in IL-1ra –/– mice seems to be dependent on T cells, in
line with the strong genetic restriction. It is consistent with
the view that IL-1 is a crucial regulator of T cell function.
Impaired T cell activation is demonstrated in IL-1–deficient
mice, linked to low levels of CD40 ligand and OX40 expres-
sion on T cells, and it underlies the suppression of CIA in
IL-1β –/– mice. Undisturbed IL-1 action, in the absence of
IL-1ra, probably permits the activation of IL-17–producing
T cells directed against exogenous triggers or endogenous
autoantigens. The spontaneous arthritis in IL-1ra –/– mice
does not develop under germ-free conditions and is reduced
in TLR4-deficient mice. Both TNF and IL-17 deficiency
prevent the onset of arthritis.41,42
IMMUNE COMPLEX ARTHRITIS
Autoantibodies such as rheumatoid factor and anticitrul-
line antibodies are a key feature of RA, and the recent suc-
cess of treatment with an anti–B cell drug (rituximab) has
enhanced the belief in a pathogenic role. In some of the
models discussed earlier, such as CIA, proteoglycan arthri-
tis, and AIA, IC formation at joint tissues is a major ele-
ment of pathogenesis. Although excessive IC formation
can cause destructive arthritis, chronicity is limited and
may be promoted by T cells. The latter may be linked to T
cells’ need to sustain antibody production and the greater
potential of T cell–macrophage interaction to sustain joint
pathology. Minute amounts of antigen suffice to stimulate
 PART 3  |  effector mechanisms in autoimmunity and inflammation
100
One hind paw
Two hind paws
Arthritis incidence (%)
75 Hind + front paw(s)
50
25
0
0 5 10 15
Age (weeks)
T cells, whereas considerable amounts of ICs are needed
to stimulate the release of inflammatory mediators from
phagocytes. It is likely that IC models mimic part of the
RA pathology.
With availability of a range of transgenic knockouts, there
is growing interest in the use of passive IC models to iden-
tify crucial pathways of inflammation and tissue destruction.
The advantage of passive systems is the lower dependence
on genetic background, avoiding excessive backcrossing to
create transgenics in suitable, susceptible mouse strains.
Passive Collagen Arthritis
Passive transfer of collagen arthritis can be performed with
a critical mixture of a number of anti-CII monocloclonal
antibodies, including complement-binding IgG2a. Sets are
now commercially available, routinely recommending DBA
mice as sensitive recipients. Accepted concepts of inflamma-
tion pathways include IC-mediated complement activation
and Fcγ receptor triggering on phagocytes. Proteoglycan
antibodies from the proteoglycan arthritis model can induce
transient arthritis upon transfer, with concomitant proteo-
glycan loss from the cartilage but no erosive damage. IgG1
seems to be the critical subclass; the limited destructive
potential is unclear.
Passive Poly-L-Lysine–Lysozyme Arthritis
An IC model emerging from the murine AIA model and
using the principle of cationic retention is the passive transfer
of antilysozyme antibodies to mice that are locally injected
in one knee joint with poly-L-lysine (PLL)–lysozyme.
PLL-coupled lysozyme is highly cationic and sufficiently large
405
Figure 25-4  Arthritis expression in
IL-1ra –/– mice. Marked synovial villi
and joint erosions.
to be retained in the joint for prolonged periods. The associ-
ation with synovial tissue and the heavy sticking to cartilage
surfaces contribute to chronicity and cartilage destruction.
An intriguing observation is the more chronic and destruc-
tive nature of this arthritis in DBA/1j mice compared with
BALB/c mice(Fig. 25-5),43 which seems to be related to high
sustained levels of activating Fcγ receptors on macrophages
of DBA/1j mice. The model shows strong dependence on
IL-1, whereas TNF blockade is ineffective.44 FcγRI rather
than III appears to be crucial in cartilage damage.45
Passive Glucose-6-Phosphate Isomerase Arthritis
Antibodies present in the serum of arthritic KRN mice
are directly pathogenic upon systemic transfer. They rec-
ognize endogenous GPI, which associates preferentially
with the cartilage surface.30 This tendency may underlie
the dominance of joint pathology in these mice, although
GPI is also abundant at other body sites. IgG1 antibod-
ies are the major subclass and cause a sustained, erosive
arthritis after continued transfer, with high sensitivity of
BALB/c mice. Repeat injections enhance chronicity and
joint destruction. The model comes close to passive CIA
and IC arthritis with planted cartilage-associated antigen;
all these types have IC formation at the cartilage surface
as a salient feature.
Recently, an arthritis model was developed by immu-
nization with GPI in Freund’s adjuvant. Serum from this
model was not capable of transferring arthritis, indicating
that antibodies alone are poorly arthritogenic. Upscaling or
the use of a critical mixture of multiple epitope-recognizing
antibodies is needed, in line with observations related to
anti-CII antibodies.
 406 van den Berg  | Animal Models of Inflammatory Arthritis
Figure 25-5  Histology of day 7 passive poly-L-lysine–
lysozyme arthritis in two mouse strains (BALB/c and
DBA). The upper panel shows almost resolved arthritis,
with limited cartilage damage. The lower panel shows
aggressive cartilage surface erosion and bone erosion.
Passive Citrulline Arthritis
After the identification of anticitrulline antibodies as an early
marker of the RA process, many groups attempted to induce
citrulline arthritis. So far, anticitrulline antibodies are poorly
arthritogenic on their own but can amplify existing IC arthri-
tis.46 This amplifying principle also applies to rheumatoid
factor, as discovered previously. Intriguingly, mice tolerized to
citrulline responses show reduced collagen arthritis, suggest-
ing a contribution of citrulline responses in CIA.
Strain and Cytokine Dependency of Passive
Immune Complex Arthritis
As mentioned earlier for passive IC complex models, sever-
ity is dependent on complement factors and Fcγ receptors.
Activation of complement through the alternative pathway
is crucial in GPI arthritis, consistent with the dominance
of IgG1. Because the activity of complement and the level
of expression of Fcγ receptors on phagocytes are different
in various mouse strains, such factors largely determine
the variable susceptibility. BALB/c mice are hyperreac-
tive, whereas DBA/1 and C57Bl6 mice are less susceptible;
minor responsiveness is seen in 129/Sv mice, in strong con-
trast to the low sensitivity of BALB/c mice in other pas-
sive IC arthritis models. GPI antibodies are found in some
but not all RA patients, and levels are moderate. Their role
in RA remains to be identified.47 The involvement of IL-
1 and TNF follows earlier observations in similar models.
IL-1 is obligatory, with no arthritis in IL-1–deficient mice.
TNF is also essential but is less critical than IL-1, because a
proportion of TNF-deficient mice develop robust arthritis.
Compared with the passive arthritis induced with antibod-
ies to the cationic antigen PLL-lysozyme, a higher TNF sen-
sitivity is apparent, as well as a dependence on mast cells.
These findings suggest a role of environmental initiating
elements. It is likely that the onset of mild GPI arthritis is
facilitated by local TNF generation and mast cell–depen-
dent histamine release, whereas a model with an injected
cationic antigen in the joint generates sufficient nonspecific
inflammation to initiate arthritis without the need for addi-
tional facilitating mediators such as TNF. Once the joint is
affected, the role of TNF is limited; this is also the case in
GPI arthritis. T cell IL-17 can accelerate and enhance the
expression of GPI arthritis,48 making TNF redundant. This
illustrates the remarkable potency of the combination of IC
and T cell activation in determining the severity and chro-
nicity of arthritis.
CYTOKINES AS TARGETS IN
SUSCEPTIBILITY AND DESTRUCTION
Findings supporting TNF, IL-1, and IL-17 involvement in
arthritis pathogenesis have been partly addressed under the
headings of the various models. TNF is a major mediator in
early stages of joint inflammation in every model. Although
IL-1 is not a dominant early inflammatory cytokine in all
models, it is the pivotal cytokine in the inhibition of chon-
drocyte proteoglycan synthesis in all models studied so far,
and blocking of IL-1 has a great beneficial impact on net
cartilage destruction.49 In line with this, chronic destructive
arthritis cannot be induced in IL-1–deficient mice using any
of the classic arthritis models. In contrast, TNF deficiency
reduces the incidence of autoimmune arthritis expression,
but once joints become afflicted, full progression to erosive
arthritis occurs (see Fig. 25-2).50 It is still unclear why IL-1
is such a dominant target in IC and T cell–driven murine
models, as well as a pivotal secondary mediator in TNF
transgenic arthritis; the role of IL-1 in autoimmune RA is
still being debated. The novel T cell cytokine IL-17 pro-
vides an additional target apart from TNF and IL-1. Local
 PART 3  |  effector mechanisms in autoimmunity and inflammation
o­ verexpression shows that it can accelerate inflammation
and tissue destruction in CIA, independent of IL-1, and
IL-17 blocking appears to be superior in the T cell flare of
AIA.26 In addition, the macrophage-derived cytokines IL-
15, IL-18, and IL-23 are abundant in RA synovia; they can
contribute to T cell maturation and activation and were
shown to promote collagen arthritis.51,52 Recently, it was
discovered that IL-23 rather than IL-12 stimulates Th17
cells and drives arthritis,53,54 making this cytokine an attrac-
tive therapeutic target.
REGULATION OF ARTHRITIS SUSCEPTIBILITY
Apart from insight into the role of arthritogenic cytokines,
models are well suited to identify modulatory cytokines.
IL-4, IL-10, transforming growth factor-β, and IL-27 appear
to be of prime importance. In general, the endogenous Th2
cytokines IL-4 and IL-10 are protective, although at the
local level, IL-4 can be inflammatory as well. An enhanced
incidence of CIA and proteoglycan arthritis is seen in
IL-4– and IL-10–deficient mice, and treatment with IL-4
or IL-10 suppresses arthritis.55 In addition, IL-12 and IL-18
promote such diseases through the enhancement of T cell
reactivity, and strong immunization with high or repeated
adjuvant exposure makes seemingly resistant mouse strains
susceptible. Enhanced expression of autoimmune arthritis
can be induced with a single lipopolysaccharide or bacte-
rial fragment injection shortly before the expected onset
through the generation of IL-12, the promotion of TNF
and IL-1 production, and the boosting of T cell responses.
It reflects the potential impact of environmental pressure.
Of note, TLR4 blocking ameliorates collagen arthritis, and
TLR4 deficiency reduces erosive arthritis in IL-1ra –/– mice
(Fig. 25-6). A similar example is provided by the promoting
effect of fungal glucans in the induction of SKG arthritis.32
Much attention has been given to identifying the role of
regulatory T cells in arthritis. It is claimed that this ­function
407
is defective in RA. Animal model studies show that the
engineered absence of regulatory T cells allows arthritis in
K/BxN mice to spread to joints not normally affected and
to become more destructive.56 Another intriguing exam-
ple is the demonstration of the immunoregulatory role of
a subset of IL-10–producing B cells in collagen arthritis.57
Of note, rituximab appears to be efficacious in RA patients
when it induces a strong IL-10 response shortly after treat-
ment. Finally, the local architecture of the synovial tissue
is of prime importance for arthritis expression. Liposomal
depletion of synovial lining cells,58 as well as disturbed syno-
vial architecture in cadherin-11–deficient mice,59 prevents
arthritis. The presence of mast cells makes a joint suscepti-
ble to GPI arthritis,60 explaining the preferential expression
at distinct joints.
CARTILAGE AND BONE DESTRUCTION
Animal models are excellent tools to characterize destruc-
tive pathways. Cartilage damage observed in models ranges
from a reversible loss of proteoglycans to collagen damage,
cell death, and complete surface erosion (see Fig. 25-1).
This underlines that arthritic processes can be more or less
destructive, depending on the underlying (immune) process
and cytokine mixture. Collagen breakdown and aggressive
cartilage loss are noted predominantly in the presence of
IC deposition, whereas milder, more gradual forms of dam-
age are seen in models driven by macrophage or T cell
activation (see Table 25-1). Large variations in progressive
destruction are also observed in patients with RA, and the
presence or absence of autoantibodies (RF, anti-CCP) make
a difference. Studies in ADAMTS4 and ADAMTS5 knock-
out mice identified the crucial role of ADAMTS5 in early
proteoglycan loss in AIA.61 This makes ADAMTS5 a prom-
ising target in RA, potentially preventing cartilage damage
if treatment is started early enough. Studies in immune
models in various Fcγ receptor knockout mice found that
Figure 25-6  Hematoxylin and eosin stain (upper) and
safranin O stain (lower) of IL-1ra –/– mice, crossed with
wild-type BALB/c (left) or TLR4 –/– mice. Note the marked
protection from arthritis and damage in TLR4 –/–.
 408 van den Berg  | Animal Models of Inflammatory Arthritis
Figure 25-7  VDIPEN neoepitope
staining in cartilage from antigen-in-
duced arthritis on day 7 in wild-type
mice (left) and FcγRI –/– mice (right),
indicative of metalloproteinase acti-
vation. Note that activation is almost
fully absent in FcγRI –/–.
Fcγ receptor type I is pivotal in driving IC-mediated cell
activation, metalloproteinase activation, and concomitant
cartilage erosion62,63 (Fig. 25-7).
Cytokines such as TNF, IL-1, and IL-17 cause bone ero-
sion through the upregulation of RANKL. Like cartilage
damage, bone erosion may occur in the absence of inflam-
mation. Initially, local gene transfer with IL-4 seemed to be
ineffective because it did not suppress joint inflammation;
however, it markedly reduced cartilage and bone destruc-
tion in CIA through the suppression of RANKL.64 Likewise,
in RANKL-deficient mice, joint inflammation continues in
the passive GPI-type IC arthritis, but bone erosion is pre-
vented. Similarly, when TNF transgenic mice were crossed
with c-fos–deficient mice, joint inflammation continued,
but bone erosion was absent. C-fos mice lack functional
osteoclasts, and in this background, TNF transgenic mice
show a nondestructive phenotype. Treatment with osteo-
protegerin, which is the natural inhibitor of RANKL, does
not reduce inflammation in AA and TNF transgenic mice,
but bone erosion is reduced.8,65
These examples show that inflammation, bone erosion,
and cartilage erosion are separate processes that may require
selective therapy. Improvements in therapy, including bone
repair, can be achieved by the combined blocking of cyto-
kines and RANKL, as well as by providing an additional bone
anabolic stimulus such as parathyroid hormone.66,67 Models
also show that bone repair is possisble, but cartilage repair is
more difficult. Anabolic stimulation and tissue engineering
approaches are needed when structural damage is advanced.
A final remark about bone erosion versus bone apposi-
tion is in order. Unlike the situation in RA patients, many
arthritis models exhibit bone erosion as well as pronounced
new bone formation at distinct sites. The latter is virtu-
ally absent in TNF transgenic mice, and a TNF-inducible
regulator of bone apposition, DKK-1, was recently identi-
fied.68 It might be argued that most murine arthritis models
are relatively devoid of TNF compared with human RA.
Intriguingly, IL-32, a novel human cytokine driving TNF,
was recently discovered. It induces arthritis in mice,69 but
an endogenous murine analogue has not been found yet.
SUMMARY
A variety of animal arthritis models have been described
that facilitate the investigation of pathogenetic mecha-
nisms of inflammatory arthritis. Their variety clearly
illustrates the need for a broad approach in determining
their utility as predictive models for therapeutic target-
ing. That being said, their major role is in unraveling the
complex inflammatory and matrix modeling, and their
use on this basis remains critical in attempts to elucidate
pathology and treatment.
REFERENCES
  1. Wooley PH: Animal models of rheumatoid arthritis. Curr Opin
Rheumatol 3:407-420, 1991.
  2. Pearson CM: Development of arthritis, periarthritis and periostitis in
rats given adjuvants. Proc Soc Exp Biol (NY) 91:95-101, 1956.
  3. Kleinau S, Erlandsson H, Holmdahl R, et al: Adjuvant oils induce
arthritis in the DA rat. I. Characterization of the disease and evidence
for an immunological involvement. J Autoimmun 4:871-880, 1991.
  4. Wooley PH, Seibold JR, Whalen JD, et al: Pristane induced arthri-
tis: The immunologic and genetic features of an experimental murine
model of autoimmune disease. Arthritis Rheum 32:1022-1030, 1989.
  5. Van Eden W, van der Zee R, Prakken B: Heat shock proteins induce
T cell regulation of chronic inflammation. Nat Rev Immunol Rev 5:
318-330, 2005.
  6. Holmdahl R, Lorentzen JC, Lu S, et al: Arthritis induced in rats
with nonimmunogenic adjuvants as models for RA. Immunol Rev
184:184-202, 2001.
  7. Kong YY, Feige U, Sarosi I, et al: Activated T cells regulate bone
loss and joint destruction in adjuvant arthritis through osteoprote-
gerin ligand. Nature 402:304-309, 1999.
  8. Pettit AR, Ji H, von Stechow D, et al: TRANCE/RANKL knockout
mice are protected from bone erosion in a serum transfer model of
arthritis. Am J Pathol 159:1689-1699, 2001.
  9. Bendele AM, Chlipala ES, Scherrer J, et al: Combination benefit of
treatment with the cytokine inhibitors IL-1ra and PEGylated solu-
ble TNF receptor type I in animal models of RA. Arthritis Rheum
43:2648-2659, 2000.
10. Trentham DE, Townes AS, Kang AH: Autoimmunity to type II col-
lagen: An experimental model of arthritis. J Exp Med 146:857-868,
1977.
11. Holmdahl R, Malmstrom V, Vuorio E: Autoimmune recognition of
cartilage collagens. Ann Med 25:251-264, 1993.
12. Finnegan A, Mikecz K, Tao P, et al: Proteoglycan (aggrecan)-induced
arthritis in BALB/c mice is a Th1-type disease regulated by Th2 cyto-
kines. J Immunol 163:5383-5390, 1999.
13. Otto JM, Chandrasekeran R, Vermes C, et al: A genome scan using
a novel genetic cross identifies new susceptibility loci and traits in a
mouse model of RA. J Immunol 165:5278-5286, 2000.
14. Joosten LAB, Helsen MMA, van de Loo FAJ, et al: Anticytokine
treatment of established type II collagen-induced arthritis in DBA/1
mice: A comparative study using anti-TNFα, anti-IL-1α/β and
IL-1ra. Arthritis Rheum 39:797-809, 1996.
15. Cromartie WJ, Craddock JG, Schwab JH, et al: Arthritis in rats
after systemic injection of streptococcal cell walls. J Exp Med 146:
1485-1602, 1977.
16. Hazenberg MP, Klasen IS, Kool J, et al: Are intestinal bacteria involved
in the etiology of rheumatoid arthritis? APMIS 100:1-9, 1992.
 PART 3  |  effector mechanisms in autoimmunity and inflammation
17. Lafyatis R, Thompson NL, Remmers EF, et al: Transforming growth
factor-β production by synovial tissues from rheumatoid patients and
streptococcal cell wall arthritic rats. J Immunol 143:1142-1148, 1989.
18. Kuiper S, Joosten LAB, Bendele AM, et al: Different roles of TNFα
and IL-1 in murine streptococcal cell wall arthritis. Cytokine
10:690-702, 1998.
19. Lubberts E, Schwarzenberger P, Huang W, et al: Requirement of
IL-17 receptor signaling in resident synoviocytes for development of
full blown destructive arthritis. J Immunol 175:3360-3368, 2005.
20. Joosten LA, Koenders MI, Smeets RL, et al: TLR2 pathways drives
SCW induced joint inflammation: Critical role of MyD88. J Immunol
171:6145-6153, 2003.
21. Deng GM, Tarkowski A: Synovial cytokine mRNA expression dur-
ing arthritis triggered by CpG motifs of bacterial DNA. Arthritis Res
3:48-53, 2001.
22. Dumonde DC, Glynn LE: The production of arthritis in rabbits by an
immunological reaction to fibrin. Br J Exp Pathol 43:373-383, 1962.
23. Cooke TDV, Hird ER, Ziff M, et al: The pathogenesis of chronic
inflammation in experimental antigen induced arthritis. J Exp Med
135:323-338, 1972.
24. Van den Berg WB, van de Putte LBA, Zwarts WA, et al: Electrical
charge of the antigen determines intraarticular antigen handling and
chronicity of arthritis in mice. J Clin Invest 74:1850-1859, 1984.
25. Van Meurs JBJ, van Lent PLEM, Singer II, et al: IL-1ra prevents
expression of the metalloproteinase-generated neoepitope VDIPEN in
antigen-induced arthritis. Arthritis Rheum 41:647-656, 1998.
26. Koenders MI, Lubberts E, Oppers-Walgreen B, et al: Blocking of IL-17
during reactivation of experimental arthritis prevents joint inflamma-
tion and bone erosion by decreasing RANKL and IL-1. Am J Pathol
167:141-149, 2005.
27. Van den Broek MF, van den Berg WB, van de Putte LBA, et al: Strep-
tococcal cell wall induced arthritis and flare-up reactions in mice
induced by homologous and heterologous cell walls. Am J Pathol
133:139-149, 1988.
28. Iwakura Y, Tosu M, Yoshida E, et al: Induction of inflammatory
arthropathy resembling rheumatoid arthritis in mice transgenic for
HTLV-I. Science 253:1026-1028, 1991.
29. Korganow AS, Ji H, Mangialaio S, et al: From systemic T cell self-
reactivity to organ-specific autoimmune disease via immunoglobulins.
Immunity 10:451-461, 1999.
30. Maccioni M, Zeder-Lutz G, Huang H, et al: Arthritogenic monoclonal
antibodies from K/BxN mice. J Exp Med 195:1071-1077, 2002.
31. Hata H, Sakaguchi N, Yoshitomi H, et al: Distinct contribution of
IL-6, TNF-alpha, IL-1 and IL-10 to T cell-mediated spontaneous auto-
immune arthritis in mice. J Clin Invest 114:582-588, 2004.
32. Yoshitomi H, Sakaguchi N, Kobayashi K, et al: A role for fungal β-
glucans and their receptor Dectin-1 in the induction of autoimmune
arthritis in genetically susceptible mice. J Exp Med 201:949-960, 2005.
33. Atsumi T, Ishihara K, Kamimura D, et al: A point mutation of Tyr-759
in IL-6 family cytokine receptor subunit gp130 causes autoimmune
arthritis. J Exp Med 196:979-990, 2002.
34. Sawa S, Kamimura D, Jin GH, et al: Autoimmune arthritis associated with
mutated IL-6 receptor gp130 is driven by STAT3/IL-7-dependent homeo-
static proliferation of CD4+ T cells. J Exp Med 203:1459-1470, 2006.
35. Keffer J, Probert L, Cazlaris H, et al: Transgenic mice expressing
human tumor necrosis factor: A predictive genetic model of arthri-
tis. EMBO J 13:4025-4031, 1991.
36. Kontoyiannis D, Pasparakis M, Pizarro TT, et al: Impaired on/off
regulation of TNF biosynthesis in mice lacking TNF AU-rich ele-
ments: Implications for joint and gut-associated immunopathologies.
­Immunity 10:387-398, 1999.
37. Kollias G, Douni E, Kassiotis G, et al: On the role of TNF and recep-
tors in models of multiorgan failure, RA, multiple sclerosis and inflam-
matory bowel disease. Immunol Rev 169:175-194, 1999.
38. Kassiotis G, Kollias G: Uncoupling the proinflammatory from the
immunosuppressive properties of TNF at the p55 TNF receptor level:
Implications for pathogenesis and therapy of autoimmune demyelin-
ation. J Exp Med 193:427-434, 2001.
39. Niki Y, Yamada H, Seki S, et al: Macrophage- and neutrophil-domi-
nant arthritis in human IL-1 alpha transgenic mice. J Clin Invest
107:1127-1135, 2001.
40. Horai R, Saijo S, Tanioka H, et al: Development of chronic inflam-
matory arthropathy resembling RA in IL-1ra-deficient mice. J Exp
Med 191:313-320, 2000.
409
41. Nakae S, Saijo S, Horai R, et al: IL-17 production from activated
T cells is required for the spontaneous development of destructive
arthritis in mice deficient in IL-1 receptor antagonist. Proc Natl Acad
Sci U S A 100:5986-5990, 2003.
42. Horai R, Nakajima A, Habiro K, et al: TNFα is crucial for the devel-
opment of autoimmune arthritis in IL-1 receptor antagonist-deficient
mice. J Clin Invest 114:1603-1611, 2004.
43. Blom AB, van Lent PLEM, van Vuuren H, et al: FcgammaR
expression on macrophages is related to severity and chronicity of
synovial inflammation and cartilage destruction during experimen-
tal immune-complex-mediated arthritis (ICA). Arthritis Res 2:
489-503, 2000.
44. Van Lent PLEM, van de Loo FAJ, Holthuysen AEM, et al: Major role
for IL-1 but not for TNF in early cartilage damage in immune complex
arthritis in mice. J Rheumatol 22:2250-2258, 1995.
45. Nabbe KCAM, Boross P, Holthuysen AEM, et al: Joint inflamma-
tion and chondrocyte death become independent of FcγrIII by local
overexpression of IFN-γ during immune complex mediated-arthritis.
Arthritis Rheum 52:967-974, 2005.
46. Kuhn KA, Kulik L, Tomooka B, et al: Antibodies against citrullinated
proteins enhance tissue injury in experimental autoimmune arthritis.
J Clin Invest 116:961-973, 2006.
47. Monach PA, Benoist C, Mathis D: The role of antibodies in mouse
models of rheumatoid arthritis, and relevance to human disease.
Adv Immunol 82:217-248, 2004.
48. Koenders MI, Lubberts E, van de Loo FAJ, et al: Interleukin-17
acts independently of TNF-α under arthritic conditions. J Immunol
176:6262-6269, 2006.
49. Van den Berg WB: What we learn from arthritis models to benefit
arthritis patients. Baillieres Clin Rheumatol 14:599-616, 2000.
50. Campbell IK, O’Donnell K, Lawlor KE, et al: Severe inflammatory
arthritis and lymphadenopathy in the absence of TNF. J Clin Invest
107:1519-1527, 2001.
51. Gracie JA, Forsey RJ, Chan WL, et al: A proinflammatory role for
IL-18 in rheumatoid arthritis. J Clin Invest 104:1393-1401, 1999.
52. McInnes IB, Liew FY: Interleukin-15: A proinflammatory role in rheu-
matoid arthritis synovitis. Immunol Today 19:75-79, 1998.
53. Murphy CA, Langrish CL, Chen Y, et al: Divergent pro and anti-
inflammatory roles for IL-23 and IL-12 in joint autoimmune inflam-
mation. J Exp Med 198:1951-1957, 2003.
54. Cho ML, Kang JW, Moon YM, et al: STAT3 and NF-kappaB signal
pathways required for IL-23 mediated IL-17 production in spontane-
ous arthritis model IL-1ra deficient mice. J Immunol 176:5652-5661,
2006.
55. Joosten LAB, Lubberts E, Durez P, et al: Role of IL-4 and IL-10 in
murine collagen-induced arthritis: Protective effect of IL-4 and
IL-10 treatment on cartilage destruction. Arthritis Rheum 40:
249-260, 1997.
56. Nguyen LT, Jacobs J, Mathis D, et al: Where FoxP3-dependent regu-
latory T cells impinge on the development of inflammatory arthritis.
Arthritis Rheum 56:509-520, 2007.
57. Evans JG, Chavez-Rueda KA, Eddaoudi A, et al: Novel suppressive
function of transitional 2 B cells in experimental arthritis. J Immunol
178:7868-7878, 2007.
58. van Lent PL, van den Hoek AE, van den Bersselaar LA, et al: In vivo
role of phagocytic synovial lining cells in onset of experimental arthri-
tis. Am J Pathol 143:1226-1237, 1993.
59. Lee DM, Kiener HP, Agarwal SK, et al: Cadherin-11 in synovial lining
formation and pathology in arthritis. Science 315:1006-1010, 2007.
60. Nigrovic PA, Binstadt BA, Monach PA, et al: Mast cells contribute to
initiation of autoantibody-mediated arthritis via IL-1. Proc Natl Acad
Sci U S A 104:2325-2330, 2007.
61. Stanton H, Rogerson FM, East CJ, et al: ADAMTS5 is the major
aggrecanase in mouse cartilage in vivo and in vitro. Nature 434:
648-652, 2005.
62. Van den Berg WB: Uncoupling of inflammatory and destructive mech-
anisms in arthritis. Semin Arthritis Rheum 30(Suppl 2):7-16, 2001.
63. van Lent PL, Grevers L, Lubberts E, et al: FcgR mediate cartilage
but not bone destruction in murine AIA: Uncoupling of cartilage
damage from bone erosion and joint inflammation. Arthritis Rheum
54:3868-3877, 2006.
64. Lubberts E, Joosten LAB, Chabaud M, et al: IL-4 gene therapy for col-
lagen arthritis suppresses synovial IL-17 and osteoprotegerin ligand
and prevents bone erosion. J Clin Invest 105:1697-1710, 2000.
 410 van den Berg  | Animal Models of Inflammatory Arthritis

65. Redlich K, Hayer S, Ricci R, et al: Osteoclasts are essential for TNFα- 68. Diarra D, Stolina M, Polzer K, et al: Dickkopf-1 is a master regulator of
mediated joint destruction. J Clin Invest 110:1419-1427, 2002. joint remodeling. Nat Med 13:156-163, 2007.
66. Zwerina J, Hayer S, Tohidast-Akrad M, et al: Single and combined 69. Joosten LA, Netea MG, Kim SH, et al: IL-32 a proinflammatory cyto-
inhibition of TNF, IL-1 and RANKL pathways in TNF-induced kine in RA. Proc Natl Acad Sci U S A 103:3298-3303, 2006.
arthritis: Effects on synovial inflammation, bone erosion, and cartilage
destruction. Arthritis Rheum 50:277-290, 2004.
67. Redlich K, Gortz B, Hayer S, et al: Repair of local bone erosions and
reversal of systemic bone loss upon therapy with anti-TNF in combi-
nation with osteoprotegerin or parathyroid hormone in TNF-medi-
ated arthritis. Am J Pathol 164:543-555, 2004.
 26
Key Points
Neurogenic inflammation is defined as release of inflamma-
tory substances from the peripheral terminals of primary
afferent fibers.
Neuropeptides, substance P and calcitonin gene–related pep-
tide, released from peripheral terminals of nociceptors cause
plasma extravasation and vasodilation.
The neurotransmitter glutamate increases in joint inflamma-
tory conditions, increasing blood flow, extravasation, swell-
ing, and inflammation.
Increased release of glutamate activates transcription factors
to turn on gene transcription.
The axon reflex is a local reflex that contributes to neurogenic
inflammation when the nociceptor is activated.
The dorsal root reflex, which occurs after prolonged intense
stimulation, originates in the spinal cord dorsal horn and
results in antidromic activation of primary afferent fibers and
peripheral release of neuropeptides and excitatory amino
acids.
The sympathetic nervous system contributes to neurogenic
inflammation through activation of α1-adrenoreceptors or
neuropeptide Y2 receptors.
Arthritis is a major debilitating disease that involves inflam-
mation of a joint. Arthritis may take numerous forms, but
all varieties result in severe destruction of the synovial
tissue, cartilage, and bone microenvironment. Although
arthritis is a multifactorial disease, the hallmark, similar to
all inflammatory reactions, is release of proinflammatory
immune mediators, such as tumor necrosis factor (TNF)-α
and interleukin (IL)-1. There is now consensus concerning
the triggers of the inflammatory cascade, but the continuing
inflammation of the chronic disease flares repeatedly despite
aggressive therapy with steroids and nonsteroidal anti-
inflammatory drugs suggesting the presence of a persistent
trigger. Blood-borne and local factors have been considered
the primary players in the development of inflammation,
although early studies suggested a neurogenic component.1
In addition to blood-borne or local factors, the nervous sys-
tem plays a significant role in the amplification of inflam-
mation and the consequent pain.2-4 The involvement of
the nervous system in inflammation is termed neurogenic
inflammation and is defined as release of inflammatory sub-
stances from the peripheral terminals of primary afferent
fibers. Peripheral and central neuronal mechanisms control
and contribute to neurogenic inflammation. This chapter
reviews the evidence and mechanisms that account for neu-
rogenic inflammation.
Neurologic Regulation
of Inflammation
Kathleen A. Sluka  • 
Karin N. Westlund-High
The cardinal signs of inflammation include heat, redness,
swelling, pain, and decreased function. These five cardi-
nal signs are interrelated and serve as markers to assess the
impact of inflammation on an individual’s quality of life. The
most sensitive factors are swelling, pain, and decreased func-
tion. The swelling associated with inflammation results from
vasodilation and plasma extravasation of proteins to the site
of injury. The pain results from activation of primary afferent
nociceptors that transmit information to the spinal cord and
to higher brain centers, where perception occurs. The degree
of pain is related to the severity of the inflammation, and
if inflammation decreases, pain also is expected to decrease.
Decreased function is the physical consequence of increases
in inflammation and pain. This chapter discusses data not
only regarding the inflammation, but also regarding the pain
associated with inflammation in an attempt to determine
mediators and mechanisms of neurogenic inflammation.
PRIMARY AFFERENT FIBERS
In the peripheral nervous system, primary afferent fibers
release neuropeptides that result in plasma extravasation
and vasodilation at the site of inflammation. Multiple clini-
cal reports have appeared that indicate damage to nervous
pathways and peripheral nerves owing to neurologic insults,
such as stroke and peripheral nerve damage, can provide
a protective effect against development of arthritis on the
affected side, proportional to the magnitude of the neuro-
logic insult.5-13 These clinical reports show that primary
afferent fibers and the central nervous system play key roles
in the development of arthritis.
Primary afferent nerves are key components of neuro-
genic inflammation. Neurogenic inflammation is a form of
inflammation that is initiated by activation of afferent sen-
sory nerves.14,15 Activation of thinly myelinated, Aδ or type
III, and unmyelinated, C fiber or type IV, primary afferent
fibers results in vasodilation and plasma extravasation.16-19
Electric stimulation at intensities that activate Aδ fibers
produces vasodilation; increasing the intensity to include
C fibers also produces plasma extravasation.16,19,20 In con-
trast, removal of C fibers by treating the neonatal rat with
capsaicin decreases plasma extravasation and the severity of
the pathologic changes induced by inflammation.21,22 Acute
depletion of neuropeptides with capsaicin similarly reduces
plasma extravasation.23 Selective destruction of Aδ fibers
by neonatal treatment with nerve growth factor decreases
vasodilation that results from antidromic stimulation of Aδ
or C fibers.17 Aδ and C fiber activation is necessary for gen-
eration of neurogenic inflammation.
Neurogenic inflammation is known to contribute to nu­­­­
merous pathophysiologic states. Clinically relevant examples
411
 412 SLUKA  |  Neurologic Regulation of Inflammation

include arthritis; gastrointestinal nausea, emesis, disten-
tion, and pain; asthma; and urinary bladder incontinence.24
­Neurogenic inflammation was first described, however, for
skin because it can be observed easily there as the erythemic
flare after bee stings. The term neurogenic implies that this
form of inflammation is generated through mechanisms that
depend on the innervation of an organ. This dependence
is shown by the fact that denervation prevents this type of
inflammation.25,26
PERIPHERAL NEUROTRANSMITTERS
Neuropeptides
The neuropeptides found in Aδ and C fibers include sub-
stance P and calcitonin gene–related peptide (CGRP) and
promote plasma extravasation (substance P) and vasodila-
tion (CGRP) on their release. Electric stimulation of pri-
mary afferent fibers releases substance P.27,28 Substance P
infused into rat knee joint results in pathologic changes
associated with arthritis, vasodilation, and plasma extrava-
sation (Fig. 26-1).23,29,30 Substance P has been reported to
increase in rat arthritic joints31 and to increase the severity
of arthritis if added to the joint by increasing protein/plasma
extravasation2,32 in experimentally induced animal models
of inflammation, and substance P and CGRP are found in
inflammatory exudates in human subjects.33-35 In contrast,
substance P antagonists delivered at the site of inflamma-
tion reduce the plasma extravasation and the severity of
the inflammation.16,29,30,36 Similarly, direct application of a
substance P antagonist to an inflamed knee joint reduces
hyperalgesia.37 Substance P plays a role in the hyperalge-
sia associated with joint inflammation. A single dose of a
substance P receptor antagonist has no effect, however, on
the joint swelling when it is fully developed.37 Substance P
and CGRP are found in human subjects with arthritis, are
released peripherally from primary afferent nerve fibers, and
contribute to plasma extravasation and vasodilation ulti-
mately to enhance inflammation.
CGRP is found almost exclusively in primary afferent
nerves.38 CGRP has been shown to block the degradation
of substance P39 and contributes to the enhanced effects of
substance P during inflammation. Increases in neuropep-
tide content in afferent endings in the dorsal horn, and

A B C

Vasodilation 40 Plasma protein extravasation

80 Plasma protein extravasation
2.0 1nmol SP 1 µM SP 75 pmol SP
1nmol SP + 10 nmol FK888 1 µM SP +1 00 µM FK888 75 pmol SP + 6.0 pmol CGRP
30
1.5 *τ 60 * #
Voltage

40 20
1.0 *
#
10 *
0.5 20
# #

0.0 0 0
D Normal Inflammation E F
Figure 26-1  A, Radiograph of a normal knee joint. B, Radiograph after infusion of substance P in an arthritic joint (arrow). C, Radiograph after 
infusion of a substance P antagonist into an arthritic joint (arrow). D, Amount of vasodilation from direct application of substance P (SP) onto the knee
joint measured with laser Doppler imaging in a normal animal and an animal with knee joint inflammation. The vasodilation was greater in animals 
with inflammation than in animals without and was reduced by the substance P antagonist FK888. *, significantly increased from control; #, signifi-
cantly less than infusion of substance P; τ, significantly greater than normals. E, Plasma protein extravasation is produced by infusion of substance P
into the knee joint (*), and this is prevented by blockade of substance P receptors with FK888 (#). F, Plasma protein extravasation by substance P (*) is 
potentiated by combining with calcitonin gene–related peptide (CGRP) (#). (A from Levine JD, Clark R, Devor M, et al: Intraneuronal substance P ­contributes
to the severity of experimental arthritis. Science 226:547-549, 1984; D and E redrawn from Lam FY, Ferrell WR, Scott DT: Substance P-induced ­inflammation
in the rat knee joint is mediated by neurokinin 1 (NK1) receptors. Regul Peptides 46:198-201, 1993; F redrawn from Gamse R, Saria A: Eur J Pharmacol 115:
61-66, 1985.)
 PART 3  |  effector mechanisms in autoimmunity and inflammation
α-CGRP mRNA in spinal cord also are observed in arthritic
animals.40-43 There are related neurogenic contributions to
increased blood flow and vasodilation already described for
substance P and CGRP in the knee joint.2,30,44,45 Admin-
istration of substance P and CGRP simultaneously poten-
tiates the plasma extravasation (see Fig. 26-1).46,47 It is
concluded that substance P and CGRP found in the joints
of human subjects with arthritis are released peripher-
ally from primary afferent fibers and contribute to plasma
extravasation and vasodilation that ultimately enhance
inflammation.
The neuropeptide substance P also has been shown to
potentiate the release of glutamate in the spinal cord dorsal
horn.48,49 Substance P interacts with glutamate at periph-
eral nerve endings to enhance and increase the duration of
glutamate-induced behavioral responses.31 Substance P also
promotes release of histamine by degranulating mast cell
and leukocyte migration.50 Various lines of evidence have
implicated glutamate and substance P in peripheral interac-
tive events.
Colocalization of glutamate, substance P, and CGRP
occurs in some dorsal root ganglia soma and primary affer-
ent nerve endings.51-54 Corelease and interactions between
these neuromodulators have been reported, including
(1) potentiation of glutamate receptor effects,55,56 (2) acti-
vation of second messenger systems increasing intracellular
calcium levels,57 and (3) increased release of glutamate into
the extracellular space.58 The modification of receptor and
cellular events produces long-term facilitation of spinal cord
neuronal responses to peripheral stimuli leading to central
sensitization; this can be mimicked experimentally with
intrathecal injection of substance P or N-methyl-d-aspar-
tate (NMDA), which produces an immediate increase in
prostaglandin (substance P) or cyclooxygenase-2 (NMDA)
release.59 The plasma membrane substance P receptors on
dorsal horn neurons that may be spinothalamic tract cells
undergo internalization after noxious stimulation.60
Glutamate
In addition to the neuropeptides, the excitatory neurotrans-
mitter glutamate plays a significant role in the inflamma-
tory process and is thought to interact with substance P.
Glutamate receptors have been described peripherally on
lymphocytes, fibroblasts, and nerve endings in the skin.61,62
Increased content in primary afferent fibers of the excit-
atory neurotransmitter glutamate (well above metabolic
levels) in primary afferent fibers is found in twice as many
type III (Aδ) and type IV (C) fibers in the medial ­articular
nerve innervating inflamed knee joints compared with con-
trols (Fig. 26-2).63 Direct knee joint injection of ­glutamate
receptor agonists increases joint blood flow, ­extravasation,
and swelling.64 Excitatory amino acids released into the
joint are doubled during knee joint inflammation (see
Fig. 26-2)65 and can be reduced by lidocaine. Direct knee
joint administration of the excitatory amino acids glutamate
and aspartate induces persistent nociceptive behavior in rats
(see Fig. 26-2).66 Substance P interacts with excitatory amino
acids at peripheral nerve endings to enhance and increase
the duration of glutamate-induced behavioral responses.31
In contrast, administration of NMDA or non-NMDA glu-
tamate receptor antagonists directly into the knee joint
413
a­ ttenuates hyperalgesic responses induced by either the
acute kaolin and carrageenan (K/C) model or the chronic
CFA model.66
In human clinic patients with arthritis, increases in gluta-
mate and aspartate also are found in the inflammatory exu-
dates.67 Exogenously applied glutamate increases TNF-α in
human synovial cultures.68 In human subjects with arthritis,
increases in glutamate and aspartate initiate inflammatory
mediator cascades resulting in inflammation of the knee
joint in conjunction with other neuromediators.
POTENTIAL MECHANISMS
Signal Transduction
Extracellular and intracellular neurogenic environmental
changes that occur in response to inflammation lead to
changes induced by signal transduction elements regulat-
ing the transcription of target genes. These factors usually
increase (although they sometimes decrease) the rate of
gene transcription, increasing the formation of mRNA and
protein. One of these glutamate receptor–mediated effects
is to promote rapid activation of nuclear factor κB (NFκB)
for coordinated activation of target genes.69,70 Activation
of NFκB occurs in many pathogenic settings, including
exposure to glutamate and its agonists.71,72 NFκB regulates
the expression of many immune and inflammatory activa-
tors forming an intracellular amplification loop involving
glutamate, NFκB, and inflammatory activators. Glutamate
receptor agonists induce nuclear translocation of NFκB
in the developing cerebellum, whereas glutamate recep-
tor antagonists abolish NFκB binding activity.73 Activa-
tion in astrocytes requires the cytokines IL-1α and TNF-α.
An endogenous loop of glutamate receptor activation and
subsequent NFκB activation is suggested, with particular
involvement of neuronal NMDA glutamate receptors.
There are related increases in nitric oxide seen in the joints
of arthritic rats,74 along with the increased glutamate, owing
to concomitant increases in cytoplasmic calcium, which
upregulates nitric oxide synthase.75 In an animal model,
a nitric oxide synthase inhibitor reverses the behavioral
changes and attenuates the joint circumference increases.76
Increased nitric oxide has been shown in a human model
to increase chemokine and cytokine production, specifically,
MIP-1α, MCP-1, IL-8, IL-1β, and TNF-α.77
Axon Reflex
Two potential mechanisms have been proposed that can
account for the response of the nervous system to insult
that precipitates the release of neurotransmitters and neu-
romodulators and inflammation. The classic theory used
to explain that the release of inflammatory neuropeptides
from the peripheral terminals of primary afferent fibers is
the axon reflex. This theory suggests that nociceptive affer-
ent fibers bifurcate peripherally, with one branch forming
the sensory receptor and the other supplying a blood vessel.
When the sensory receptors are activated in the periphery,
action potentials are propagated not only centrally to the
spinal cord for transmission of nociceptive information,
but also peripherally to branches supplying blood vessels to
release neuropeptides (Fig. 26-3).1,16,18,78-80
 414 SLUKA  |  Neurologic Regulation of Inflammation

0.2 ASP
12

0.1
10

Spikes/s
Latency (s)
0.0
8
** * * –0.1
* * * *
6 * * ** * * –0.2
* ** **
Base 4 5 6 7 8 0 50 100 150 200 250
A C Drug E Time (s)
Time (h)

14
n=5
12 * 0.2
ACPD

Withdrawal threshold (mN)

120
Concentration (µm)

10 * /ASP
8 0.1
* * * * 90
*

Spikes/s
6
* * * 0.0
4 * * * * 60 ** * **
* * ** *
2 –0.1
30 * * ** ** **
0 K/C
0 –0.2
Base 1 2 3 4 5 6 7 8 Base 4 5 6 7 8 0 250 500 750 1000
Drug
Time (hrs) F Time (s)
Time (h)
GLU
ASP ARTH only
ARG ARTH + AP7
CTN ARTH + CNOX
ARTH + Ketamine
ARTH + PB
B D
Figure 26-2  A, Electron micrograph showing an unmyelinated glutamate axon in the medial articular nerve of the knee joint innervating an inflamed
knee joint. Glutamate immunoreactivity was identified using postembedding immunogold labeling. Adjacent unmeylinated axons are unlabeled. Bar =
0.2 mm. B, Release of glutamate into the knee joint measured with microdialysis and high-performance liquid chromatography before and for 8 hours
after induction of inflammation with 3% kaolin and 3% carrageenan. The increases occur immediately after injection and last for approximately 3 hours.
ARG, arginine; ASP, aspartate; CTN, citrulline; GLU, glutamate. C, Withdrawal latency to heat before and after induction of inflammation with kaolin and
carrageenan into the knee joint (closed circles). In rats treated intra-articularly with glutamate receptor antagonists to N-methyl-d-aspartate (NMDA)
receptors (AP7, open circles; ketamine, open triangles) or to non-NMDA receptors (CNQX, closed triangles), there was a delayed onset in the development
of hyperalgesia. D, Withdrawal threshold to mechanical stimuli before and after induction of inflammation with kaolin and carrageenan into the knee
joint (closed circles). In rats treated intra-articularly with glutamate receptor antagonists to NMDA receptors (AP7, open circles; ketamine, open triangles)
or to non-NMDA receptors (CNQX, closed triangles), there was a delayed onset in the development of hyperalgesia. E, Rate histograms showing the
responses of primary articular afferents to infused activators of glutamate receptors (1 mM, intra-arterial). Coactivation with two glutamate receptor
agonists produced an amplified response compared with the limited duration excitatory response produced by a single glutamate receptor agonist
shown in E. Time of injections is indicated by the lines above the traces. ACPD, (1S, 3R)-1-aminocyclopentane-1, 3-dicarboxylic acid; ASP, aspartate.  
(A from Westlund KN, Sun YC, Sluka KA, et al: Neural changes in acute arthritis in monkeys, II: increased glutamate immunoreactivity in the medial articular
nerve. Brain Res Rev 17:15-27, 1992; B from Lawand NB, Willis WD, Westlund KN: Excitatory amino acid receptor involvement in peripheral nociceptive trans-
mission in rats. Eur J Pharmacol 324:169-177, 1997; C and D from Lawand NB, McNearney T, Westlund KN: Amino acid release into the knee joint: Key role in
nociception and inflammation. Pain 86:69-74, 2000; E and F from Lawand NB, Willis WD, Westlund KN: unpublished data, 1998.)

model of skin inflammation.87 After acute or chronically

Dorsal Root Reflex
induced inflammation in rats, dorsal root reflexes develop
It has been known for a century that activity in primary in articular afferent fibers,88,89 including Aδ and C fibers
afferent fibers supplying the skin can cause “antidromic (Fig. 26-4).90 Dorsal rhizotomy, peripheral application of
vasodilation,”81 and it has been assumed that the activity is lidocaine, or crushing the medial articular nerve ­proximal to
generated locally (axon reflex). Depolarization of the cen- the recording site also eliminates dorsal root reflex ­activity,
tral end of primary afferent fibers in the spinal cord also can showing that the action potentials involve dorsal roots and
occur, however, with “sufficient and prolonged” activation are generated in the central nervous system. Generation
resulting in the generation of action potentials that propa- of dorsal root reflex activity occurs when the dorsal horn
gate outward toward the periphery (see Fig. 26-3)82-85 and becomes sensitized, and there is an associated release of
play a significant role in neurogenic inflammation.4 Initial neurotransmitters from the central terminals of the primary
studies show that severing the dorsal nerve roots on the side afferent fibers.4,85,91,92
of inflammation reduces joint inflammation by half in the Blockade of non-NMDA glutamate and γ-aminobutyric
rat model86 and reduces neutrophilic infiltration in a rat acid (GABAA) receptors in the spinal cord eliminates the
 PART 3  |  effector mechanisms in autoimmunity and inflammation
A
Blood
vessel
Muscle
Nerve terminals
are activated locally
by axon reflex
Spinal cord
DRG
Inflammation
Thalamus
Sp CCRP
DRG
Spinal cord
+ induced by joint inflammation in cats and monkeys. J Neurophysiol 74:
DRG
–
+ +
Figure 26-3  A, The axon reflex is a local reflex that is initiated when a
nociceptor is activated. The nociceptor sends information through the
spinal cord for transmission of nociceptive information to higher brain
centers. The peripheral branches of the primary afferent also are depo-
larized resulting in release of substance P and calcitonin gene–related
peptide from the peripheral terminals. This results in vasodilation and
plasma extravasation to enhance inflammation. B, The dorsal root reflex
is a mechanism by which neurogenic inflammation is initiated in the spi-
nal cord. Antidromic action potentials are created in the central terminals
of primary afferent fibers through activation of non–N-methyl-d-aspar-
tate glutamate receptors on GABAergic neurons, and GABAA receptors
on the central terminals of primary afferent fibers. Peripheral release of
substance P (SP) and calcitonin gene–related peptide (CGRP) from the
primary afferent terminals enhances the inflammatory response. DRG,
dorsal root ganglion.
dorsal root reflex activity recorded in the medial articular
nerve of inflamed joints (Fig. 24-5).88 In parallel, blockade
of non-NMDA glutamate and GABAA receptors in the spi-
nal cord reduces inflammation by approximately 50%.86,93,94
Spinal blockade of adenosine-1 receptors decreases neutro-
philic infiltration and bone and cartilage destruction asso-
ciated with inflammation.87,95 These findings suggest that
dorsal root reflexes are an important mechanism for the
amplification and persistence of inflammation.
The dorsal root reflex involves a loop initiated by pri-
mary afferent fibers ending at the site of tissue damage and
evolving inflammation. It is proposed that persistent affer-
ent nerve fiber activity occurs in response to peripheral
insult. The afferent fibers transmit information through the
spinal cord circuitry, and if the activation is of sufficient
strength and persists long enough in the sensitized dorsal
horn, nerve activity is generated in recruited primary affer-
ent fibers sending this activity back out to the site of inflam-
mation (see Fig. 26-3). When sensitized dorsal horn neurons
act as a neurogenic drive, they contribute to inflammation
415
A B
200 ms 40 ms
Figure 26-4  A and B, The dorsal root reflexes shown are from an Aδ
(A) or a C fiber (B) in animals with knee joint inflammation. Samples of
dorsal root reflexes were recorded from two electrodes spaced 20 mm
(A) or 10 mm (B) apart. The top traces are from the proximal electrode,
and the bottom traces are from the distal electrode. (From Sluka KA,
Rees H, ­ Westlund KN, et al: Fiber types contributing to dorsal root reflexes
981-989, 1995.)
in the joint and the accompanying persistent pain state.
Excessive stimulation sensitizes the dorsal horn circuitry
through ionotropic non-NMDA glutamate, GABAA, and
adenosine-1 receptors, initiating dorsal root reflexes (i.e.,
neuronal activity that travels back out afferent nerves to the
periphery). The dorsal root reflex likely is responsible for
release of glutamate and vasoactive peptides into the joint,
which triggers release of inflammatory cytokines, which can
elicit increased firing in nociceptive afferent fibers to result
in increased hyperalgesia and inflammation.68
Sympathetic Nervous System
It is evident that there is some neural linkage between the
spinal cord dorsal horn and the knee joint that is impor-
tant in the development of the inflammation. The linkage
between the spinal cord dorsal horn and the knee joint that
contributes to the development of the inflammation does
not seem to be the sympathetic nervous system because a
surgical sympathectomy does not affect the development of
the inflammation.96 Spinal administration of bicuculline94
or dorsal rhizotomies96 also prevented half of the swelling
of the knee joint and the temperature increase. Interrupt-
ing the output of preganglionic sympathetic neurons also
does not have these effects.97 In animals that have an intact
sympathetic outflow, antagonists of α1 adrenoreceptors or of
neuropeptide Y2 receptors reduce the flare and the dorsal
root reflexes evoked by a capsaicin injection, but antago-
nists of α2 adrenoreceptors or of neuropeptide Y1 receptors
do not.98 Evidently, release of norepinephrine acting at α1
adrenoreceptors and of neuropeptide Y acting at neuro-
peptide Y2 receptors helps maintain the responsiveness of
nociceptive afferents to capsaicin and their ability to trigger
dorsal root reflexes and flare.98 The mechanism for this is
unclear, although it seems possible that the effects of these
neurotransmitters could be mediated indirectly by actions
on transient receptor potential vanilloid-1 (TRPV1).
 416 SLUKA  |  Neurologic Regulation of Inflammation

Control Control
C
10 100 3.0

Rate (spikes s-1)

a b c d e a b c d e Joint circumference
2.5
2.0 *
*
1.5 #
0 1.0 *
0
0.5
AP7 CNQX
40 0.0
00
Rate (spikes s-1)

Control AP7 CNQX

D 15
14 Response to heat # #
12
0 0 10
0 60 120 0 60 120 8 *
A Time (s) B Time (s) 6
4
b e 2
c a 0
d Baseline inflammation AP7 CNQX
Figure 26-5  A and B, Histograms show the dorsal root reflex responses to mechanical stimuli applied to the lower limb (points a-e) in animals with knee
joint inflammation (control). A, Treatment with 2 mM of AP7 to block N-methyl-d-aspartate (NMDA) glutamate receptors in the spinal cord had no effect
on the dorsal root reflex activity. B, Treatment of the spinal cord with 0.27 mM of CNQX to block non-NMDA receptors prevented the dorsal root reflex
responses in the medial articular nerve. C, Joint circumference difference from baseline increase by approximately 2 cm in animals with knee joint inflam-
mation (*). Treatment of the spinal cord with 2 mM of AP7 had no effect on the development of joint swelling. Treatment of the spinal cord with 1 mM of
CNQX reduced the development of inflammation by approximately 50%. D, Behavioral responses to heat decreased after induction of inflammation, an
indicator that heat hyperalgesia had developed (*). Treatment of the spinal cord with 2 mM of AP7 or 1 mM of CNQX prevented the development of heat
hyperalgesia (#). * , significantly increased from baseline; #, significantly less than controls. (A and B from Rees H, Sluka KA, Westlund KN, et al: The role of glu-
tamate and GABA receptors in the generation of dorsal root reflexes by acute arthritis in the anaesthetized rat. J Physiol 484:437-445, 1995; C and D from Sluka
KA, Westlund KN: Centrally administered non-NMDA but not NMDA receptor antagonists block peripheral knee joint inflammation. Pain 55:217-225, 1993.)

Although the neural links between the knee joint and
the spinal cord include sensory afferent fibers and effects
of neuropeptides on vasomotor tone, sympathetic output is
not responsible for the neurogenic inflammation observed
for the knee joint as it is in the skin. The composition of the
knee joint nerve is almost entirely small unmyelinated and
lightly myelinated sensory nerves.99 Somatic motor activity
also is not involved because inflammation could be produced
even when the musculature was paralyzed. The neurogenic
link for joint tissues has to be the sensory axons.
The sympathetic nervous system plays a role in the inflam-
matory process. Specifically, it is thought that the sympathetic
nervous system is more involved in chronic inflammation
than acute inflammation. Acutely, adrenergic blockers have
no effect on inflammation associated with antidromic stimu-
lation of primary afferent fibers.18 Surgical and chemical
sympathectomy or systemic depletion of catecholamines sim-
ilarly has no effect on the acute inflammation induced by car-
rageenan.96,100 Sympathectomy reduces the severity of injury
induced in chronic adjuvant arthritis.2,101 These studies sug-
gest that involvement of the sympathetic nervous system is a
key difference between acute and chronic inflammation.
CLINICAL SIGNIFICANCE
Clinical reports have appeared in support of neurogenic
modulation of the arthritic process. Bland and Eddy9 pointed
out in 1968 that hemiplegia can improve arthritic conditions.
When arthritis develops after hemiplegia, there is a protec-
tive effect on the hemiplegic side. The degree of protection
is proportional to the magnitude of the paralysis, but is not
attributable to disuse.9,102,103 The cases reported include two
in which there was nearly complete reversal of the arthritic
changes after stroke on the affected side. These reports
include radiologic evidence for extensive disease only on
the patients’ unaffected side, including gross osteoporosis,
gross subluxation, typical massive rheumatoid erosions, and
absorption and loss of bone. On the paralyzed side, however,
radiology documented incomplete reversal of the disease
process, with resolution of erosions and joint space narrow-
ing. Rheumatoid nodules, evidence of more severe systemic
involvement, were confined to the unaffected side in these
patients. Four additional reports documented protection of
a single limb after a lesion of the peripheral nerve.5,104-106
The clinical reports of protection support the notion of a
neurogenic contribution to arthritis in humans.
Box 26-1
Unresolved Issues in the Field with Clinical
Relevance in Years to Come
1. Treatment with anti-inflammatory agents does not
completely resolve arthritic pain.
2. Neurogenic initiators are not addressed by current
treatment regimens.
3. Treatments with neurotransmitter receptor antago-
nists affect other neuronal systems.
4. Central neuronal sensitization is not reduced by cur-
rent medications.
5. Intracellular cascades and signal transduction are
only beginning to be defined.
 PART 3  |  effector mechanisms in autoimmunity and inflammation
Box 26-2
Future Experimental Directions
1. Role of neuronal ion channels and receptors located
on inflammatory cells
2. Interactions of neurotransmitters with non-neuronal
inflammatory mediators
3. Interactions of the sympathetic nervous system with
peripheral neurotransmitters
4. Continued efforts to resolve intracellular signaling to
abrogate neuronal initiators effectively
5. Consideration of neuronal initiators of peripheral in-
flammation
6. Consideration of neuronal receptor–mediated events
affecting joint inflammation
Acknowledgments
The authors wish to thank Jacob Sluka for help with illustrations and carol
leigh for secretarial support.
REFERENCES
  1. Lewis T: The Blood Vessels of the Human Skin and Their Responses.
London, Shaw & Sons, 1927.
  2. Levine JD, Moskowitz MA, Basbaum AI: The contribution of neu-
rogenic inflammation in experimental arthritis. J Immunol 135:843s-
847s, 1985.
  3. Levine JD, Collier DH, Basbaum AI, et al: Hypothesis—the nervous
system may contribute to the patho-physiology of rheumatoid arthri-
tis. J Rheumatol 12:406-411, 1985.
  4. Sluka KA, Willis WD, Westlund KN: The role of dorsal root reflexes
in neurogenic inflammation. Pain Forum 4:141-149, 1995.
  5. McEwen C: Heberden’s nodes: Heredity in hypertrophic arthritis of
the finger joints (a discussion). JAMA 115:2025, 1940.
  6. Nakamura K, Akizuki M, Kimura A, et al: A case of polyarthritis
developed on the non-paralytic side in a hemiplegic patient. Ryuma-
chi 34:656-661, 1994.
  7. Thomason M, Bywater E: Unilateral rheumatoid arthritis following
hemiplegia. Ann Rheum Dis 21:370-377, 1962.
  8. Velayos E, Cohen S: The effect of stroke on well established rheuma-
toid arthritis. Md State Med J 21:38-42, 1972.
  9. Bland JH, Eddy WM: Hemiplegia and rheumatoid hemiarthritis.
Arthritis Rheum 11:72-80, 1968.
10. Glyn J, Clayton M: Sparing effect of hemiplegia on tophaceous gout.
Ann Rheum Dis 35:534-535, 1976.
11. Sethis S, Sequiera W: Sparing effect of hemiplegia on scleroderma.
Ann Rheum Dis 49:999-1000, 1990.
12. Winter S: Unilateral Heberden’s nodes in a case of hemiplegia. N Y
State J Med 52:349-350, 1952.
13. Veale D, Farrell M, Fitzgerald O: Mechanism of joint sparing in a
patient with unilateral psoriatic arthritis and a longstanding hemi-
plegia. Br J Rheumatol 32:413-416, 1993.
14. Lynn B, Schutterle S, Pierau FK: The vasodilator component of neu-
rogenic inflammation is caused by a special subclass of heat-sensitive
nociceptors in the skin of the pig. J Physiol (Lond) 494:587-593,
1996.
15. Holzer P: Implications of tachykinins and calcitonin gene-related
peptide in inflammatory bowel disease. Digestion 59:269-283, 1998.
16. Kenins P: Identification of the unmyelinated sensory nerves which
evoke plasma extravasation in response to antidromic stimulation.
Neurosci Lett 25:137-141, 1981.
17. Lewin GR, Lisney SJW, Mendell LM: Neonatal anti-NGF treatment
reduces the A delta and C fiber evoked vasodilator responses in rat
skin: Evidence that nociceptor afferents mediate antidromic dilata-
tion. Eur J Neurosci 4:1213-1218, 1992.
18. Ferrell WR, Russell JW: Extravasation in the knee induced by
­antidromic stimulation of articular C fibre afferents of the anaes-
thetized cat. J Physiol 379:407-416, 1986.
417
19. Evans RH, Long SK: Primary afferent depolarization in the rat spi-
nal cord is mediated by pathways utilising NMDA and non-NMDA
receptors. Neurosci Lett 100:231-236, 1989.
20. Janig W, Lisney SJW: Small diameter myelinated afferents produce
vasodilatation but not plasma extravasation in rat skin. J Physiol
415:477-486, 1989.
21. Colpaert FC, Donnerer J, Lembeck F: Effects of capsaicin on inflam-
mation and on substance P content of nervous tissues in rats with
adjuvant arthritis. Life Sci 32:1827-1834, 1983.
22. Gamse R, Holzer P, Lembeck F: Decrease of substance P in primary
afferent neurones and impairment of neurogenic plasma extravasa-
tion by capsaicin. Br J Pharmacol 68:207-213, 1980.
23. Lam FY, Ferrell WR: Capsaicin suppresses substance P-induced joint
inflammation in the rat. Neurosci Lett 105:155-158, 1989.
24. Geppetti P, Holzer P: Neurogenic Inflammation. Boca Raton, Fla,
CRC Press, 1996.
25. Jansco N, Jansco-Gabor A, Szolcsanyi J: Direct evidence of neurogenic
inflammation and its prevention by denervation and by pretreatment
with capsaicin. Br J Pharmacol Chemother 31:138-151, 1967.
26. Gamillscheg A, Holzer P, Donnerer J, et al: Effect of neonatal treat-
ment with capsaicin on carrageenan-induced paw edema in the rat.
Naunyn-Schmiedebergs Arch Pharmacol 326:340-342, 1984.
27. White DM, Helme RD: Release of substance P from peripheral
nerve terminals following electrical stimulation of the sciatic nerve.
Brain Res 336:27-31, 1985.
28. Yaksh TL, Bailey J, Roddy DR, et al: Peripheral release of substance P
from primary afferents. In Gebhart GF, Bond MR (eds): Proceedings
from the Vth World Congress on Pain, Amsterdam, Elsevier, 1988,
pp 51-54.
29. Levine JD, Clark R, Devor M, et al: Intraneuronal substance
P contributes to the severity of experimental arthritis. Science
226:547-549, 1984.
30. Lam FY, Ferrell WR, Scott DT: Substance P-induced inflammation
in the rat knee joint is mediated by neurokinin 1 (NK1) receptors.
Regul Peptides 46:198-201, 1993.
31. Bileviciute I, Lundegerg T, Ekblom A, et al: Substance P-, neuro-
kinin A-, calcitonin gene-related peptide- and neuropeptide Y-like
immunoreactivity (-LI) in the rat knee joint synovial fluid during
acute monoarthritis is not correlated with concentrations of neuro-
peptide-LI in cerebrospinal fluid and plasma. Neurosci Lett 167:1
45-148, 1994.
32. Lembeck F, Holzer P: Substance P as a neurogenic mediator of
antidromic vasodilation and neurogenic plasma extravasation.
Naunyn-Schmiedebergs Arch Pharmacol 310:175-183, 1979.
33. Appelgren A, Applegren B, Eriksson S, et al: Neuropeptides in tem-
poromandibular joints with rheumatoid arthritis: A clinical study.
Scand J Dent Res 99:519-521, 1991.
34. Larsson J, Ekblom A, Henriksson K, et al: Immunoreactive tachykinins,
calcitonin gene-related peptide and neuropeptide Y in human synovial
fluid from inflamed knee joints. Neurosci Lett 100:326-330, 1989.
35. Larsson J, Ekblom A, Henriksson K, et al: Concentration of substance
P, neurokinin A, calcitonin gene-related peptide, neuropeptide Y and
vasoactive intestinal polypeptide in synovial fluid from knee joints
in patients suffering from rheumatoid arthritis. Scand J Rheumatol
20:326-335, 1991.
36. Birch PJ, Harrison SM, Hayes AG, et al: The non-peptide NK1
receptor antagonist, (+)-CP-96,345, produces antinociceptive
and anti-oedema effects in the rat. Br J Pharmacol 105:508-510,
1992.
37. Sluka KA, Milton MA, Westlund KN, et al: Differential roles of
neurokinin 1 and neurokinin 2 receptors in the development and
maintenance of heat hyperalgesia induced by acute inflammation. Br
J Pharmacol 120:1263-1273, 1997.
38. Mcneill DL, Chung K, Carlton SM, et al: CGRP-immunoreactive
primary afferent-fibers in dorsal and dorsolateral funiculi of the rat
spinal-cord. Anat Rec 220:A68, 1988.
39. Mao JR, Coghill RC, Kellstein DE, et al: Calcitonin gene-related
peptide enhances substance-P-induced behaviors via metabolic inhi-
bition—in vivo evidence for a new mechanism of neuromodulation.
Brain Res 574:157-163, 1992.
40. Takahashi O, Traub RJ, Ruda MA: Demonstration of calcitonin gene-
related peptide immunoreactive axons contacting dynorphin A(1-8)
immunoreactive spinal neurons in a rat model of peripheral inflam-
mation and hyperalgesia. Brain Res 475:168-172, 1988.
 418 SLUKA  |  Neurologic Regulation of Inflammation
41. Donaldson LF, Harmar AJ, Mcqueen DS, et al: Increased expression
of preprotachykinin, calcitonin gene-related peptide, but not vasoac-
tive intestinal peptide messenger RNA in dorsal root ganglia during
the development of adjuvant monoarthritis in the rat. Mol Brain Res
16:143-149, 1992.
42. Sluka KA, Westlund KN: Behavioral and immunohistochemical
changes in an experimental arthritis model in rats. Pain 55:367-377,
1993.
43. Kuraishi Y, Nanayama T, Ohno H, et al: Calcitonin gene-related
peptide increases in the dorsal-root ganglia of adjuvant arthritic rat.
Peptides 10:447-452, 1989.
44. Lam FY, Ferrell WR: Specific neurokinin receptors mediate plas-
ma extravasation in the rat knee joint. Br J Pharmacol 103:1263-
1267, 1991.
45. Lam FY, Ferrell WR: Acute inflammation in the rat knee joint
attenuates sympathetic vasoconstriction but enhances neuropeptide-
mediated vasodilation assessed by laser Doppler perfusion imaging.
Neuroscience 52:443-449, 1993.
46. Gamse R, Posch M, Saria A, et al: Several mediators appear to inter-
act in neurogenic inflammation. Acta Physiol Hung 69:343-354,
1987.
47. Brain SD, Williams TJ: Inflammatory oedema induced by ­synergism
between calcitonin gene-related peptide (CGRP) and mediators
of increased vascular permeability. Br J Pharmacol 86:855-860,
1985.
48. Kangrga I, Randic M: Tachykinins and calcitonin gene-related pep-
tide enhance release of endogenous glutamate and aspartate from the
rat spinal dorsal slice. J Neurosci 10:2026-2038, 1990.
49. Smullin DH, Skilling SR, Larson AA: Interactions between sub-
stance-P, calcitonin gene-related peptide, taurine and excitatory
amino acids in the spinal cord. Pain 42:93-101, 1990.
50. Kawana S, Liang ZF, Nagano M, et al: Role of substance P in stress-
derived degranulation of dermal mast cells in mice. J Dermatol Sci
42:47-54, 2006.
51. Battaglia G, Rustioni A: Coexistence of glutamate and substance P
in dorsal root ganglion neurons of the rat and monkey. J Comp Neu-
rol 277:302-312, 1988.
52. DeBiasi S, Rustioni A: Glutamate and substance P coexist in primary
afferent terminals in the superficial laminae of spinal cord. Proc Natl
Acad Sci U S A 85:7820-7824, 1988.
53. Jessell TM, Dodd J: Neurotransmitters and differentiation antigens
in subsets of sensory neurons projecting to the spinal dorsal horn. In
Martin B, Barchas JD (eds): Neuropeptides in Neurologic and Psy-
chiatric Disease, New York, Raven Press, 1986, pp 111-131.
54. Wiesenfeld-Hallin Z, Hokfelt T, Lundberg JM, et al: Immunoreactive
calcitonin gene-related peptide and substance P coexist in sensory
neurons to the spinal cord and interact in spinal behavioral responses
of the rat. Neurosci Lett 52:199-204, 1984.
55. Dougherty PM, Willis WD: Enhancement of spinothalamic neu-
ron responses to chemical and mechanical stimuli following com-
bined micro-iontophoretic application of N-methyl-D-aspartic acid
and substance-P. Pain 47:85-93, 1991.
56. Murase K, Ryu PD, Randic M: Excitatory and inhibitory amino acids
and peptide-induced responses in acutely isolated rat spinal dorsal
horn neurons. Neurosci. Lett 103:56-63, 1989.
57. Womack MD, MacDermott AB, Jessell TM: Sensory transmit-
ters regulate intracellular calcium in dorsal horn neurons. Nature
334:351-353, 1988.
58. Sorkin LS, Westlund KN, Sluka KA, et al: Neural changes in acute
arthritis in monkeys, IV: time-course of amino-acid release into the
lumbar dorsal horn. Brain Res Rev 17:39-50, 1992.
59. Dirig DM, Isakson PC, Yaksh TL: Spinal and systemic cyclooxy-
genase (COX) inhibitors suppress paw carrageenan-evoked thermal
hyperalgesia in rats. Anesthesiology 87:A721, 1997.
60. Mantyh PW, Demaster E, Malhotra A, et al: Receptor endocytosis
and dendrite reshaping in spinal neurons after somatosensory stimu-
lation. Science 268:1629-1632, 1995.
61. Lombardi G, Dianzani C, Miglio G, et al: Characterization of iono-
tropic glutamate receptors in human lymphocytes. Br J Pharmacol
133:936-944, 2001.
62. Boldyrev AA, Kazey VI, Leinsoo TA, et al: Rodent lymphocytes
express functionally active glutamate receptors. Biochem Biophys
Res Commun 324:133-139, 2004.
63. Westlund KN, Sun YC, Sluka KA, et al: Neural changes in acute
arthritis in monkeys, II: increased glutamate immunoreactivity in the
medial articular nerve. Brain Res Rev 17:15-27, 1992.
64. Lawand NB, Reddig WJ, Cashin AE, et al: NMDA receptors and
associated signaling pathways: A role in knee joint blood flow regula-
tion. Eur J Pharmacol 499:155-161, 2004.
65. Lawand NB, McNearney T, Westlund KN: Amino acid release into
the knee joint: Key role in nociception and inflammation. Pain
86:69-74, 2000.
66. Lawand NB, Willis WD, Westlund KN: Excitatory amino acid recep-
tor involvement in peripheral nociceptive transmission in rats. Eur J
Pharmacol 324:169-177, 1997.
67. McNearney T, Speegle D, Lawand N, et al: Excitatory amino acid
profiles of synovial fluid from patients with arthritis. J Rheumatol
27:739-745, 2000.
68. McNearney T, Baethge BA, Cao S, et al: Excitatory amino acids,
TNF-alpha, and chemokine levels in synovial fluids of patients with
active arthropathies. Clin Exp Immunol 137:621-627, 2004.
69. Guerrini L, Blasi F, Denisdonini S: Synaptic activation of Nf-kappa-B
by glutamate in cerebellar granule neurons in vitro. Proc Natl Acad
Sci U S A 92:9077-9081, 1995.
70. Barnes PJ, Karin M: Nuclear factor-kappa B: A pivotal transcription
factor in chronic inflammatory diseases. N Engl J Med 336:1066-
1071, 1997.
71. Kaltschmidt C, Kaltschmidt B, Baeuerle PA: Stimulation of iono-
tropic glutamate receptors activates transcription factor Nf-kappa-B
in primary neurons. Proc Natl Acad Sci U S A 92:9618-9622, 1995.
72. Kopp EB, Ghosh S: NF-kappa B and related proteins in innate immu-
nity. Adv Immunol 58:1-27, 1995.
73. Guerrini L, Molteni A, Wirth T, et al: Glutamate-dependent activa-
tion of NF-kappa B during mouse cerebellum development. J Neuro-
sci 17:6057-6063, 1997.
74. Willis WD, Sluka KA, Rees H, et al: Cooperative mechanisms of
neurotransmitter action in central nervous sensitization. Prog Brain
Res 110:151-166, 1996.
75. Wu J, Lin Q, Lu Y, et al: Changes in nitric oxide synthase isoforms
in the spinal cord of rat following induction of chronic arthritis. Exp
Brain Res 118:457-465, 1998.
76. Lawand NB, Willis WD, Westlund KN: Blockade of joint inflamma-
tion and secondary hyperalgesia by L-NAME, a nitric oxide synthase
inhibitor. Neuroreport 8:895-899, 1997.
77. Beck KF, Eberhardt W, Frank S, et al: Inducible NO synthase: Role
in cellular signalling. J Exp Biol 202:645-653, 1999.
78. Lewis T: Experiments relating to cutaneous hyperalgesia and its
spread through somatic nerves. Clin Sci 2:373-417, 1936.
79. Szolcsanyi J: Antidromic vasodilatation and neurogenic inflamma-
tion. Agents Actions 23:4-11, 1988.
80. Pierau FK, Szolcsanyi J: Neurogenic inflammation—axon reflex in
pigs. Agents Actions 26:231-232, 1989.
81. Bayliss SW: The Vaso-Motor System. London, Longmans, Green &
Co, 1923.
82. Gotch F, Horseley V: On the mammalian nervous system, its func-
tions and their localizations, determined by an electrical method.
Philos Trans B 182:267-326, 1891.
83. Toennies JF: Reflex discharge from the spinal cord over the dorsal
roots. J Neurophysiol 1:378-390, 1938.
84. Barron DH, Matthews BHC: The interpretation of potential changes
in the spinal cord. J Physiol 92:276-321, 1938.
85. Eccles JC, Kozak W, Magni F: Dorsal root reflexes of muscle group I
afferent fibres. J Physiol 159:128-146, 1961.
86. Sluka KA, Jordan HH, Westlund KN: Reduction in joint swelling
and hyperalgesia following post treatment with a non-NMDA gluta-
mate receptor antagonist. Pain 59:95-100, 1994.
87. Sorkin LS, Moore J, Boyle DL, et al: Regulation of peripheral inflam-
mation by spinal adenosine: Role of somatic afferent fibers. Exp Neu-
rol 184:162-168, 2003.
88. Rees H, Sluka KA, Westlund KN, et al: The role of glutamate and
GABA receptors in the generation of dorsal root reflexes by acute
arthritis in the anaesthetized rat. J Physiol 484:437-445, 1995.
89. Rees H, Sluka KA, Lu Y, et al: Dorsal root reflexes in articular affer-
ents occur bilaterally in a chronic model of arthritis in rats. J Neuro-
physiol 76:4190-4193, 1996.
90. Sluka KA, Rees H, Westlund KN, et al: Fiber types contributing
to dorsal root reflexes induced by joint inflammation in cats and
monkeys. J Neurophysiol 74:981-989, 1995.
91. Eccles RM, Willis WD: Presynaptic inhibition of the monosynaptic
reflex pathway in kittens. J Physiol 165:403-420, 1962.
92. Duchen MR: Excitation of mouse motoneruones by GABA-mediated
primary afferent depolarization. Brain Res 379:182-187, 1986.
 PART 3  |  effector mechanisms in autoimmunity and inflammation
93. Sluka KA, Westlund KN: Centrally administered non-NMDA but
not NMDA receptor antagonists block peripheral knee joint in-
flammation. Pain 55:217-225, 1993.
94. Sluka KA, Willis WD, Westlund KN: Joint inflammation and hyper-
algesia are reduced by spinal bicuculline. Neuroreport 5:109-112,
1993.
95. Boyle DL, Moore J, Yang L, et al: Spinal adenosine receptor acti-
vation inhibits inflammation and joint destruction in rat adjuvant-
induced arthritis. Arthritis Rheum 46:3076-3082, 2002.
96. Sluka KA, Lawand NB, Westlund KN: Joint inflammation is reduced
by dorsal rhizotomy and not by sympathectomy or spinal cord tran-
section. Ann Rheum Dis 53:309-314, 1994.
97. Lin Q, Zou XJ, Fang L, et al: Sympathetic modulation of acute cuta-
neous flare induced by intradermal injection of capsaicin in anesthe-
tized rats. J Neurophysiol 89:853-861, 2003.
98. Lin Q, Zou X, Ren Y, et al: Involvement of peripheral sympathetic
modulation neuropeptide Y receptors in of acute cutaneous flare
induced by intradermal capsaicin. Neurol Sci 123:337-347, 2004.
99. Heppelmann B, Messlinger K, Neiss WF, et al: Fine sensory innerva-
tion of the knee joint capsule by group III and group IV nerve fibers
in the cat. J Comp Neurol 351:415-428, 1995.
419
100. Lam FY, Ferrell WR: Neurogenic component of different models
of acute inflammation in the rat knee joint. Ann Rheum Dis 50:
747-751, 1991.
101. Levine JD, Dardick SJ, Roizen MF, et al: Contribution of sensory
afferents and sympathetic efferents to joint injury in experimental
arthritis. J Neurosci 6:3423-3429, 1986.
102. Thompson M, Bywaters EG: Unilateral rheumatoid arthritis follow-
ing hemiplegia. Ann Rheum Dis 21:370-377, 1962.
103. Velayos EE, Cohen BS: The effect of stroke on well-established rheu-
matoid arthritis. Md State Med J 21:38-42, 1972.
104. Hench PS: Heberden’s nodes: Heredity in hypertrophic arthritis of
the finger joints (a discussion). JAMA 115:2050, 1940.
105. Stecher RM, Karnosh LJ: Heberden’s nodes, IV: The effect of nerve
injury upon formation of degenerative joint disease of the fingers.
Am J Med Sci 213:181-189, 1947.
106. Kamermann JS: Protective effects of traumatic lesions on rheumatoid
arthritis. Ann Rheum Dis 25:361-373, 1966.
 27
KEY POINTS
Patients with rheumatoid arthritis (RA) are at an elevated
­(approximately 1.5-fold to 2-fold) risk for vascular events.
Patients with RA tend to experience less angina, but may
experience more sudden deaths and unrecognized
myocardial infarctions.
The excess vascular risk is driven principally by systemic
inflammation via direct and indirect mechanisms.
Current evidence suggests disease-modifying antirheumatic
drugs and biologics lessen vascular risk, whereas chronic
high-dose steroids may increase vascular risk.
All patients with RA would benefit from cardiovascular ­
disease (CVD) risk factor screening and using existing risk
scores.
Patients at high absolute CVD risk, estimated at 1.5 times
Framingham risk score or equivalent in those with longer
duration or more severe disease, would benefit from statin
therapy.
Other CVD risk factors, such as hypertension and smoking,
should be managed aggressively.
Rheumatologists may benefit from wider training on methods
of CVD risk assessment.
The excess risk of vascular disease in the rheumatic
diseases has been known for many decades,1 but at the
clinical level, coronary heart disease (CHD) or global
cardiovascular disease (CVD) risk assessment and related
preventive treatment have been less well characterized.
Increasing awareness in the 1990s of inflammation as a
novel player in the origin of CVD in the general (non-
rheumatic diseases) population2 has stimulated resurgent
interest in potential mechanisms underpinning excess
CVD risk in rheumatic diseases. This chapter summarizes
more recent evidence in this field, including discussions
on epidemiology, pathogenesis, and cardiovascular risk
screening. Extrapolating from work in the CVD arena,
we discuss how best to incorporate findings into clinical
practice to improve management of CVD risk in the rheu-
matic diseases. The focus is mainly on rheumatoid arthri-
tis (RA) because this is the rheumatic disease for which
most data are available. Increased risk in systemic lupus
erythematosus (SLE) also is considered.
Atherosclerosis
in Rheumatoid Arthritis
Naveed Sattar  •  Iain B. McInnes
CARDIOVASCULAR RISK
IN RHEUMATOID ARTHRITIS
EPIDEMIOLOGY
Extent of Coronary Heart Disease Risk
in Rheumatoid Arthritis
Van Doornum and colleagues3 summarized evidence from
21 observational studies published before and including the
year 2000 that examined CHD risk levels in RA. These
investigators noted 17 of the 21 studies show an increased
standardized morality ratio in RA, and that life expectancy
is shortened by 3 to 18 years. Pooled analysis of these studies
suggests a 70% increased risk of death in RA patients. Most
such studies examined RA patients with duration of disease of
10 years or longer. More recent studies have corroborated
and extended earlier evidence.
Solomon and associates4 compared incidence rates of
myocardial infarction and stroke in subjects with and with-
out RA among the 114,342 women in the Nurses’ Health
Study. Multivariate pooled logistic regression was used to
adjust for potential cardiovascular risk factors. During
2.4 million person-years of follow-up, 527 incident cases of
RA and 3622 myocardial infarctions and strokes were con-
firmed. The adjusted relative risk of myocardial infarction
in women with RA compared with women without RA was
2.0 (95% confidence interval [CI] 1.23 to 3.29). Women
who had RA for at least 10 years had a relative risk for
myocardial infarction of 3.10 (95% CI 1.64 to 5.87). The
end point data were prospectively gathered and validated
with record acquisition. In addition, the risk for myocardial
infarction was hardly attenuated after adjustment for most
traditional risk factors, although high-density lipoprotein
(HDL) cholesterol was not included as a potential con-
founder. Future epidemiologic studies should address this
deficiency because low HDL cholesterol is closely linked to
excess CVD risk generally and is one of the most consistent
findings in RA patients. Similarly, analysis of the relevant
data from the United Kingdom General Practice Research
Database (>2 million patients) confirms elevated CHD risk
in RA patients.3
Finally, investigators in Malmo5 reported a standard-
ized mortality ratio of 176 for myocardial infarction in RA
patients compared with the general population. Relevant
evidence from all studies suggests CHD risk is equally
elevated in men and women with RA, increases with dis-
ease severity and evidence of extra-articular disease, and
increases with disease duration.3,4
There is consistent evidence of higher CVD risk in RA
patients. It is difficult to give a precise estimate of the level
of this excess risk, but it seems to be about 1.5-fold to 2-fold
421
 422 sattar  |  Atherosclerosis in Rheumatoid Arthritis
higher. This risk is lower than the excess risk of CVD seen in
patients with type 2 diabetes, although previous comparisons
on the CVD risk in the two conditions have been made.
When Does Risk Begin and How Does It Manifest?
More recent analyses from the Minnesota residents’ cohort
have suggested that the excess CVD risk began early. In the
2 years before fulfillment of the American College of Rheu-
matology criteria, RA patients were about three times more
likely to be hospitalized for acute myocardial infarction, and
nearly six times more likely to have experienced an unrec-
ognized myocardial infarction compared with individuals
without RA.6 This excess risk did not seem to be accounted
for by traditional CHD risk factors, although adjustments
did not take account of continuous measures, but used cat­
egorical variables,6 so residual confounding is possible. In
the study by Maradit-Kremers and coworkers,6 in addition
to a higher risk of unrecognized myocardial infarction,
­sudden death was more likely in RA patients. Perhaps in
keeping with these findings, RA patients were less likely to
have a history of angina and, perhaps related to this, had
lower rates of coronary artery bypass grafting.
An analysis of all cases of first cardiovascular event in
Victoria, Australia, revealed a higher 30-day case-fatality
rate (1.6; 95% CI 1.2 to 2.2) in patients with RA compared
with patients without RA.7 This excess risk was mostly
accounted for by excess death after myocardial infarction,
although CIs were large because of small numbers of events.
Nevertheless, consistent with this finding, Solomon and
colleagues8 also noted a higher 30-day mortality rate (1.89;
95% CI 1.56 to 2.30) in RA patients after a CVD event
in their examination of relevant data from British Colum-
bia. In the latter study, if RA patients survived through this
period, their subsequent vascular risk was not elevated com-
pared with patients without RA.8 These observations merit
further investigation.
Summary of Vascular Risk in Rheumatoid Arthritis
from Epidemiologic Findings
From available epidemiologic data, the following broad con-
clusions can be made:
• CHD risk is greater in patients with RA by approxi-
mately 1.5-fold to 2-fold (i.e., relative risk). This excess
risk does not seem to be accounted for by most tradi-
tional risk factors, although more studies are needed to
clarify this.
• Absolute risk of a vascular event in RA must include
use of traditional risk factor algorithms, which include
age, sex, blood pressure, smoking, and lipids. When
determined, risk could be multiplied by 1.5 in RA
­patients with modest-to-severe disease severity and
longer duration (i.e., >5 years).
• An elevated CHD risk in RA may begin early in the
course of the disease, but on balance, relative risk
seems greater with greater evidence of systemic disease
and longer duration of RA.
• The excess vascular risk manifests differently in RA
patients compared with patients without RA, with
less angina, but more sudden deaths and unrecognized
myocardial infarctions.
• Early 30-day case-fatality after a cardiovascular event
seems to be greater in RA patients versus patients
without RA.
These findings make it clear that RA patients not only
have higher cardiovascular risk, but also they are less likely
to be symptomatic and more likely to die before getting to
the hospital if they have a myocardial infarction. Even if
RA patients with a myocardial infarction survive until they
get to the hospital, they are more likely to die in the first
30 days after myocardial infarction. These findings make
arguments for routine CHD risk screening in patients with
RA more compelling.
PATHOGENESIS
Inflammation and Atherogenesis
in the General Population
To understand fully the excess CHD risk in RA, one
first must appreciate the link between inflammation and
CHD or CVD risk in the general population. Interest in
understanding CHD risk in RA has been stimulated by rec-
ognition of the inflammatory basis for cardiovascular dis-
ease in general. Plaque composition of unstable coronary
lesions includes an abundance of inflammatory moieties
and immune cells at the shoulder region, with erosion of
the collagen cap that separates the atheromatous material
of the plaque from the lumen.2 This appearance is strik-
ingly similar to the phenotype of inflammatory synovitis
in RA.9
Although elevated systemic markers of inflammation,
albeit at considerably lower levels than those apparent
in RA, independently predict CHD events in the general
population, the level of independent prediction afforded
by, for example, C-reactive protein (CRP), is debated.10
There also remain uncertainties about causality of the
“low-grade” inflammation in the atherogenic process. Some
investigators argue that the inflammatory features within
an evolving plaque may largely be consequent to entry of
atherogenic low-density lipoprotein (LDL) species into
the vessel wall, which sets in motion a chain of molecular
events leading to macrophage recruitment (and associated
molecular and cellular features), and subsequent foam cell
formation.11 Elevated systemic inflammatory markers may
arise from diseased blood vessels—so-called reverse causa-
tion.
Alternatively, elevated (low-grade) systemic inflamma-
tion levels may stem from multiple lifestyle factors that can
increase CHD risk by mechanisms other than inflamma-
tion (Fig. 27-1). Adiposity especially,12 but also smoking,
poor diet, low physical activity, and social deprivation are
linked to elevated detectable inflammation in the general
population, manifest by systemic CRP estimation, but such
factors also are linked to many other atherogenic pathways.
Adiposity explains about 20% of the systemic inflammatory
burden in population studies, and more recent evidence
indicates obesity per se leads to excess and likely detrimen-
tal macrophage recruitment into adipose tissue.13 Further
studies are required to disentangle the link between “low-
grade” inflammation and cardiovascular disease, including
genetic studies and clinical trials determining the vascular
effects of specific anti-inflammatory agents.
 PART 3  |  effector mechanisms in autoimmunity and inflammation 423

A
Low-grade inflammation
CRP 0-6 mg/L, on average 1-3 mg/L
Adiposity
Diet
Genes Risk factor pathways,
Low physical activity e.g.,lipids, insulin resistance, CHD
Smoking hemostatic abnormalities
Deprivation

B
Statins Accelerated
atherogenesis

Atherogenic lipid pool

High-grade systemic
inflammation
RA
CRP often far in
synovitis
excess of 6 mg/L,
typically 10-50 mg/L
DMARD or Anti-inflammatory therapy
Insulin resistance
Atherogenic effects Hemostatic abnormalities CHD
on numerous risk Oxidative stress
factor pathways Endothelial progenitor
cell effects
Elevations in homocysteine
Myocardial
Endothelial dysfunction
microvascular
abnormalities
Independent
of narrowed
coronary vessels

Figure 27-1  Comparing and contrasting links between low-grade versus high-grade inflammation and coronary heart disease (CHD). A, Low-grade
i­nflammation. The complex potential links between low grade inflammation and CHD are shown. Although low-grade inflammation, stemming from
a combination of lifestyle and genetic factors, may be causally related to the pathogenesis of CHD either directly or indirectly via its influence on other
risk factors, it is possible that low-grade inflammation is a noncontributory correlate in the risk factor–CHD relationship, or a consequence rather than a
cause of blood vessel disease. B, High-grade systemic inflammation. Systemic inflammation levels in individuals with rheumatoid arthritis (RA) are gen-
erally far greater than in individuals without RA, well above levels attributed to lifestyle factors (e.g., obesity, smoking) or diseased blood vessels. Rather,
the driver to systemic acute-phase reactants in RA is predominantly synovial inflammation. Resultant concentrations of circulating cytokines alter the
function of distant tissues, including adipose, skeletal muscle, liver, and vascular endothelium, to generate a spectrum of proatherogenic changes that
include an atherogenic lipid pattern, insulin resistance, pro-oxidative and procoagulative effects, and endothelial dysfunction and damage. Myocardial
microvascular effects may contribute to the burden of CHD in RA independent of accelerated atherogenesis, but this requires further investigation. This
model predicts CHD risk reduction with inflammatory suppression, and evidence for this (see text) is emerging. Because most RA patients continue to
exhibit significant systemic inflammation despite potent therapy, however, consideration of established CHD risk reduction strategies in RA patients at
elevated risk would seem appropriate. Statins have been shown to reduce lipid levels in RA markedly and may offer a modest disease-modifying effect.
CRP, C-reactive protein; DMARD, disease-modifying antirheumatic drug. (Modified from Sattar N, McInnes IB: Vascular comorbidity in rheumatoid arthritis:
Potential mechanisms and solutions. Curr Opin Rheumatol 17:286-292, 2005.)

Inflammation and Atherogenesis Typical profile in RA patient

in Rheumatoid Arthritis
In contrast to the complexities of linking low-grade inflam-
mation and CHD pathogenesis, the inflammation-CHD
link in RA is, paradoxically, easier to establish (see Fig.
27-1).14 Systemic inflammation levels in patients with RA
are often far greater than in individuals without RA, well
above levels attributed to lifestyle factors (e.g., obesity,
smoking) or diseased blood vessels (Fig. 27-2). The major
driver explaining elevation in systemic acute-phase reac-
tants in RA is predominantly synovial inflammation and
subsequent cross-talk with the liver via cytokine release. On
the basis of several lines of evidence, we15 and others have
argued that such “high-grade” inflammation is likely pivotal
to the accelerated CHD in RA. Several prior studies and
more recent studies are described to support this hypothesis
(Table 27-1):
• CHD risk in RA is related to number of inflamed
joints3 and seems to be greatest in RA patients with
extra-articular disease or patients in tertiary referral
centers.
• Conventional risk factors do not account for excess
CHD risk in RA patients. In an analysis from the
Nurses’ Health Study, although inflammatory markers
[CRP]
50 mg/L
Uper end of ‘normal’ range
6 mg/L
High-sensitivity range: CHD risk increases
by∼50% in general population with CRP
rising from <1 mg/L to >3 mg/L
Time
Figure 27-2  The magnitude and chronicity of systemic inflam-
mation in individuals with rheumatoid arthritis (RA) are particularly
deleterious, such that, even during quiescent phases of the disease,
systemic levels of cytokines remain high relative to individuals with-
out RA and, as such, may continue to promote vascular risk. CHD,
coronary heart disease; CRP, C-reactive protein. (Modified from Sattar
N, McCarey DW, Capell H, et al: Explaining how “high-grade” systemic
inflammation accelerates vascular risk in rheumatoid arthritis. Circula-
tion 108:2957-2963, 2003.)
 424 sattar  |  Atherosclerosis in Rheumatoid Arthritis

Table 27-1  Risk Factor Alterations in Rheumatoid Arthritis including Associations with the Inflammatory  
Response in Cross-Sectional Studies, and Documented Improvements on Inflammatory Suppression*
Strength of Evidence Linking
Strength of Evidence Metabolic Perturbances Evidence for Improvement in
Coronary Heart Disease for Abnormality of Risk to Inflammatory Response Risk Factor on Inflammatory
Risk Factors Factor in Rheumatoid Arthritis in Rheumatoid Arthritis Suppression
Obesity/Insulin
Body fat redistribution + ND Steroids and SSZ may enhance
Hyperinsulinemia + + insulin sensitivity in RA; TNF
Insulin resistance ++ + blockade improves
↓ SHBG (surrogate marker) + ++
Dyslipidemia
FFAs + + Anti-inflammatory treatment
Triglyceride +/0 + increases HDL-cholesterol or
↓ HDL-cholesterol ++ ++ apolipoprotein AI; reduction
↓ HDL2 ++ + in Lp(a) lipoprotein noted with
Small, dense LDL + + TNF-α blockade
Lp(a) lipoprotein ++ +
Endothelial
sICAM-1 ++ + FMD/PWV improve with  
vWF + + anti-TNF-α therapy; sICAM
Microalbuminuria + + declines with SSZ
Impaired vasoreactivity ++ +
Arterial stiffness ++ +
Oxidative Stress
MDA + + Not examined, but ibuprofen  
↑ antioxidant levels in cancer
subjects
↓ Vitamin antioxidants + +
Hemostatic Alterations
Fibrinogen ++ ++
PAI-1 + 0 TNF blockade reduces fibrinogen
Blood pressure + ND No data
Homocysteine Pathway
↓ Vitamin B6 ++ + Steroids and TNF blockade can ↓
homocysteine­
Homocysteine ++ +
*Most data stem from small, uncontrolled studies; 0 = lack of, or conflicting, evidence; + = moderate supporting evidence; ++ = consistent evidence; ND = no data.
FFAs, free fatty acids; FMD, flow-mediated dilation; HDL, high-density lipoprotein; LDL, low-density lipoprotein; MDA, malondialdehyde; PAI-1, plasminogen-
activator inhibitor-1; PWV, pulse wave velocity; RA, rheumatoid arthritis; SHBG, sex hormone binding globulin; sICAM, soluble intracellular adhesion molecule;
SSZ, sulfasalazine; TNF, tumor necrosis factor; vWF, von Willebrand factor.
Modified from Sattar N, McCarey DW, Capell H, et al: Explaining how “high-grade” systemic inflammation accelerates vascular risk in rheumatoid arthritis.
Circulation 108:2957-2963, 2003.

(including CRP and intercellular adhesion molecule-1)
were substantially elevated in women with RA com-
pared with women without RA, most traditional CHD
risk factors were similar.16 In addition, this group and
others4,17 have shown more recently that adjustment
for most traditional risk factors minimally attenuates
the RA to non-RA difference in CHD event rates, al-
though such adjustments have not always been compre-
hensive.
• Carotid artery intima-media thickness (IMT) (and
plaque), a U.S. Food and Drug Administration–­approved
surrogate marker of vascular disease, was elevated in RA
patients in association with elevated markers of inflamma-
tion.18 More significantly, arterial thickening in ­women
with RA, as measured by change in carotid IMT over
18 to 36 months, was accelerated compared with healthy
controls, and the increase in IMT was related indepen-
dently to serum CRP concentration, but not conven-
tional risk factors.19 More recently, carotid IMT in RA
patients has been shown to be related to disease duration
so that patients with prolonged RA had more atheroscle-
rosis than patients with more recent disease onset. The
findings parallel our prior suggestion that ­atherosclerosis
accelerates during the course of RA (Fig. 27-3).
• Endothelial dysfunction has been suggested as an
early event in the atherogenic process and as a novel
predictor of CHD events; more recent longitudinal
studies in the CHD arena provide some support for
this proposition.20 Several studies employing various
­“direct” ­ measures of vascular function, such as pulse
wave analysis,21 flow-mediated vasodilation,22 and ve-
nous occlusion plethysmography,23 confirm endotheli-
al dysfunction in RA patients. Where examined, such
dysfunction has been linked to systemic inflammatory
markers. Vaudo and associates24 have provided more
recent evidence for endothelial dysfunction in young
to middle-aged patients with low disease activity (dis-
ease activity score [DAS] ≤3.2) and noted a strong
 PART 3  |  effector mechanisms in autoimmunity and inflammation
Accelerated atherogenesis in RA
(plus other immunologic conditions)
Slope or risk escalation a severity
Slope largely of systemic disease
Vascular risk
dependent on
aggregation of
conventional
risk factors
Additional major source
of systemic cytokines
from inflamed joints
Onset of RA
Time
Figure 27-3  Before onset of rheumatoid arthritis (RA), classic risk fac-
tors predict risk of an event. After onset of disease, the severity of the
systemic inflammation secondary to RA joint disease is critical in the
­acceleration of atherogenesis, and risk factors influenced by systemic
inflammation predict risk of future events. (Modified from Sattar N, McCa-
rey DW, Capell H, et al: Explaining how “high-grade” systemic inflammation
accelerates vascular risk in rheumatoid arthritis. Circulation 108:2957-2963,
2003.)
a­ ssociation to average CRP levels. This finding agrees
with the earlier observation of an early excess risk of
vascular events in RA patients. Elevated LDL choles-
terol also was an independent correlate to impaired
vascular function in RA patients in the latter study.
Other more recent studies also show impaired vessel
stiffness with the technique of pulse wave velocity,25
and we have shown impaired forearm microvascular
function using laser Doppler imaging in patients with
RA in parallel with a greater systemic inflammatory
response.26
• More recent longitudinal studies report improved
endothelial function after anti–tumor necrosis factor
(TNF)-α therapy,27,28 but this benefit seems to be tran-
sient and linked to the pattern of change in systemic in-
flammatory markers on TNF blockade. Treatment with
disease-modifying antirheumatic drugs (DMARDs)
also improves endothelial function in patients with
RA,23 so the mechanism employed to achieve inflam-
matory control may not be crucial to improvement in
endothelial function.
• Numerous other risk factors beyond endothelial dys-
function can be directly and adversely influenced by
the systemic inflammatory response in RA (see Fig.
27-1 and Table 27-1).15 The lipid profile, despite
showing lower LDL cholesterol, is more atherogenic
(e.g., low HDL cholesterol, greater preponderance of
smaller atherogenic LDL species). There also is evi-
dence of greater insulin resistance, altered body fat
distribution (peripheral wasting, central fat accu-
mulation), prothrombotic effects, and pro-oxidative
stress. In addition, some evidence suggests inflamma-
tory cytokines may elevate homocysteine concentra-
tions in RA. Anti-inflammatory treatment can at-
tenuate many of these changes, and in the context
of a randomized placebo-controlled trial, we showed
that a TNF blocking agent can reduce circulating
concentrations of homocysteine, Lp(a) lipoprotein,
and fibrinogen, and increase apolipoprotein A-I (the
425
main ­ apolipoprotein in HDL particles, ­ associated
with antiatherogenic actions) and sex hormone bind-
ing globulin, the last-mentioned used as a surrogate
marker of insulin sensitivity.29 For balance, in the lat-
ter study, increases in triglyceride and apolipoprotein
B were unexpected and require further investigation
because elevated triglyceride and apolipoprotein B
are associated with an increase in CVD risk.29 Pre-
dictions of the likely vascular end point effects on
this TNF blocking agent based solely on a panel of
biochemical parameters would seem to be difficult to
make.
• Cytokines released from inflamed synovial membrane
can exert metabolic effects via effects on distant tissues,
including adipose tissues, skeletal muscle, liver, and
vascular endothelium. One consequence of this func-
tional pleiotropy is that the intensity of the metabolic
adaptation could parallel other cytokine effects. Cyto-
kine-induced metabolic effects, which include transient
alterations in lipids and peripheral insulin resistance,
are favorable in the short-term and function as part of
the host response to infection and acute inflammation
to target specific metabolic fuels to and from essential
organs. Chronic elevation in cytokine levels, regard-
less of magnitude or cause, is deleterious, however, and
promotes accelerated atherogenesis via aggravation of
several risk factor pathways as described earlier. From
a developmental standpoint, cytokines or cytokine-like
molecules, such as interleukin-630 or leptin,31 may have
evolved to impart their systemic metabolic effects at
very low levels or in rapid transient bursts. Even minor
degrees of chronic elevation are damaging. As a result,
as argued more recently,15 the magnitude and the chro-
nicity of systemic inflammation in RA may be particu-
larly deleterious. Even during “quiescent” phases of the
disease, systemic levels of cytokines or their regulatory
components often remain dysregulated relative to pa-
tients without RA and, as such, may continue to pro-
mote vascular disease.15
• There are numerous model data to support a proath-
erogenic role for cytokines.32,33 A study in apolipopro-
tein E knockout mice showed reduced atherosclerosis
via inhibition of TNF-α.34
• Endothelial progenitor cells (EPCs) represent a pop-
ulation of bone marrow–derived cells that have the
capacity to make a significant contribution to new
blood vessels. Higher concentrations seem to be
protective, whereas lower concentrations have been
correlated with higher cardiovascular risk in patients
without RA. Grisar and associates35 reported lower
EPC concentrations in active RA and noted an in-
verse correlation between EPC quantity and disease
activity score. In line with a link between inflamma-
tion and EPCs, a single dose of infliximab has been
shown to improve the number and functional proper-
ties of EPCs.36
The systemic inflammatory response in RA seems to
be associated with a largely reversible spectrum of proath-
erogenic changes in other risk factor pathways. From such
observations, it seems that CVD risk in RA must be linked
partly to disease activity, and, as discussed previously, there
is plentiful evidence supporting this potential. CVD risk in
 426 sattar  |  Atherosclerosis in Rheumatoid Arthritis
RA patients is likely to decline with better and sustained
suppression of disease activity in RA patients. The mode
of inflammation suppression is relevant, and some anti-
­inflammatory drugs might have direct vascular damaging
properties that overwhelm vascular benefits arising indi-
rectly from their inflammatory suppression.
Inflammation-Mediated Microvascular
Dysfunction as a Cause of Myocardial
Ischemia in Rheumatoid Arthritis
Much of the more recent evidence in the general CHD area
suggests that patients with exercise-proven myocardial isch-
emia do not show obstructed coronary vessels on angiography.
Rather, as in women with cardiac syndrome X, myocardial
ischemia may manifest from dysfunctional endothelium in
coronary vessels or in the myocardial microvasculature, which
under conditions of stress vasoconstrict to cause symptoms.
We suggested that independent of obstructive coronary dis-
ease, insulin resistance could be a major contributor to myo-
cardial ischemia via endothelial dysfunction.37 We proposed
insulin sensitization as a novel mechanism to lessen anginal
symptoms in subjects with and without diabetes. Such work
is now ongoing in formal clinical trials.
Consistent with this concept, Raza and colleagues38
reported complete reversal of significant myocardial isch-
emia (proven by thallium scanning) in a 62-year-old man
with RA on intensive immunosuppression. Coronary angio­
graphy showed no significant atheroma. This group argued
that systemic inflammation–driven myocardial microvas-
cular abnormalities may be as important to the patho-
genesis of ischemic heart disease in RA as atherosclerotic
narrowing.38
Future studies will be needed to develop these important
findings, but two additional factors merit comment. First,
data from noninvasive measurement of endothelial func-
tion and carotid atheroma burden in patients with coronary
artery disease suggest that structural and functional status
of the vasculature are independent predictors of coronary
events.39 Second, conventional coronary angiography can
miss unstable lesions: Arterial wall remodeling permits
accumulation of a large atherosclerotic burden before there
is any detectable narrowing of the vessel lumen by con-
ventional angiography.40 Novel imaging modalities, such
as intravascular ultrasound, may allow better detection of
these “silent” plaques.40
LESSENING VASCULAR RISK IN RHEUMATOID
ARTHRITIS VIA INFLAMMATORY SUPPRESSION
Methotrexate
The foregoing arguments suggest that absolute and long-
term suppression of the systemic inflammatory response in
RA should lessen CHD risk by improving risk factors. The
balance of evidence favors this likelihood, including more
recent epidemiologic findings. In an 18-year follow-up of
1240 patients with RA, Choi and colleagues41 reported that
methotrexate treatment, generally considered to be the most
effective “nonbiologic” DMARD, reduced overall mortality
by 60% (95% CI 20% to 80%) primarily by reducing CHD
mortality by 70% (95% CI 30% to 80%). Non-CHD mor-
tality was not significantly altered.
Disease-Modifying Antirheumatic Drugs
Evidence suggests that DMARD use is associated with
a reduction in acute myocardial infarction risk of about
20% in patients with RA.42 Myocardial infarction risk in
this case-control study was reduced with all traditional
DMARDs and, consistent with the Choi results,41 also seen
with methotrexate use.
Steroids
It is unclear whether steroids increase or decrease risk or
have a neutral effect. Davis and coworkers43 found no inde-
pendent associations (adjusting for other cardiovascular
risk factors and disease activity) between cumulative glu-
cocorticoid exposure and CVD risk in RA patients followed
for 15 years. By contrast, two nested case-control studies
suggested an approximate 30% to 50% higher myocardial
infarction risk with steroid use.42,44 In line with an excess
risk, del Rincon and associates45 reported a higher incidence
of carotid plaque and arterial stiffness in patients with RA
on steroids. On balance, steroids may be one class of drugs
where the vascular beneficial effects of inflammatory sup-
pression are reversed by direct adverse effects on the vas-
culature.
Biologics
Given the mixed pattern of biochemical changes seen with
TNF blocking agents on risk factors, it is difficult to predict
vascular effects of these agents, even though TNF block-
ing agents consistently improve vascular function, albeit in
a transient fashion. An observational study from Sweden
compared CVD event rates in RA patients on anti-TNF
therapy versus RA patients not on such therapy.46 The
findings suggested an age-adjusted and sex-adjusted 54%
decreased risk (95% CI 15% to 75%) of CVD event in
patients on anti-TNF therapy. Solomon and colleagues44
have since reported a similar effect of biologics compared
with methotrexate on CVD risk, which if we accept that
the latter reduces CVD risk, is consistent with data from
Sweden. Further robust data are needed to address this
important issue, with appropriate attention to potential
confounders because epidemiologic findings can give rise
to incorrect conclusions (e.g., as in the case of hormone
replacement therapy and CVD risk). TNF-α blockade
may lead to other toxicities,47 including potential harm in
patients with heart failure.48
Future Studies Examining Links between Rheumatoid
Arthritis Treatments and Cardiovascular Disease Risk
Robust epidemiologic studies and larger controlled trials are
needed to expand the evidence base regarding RA therapies
and CHD risk. The latter should include, wherever possible,
a wide range of risk factors and established vascular measures.
The use of carotid IMT measurement in trials is to be encour-
aged, and, in the near future, magnetic resonance imaging
(MRI) and other novel imaging methods (e.g., intravascu-
lar ultrasound or novel noninvasive ­computed tomography
scanning) may offer new ways to advance knowledge in the
RA-CHD field. Because of multiple confounding factors
 PART 3  |  effector mechanisms in autoimmunity and inflammation
and an ethical need to limit disease activity, study design
is generally very complex in the RA arena, however. Only
short-term mechanistic and surrogate vascular marker stud-
ies with anti-inflammatory and disease-­modifying agents are
likely to be feasible.
Statins to Lessen Coronary Heart Disease Risk
in Rheumatoid Arthritis
Because complete and absolute long-term suppression of
systemic inflammation is rarely achieved in RA, even with
the advent of more potent therapies, vascular risk will con-
tinue to be elevated. As a result, there is merit in consider-
ing other proven measures to lessen CHD or CVD risk in
RA.
3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibi-
tors (statins) reduce CVD morbidity and mortality by approxi-
mately 25% to 50% and, on the basis of an excellent evidence
base, are widely used in primary and secondary prevention
of vascular disease.49 Although operating predominantly
through reduction in LDL cholesterol concentration, more
recent studies suggest broader properties for statins, particu-
larly in altering inflammatory pathways.50 Statins are poten-
tially ideal drugs to target CVD risk in disease states (e.g., RA)
associated with high-grade inflammation, and early evidence
is supportive in this respect.
In a placebo-controlled study of atorvastatin (40 mg/
day) in RA patients, we noted a significant reduction in
LDL cholesterol and triglyceride (45% and 18%), disease
activity score (disease activity score 28 [DAS28]; 10%),
acute-phase parameters (CRP [50%] and erythrocyte
sedimentation rate [28%]), and the swollen joint count
(decrease of 2.16 joints relative to placebo) in patients
presenting with active RA despite existing DMARD treat-
ment.51 Although the magnitude of change in disease activ-
ity with atorvastatin was modest, this study provided the
first clinical indication that pathways targeted by statins
may be useful in the treatment of inflammatory disease. In
parallel with the atorvastatin-induced reduction in CRP,
plasma viscosity, fibrinogen, and interleukin-6 concentra-
tions also were reduced relative to placebo. We observed
no significant liver function or muscle abnormalities with
atorvastatin,51 and the statin safety record in general is
excellent. The long-term implication of this study may lie,
however, more in showing the potential for attenuation
of inflammation and vascular risk parameters by statins,
rather than in providing a novel DMARD in available
statin agents.
Larger studies are required to corroborate the statin-
induced effects on RA disease activity and safety; our orig-
inal positive report in murine collagen-induced arthritis52
was not confirmed by other investigators,53 although posi-
tive effects for statins have been reported across a range
of murine autoimmune models. Nevertheless, in terms of
vascular risk modification, the lipid-lowering effect noted
by us51 was in keeping with the magnitude of changes
seen in the non-RA population.43 This finding is impor-
tant because, despite a tendency of statins to reduce LDL
cholesterol in RA, the overall lipid pool in RA is highly
atherogenic, and extrapolation from data from all statin
end point trials suggests that the extent of LDL choles-
terol reduction may account for most of the statin ­clinical
427
­benefit.54 Statin-induced reductions in CVD risk occur
even when starting LDL cholesterol values are considered
“average” or “low,” and optimal LDL cholesterol levels
may be 1.3 to 1.8 mmol/L.54
Van Doornum and colleagues55 confirmed that statins
substantially improve RA lipids in a smaller uncontrolled
study, which also reported statin-induced reduction in arte-
rial stiffness, previously shown to be elevated in RA. End
point studies with statins in RA would be ideal, but in the
interim phase, statin effects on progression of carotid IMT
would be informative.
Finally, existing data indicate that lipids enter and are
present in considerable quantity in diseased RA synovium.
More recent studies suggest oxidized LDL may promote
articular damage therein.56 If a statin-mediated reduction
in RA disease activity is proven, statin-induced reduc-
tion in the systemic lipid load, rather than or in addition
to direct anti-inflammatory effects, should be considered
as potentially responsible. This theory requires future
study.
ASSESSING CARDIOVASCULAR DISEASE RISK
IN RHEUMATOID ARTHRITIS
Who to Screen
The collective findings of higher CHD or CVD risk in RA
patients that begins early, with greater propensity to sud-
den death, unrecognized myocardial infarctions, and less
angina, plus greater 30-day case-fatality post-CVD, provide
a compelling reason to consider universal CVD risk factor
screening in RA patients. Given that absolute risk is low in
young individuals even with other significant risk factors,
however, it may be sensible to restrict universal screening
for RA patients to individuals older than 40 years of age.
CVD risk screening in younger subjects may be appropriate
in the face of several other risk factors or a strong family his-
tory of premature vascular disease.
How Best to Screen Presently
Generally, the following parameters are used to assess
risk in the primary prevention setting: age, sex, smoking,
systolic blood pressure, and cholesterol-to-HDL choles-
terol ratio. Diabetes is now no longer included in many
primary prevention charts because patients with type 2
diabetes who are older than 40 years of age are now con-
sidered at sufficiently high risk to warrant statin therapy.57
All that is required in RA patients in addition to readily
available clinical data (age, sex, smoking history) is the
­measurement of systolic blood pressure and cholesterol
and HDL cholesterol concentrations in serum or plasma.
With many laboratories now offering direct HDL choles-
terol in nonfasting samples (cholesterol and HDL choles-
terol levels are not appreciably altered whether fasting or
not), such assessments can be made as part of routine RA
assessment, regardless of the time of the day or whether
patients are seen in a physician’s office or in a hospital.
The collection of such data permits calculation of CHD
risk based on existing charts.
There may be some merit in considering obtaining an
electrocardiogram in some patients with RA to screen for
 428 sattar  |  Atherosclerosis in Rheumatoid Arthritis
prior silent myocardial infarctions, but relevant ­studies assess-
ing the detection rate for existing disease using electrocar-
diograms—never mind the economics of this approach—or
more detailed cardiac investigations, are lacking. Such stud-
ies would be useful, however.
Should CVD Risk Calculations Be Multiplied to
Account for Unmeasured Factors in Rheumatoid
Arthritis Patients?
One could make an argument to multiply risk based on
available CVD risk charts because most studies suggest that
the excess CVD risk in RA patients is not accounted for by
traditional risk factors. Only a few prospective RA studies
have measured traditional CHD risk factors in RA patients,
however, and even when measured, some important risk fac-
tors have been omitted (most commonly HDL cholesterol).
Statistical adjustments generally have used only categorical
data (e.g., adjusting for hypercholesterolemia), rather than
continuous measurements, leading to loss of power to detect
true confounding. There is a need for further prospective
studies to assess the extent to which comprehensively mea-
sured traditional risk factors attenuate or otherwise affect
the excess CHD risk in RA patients compared with patients
without RA.
We recommend adopting a conservative position with
regards to assessing CVD risk in RA. Although evidence
presented earlier4,5 suggests on balance 50% to 100% higher
CHD event rates in RA patients compared with patients
without RA (and perhaps higher in patients with more pro-
longed disease), rather than applying a multiplications factor
of 2 to current CVD charts when assessing CVD risk in RA,
it may be more judicious to use a conservative value of 1.5.
If traditional risk factors suggest a 10-year global CVD risk
of 10% in an individual who has had RA for several years
(>5 years), we suggest the risk could be nearer 15%. Cur-
rently in the United Kingdom, statin therapy is targeted to
patients without existing vascular disease if the global CVD
risk level is calculated to be 20% over 10 years (i.e., one in
five chance of any vascular event over the next 10 years).57
Other countries employ related algorithms for detecting
individuals at high CVD risk and are able to adopt a simi-
lar approach. Widespread CVD risk screening in RA and
multiplication of the derived absolute risk, perhaps applied
to a subgroup of RA patients with more severe disease and
longer duration, are important. These issues require further
discussion by rheumatologists and cardiologists.
A conservative multiplication factor of 1.5 rather
than 2 also is sensible because statins are not without
side effects, although these are often reversible on treat-
ment cessation, and serious side effects (e.g., rhabdomy-
olysis) are rare. More data on statin safety in RA patients
would be useful, together with a specific end point trial
in RA patients. One such trial is ongoing in the United
Kingdom and is expected to test the efficacy and safety
of atorvastatin in more than 3000 RA patients. Even in
advance of the results of this trial, the clear message is
that CVD risk screening in RA patients should become
the norm, rather than the exception. It may be useful for
RA specialists to familiarize themselves with CVD risk
screening and related issues, perhaps by attending rel-
evant courses.
Secondary Prevention—Risk Charts Not Required
Patients with RA who have prevalent vascular disease
(whether CHD, stroke, or peripheral vascular disease)
should be strongly considered for statin therapy regardless
of risk factor measures because they fulfill secondary pre-
vention categorization. There also is plentiful evidence to
indicate that risk reduction would materialize independent
of baseline cholesterol level in this case or in the context of
primary prevention.57
Cardiovascular Disease Risk Screening Case Example
Absolute risk of an event, rather than simply a high cho-
lesterol level, dictates treatment.57 Consider a 45-year-old
man with RA with modestly high cholesterol (6.2 mmol/L),
who is a nonsmoker, is normotensive (blood pressure 130/75
mm Hg), and has an average HDL cholesterol (1.3 mmol/
L). These results give a 10-year CVD risk of less than 10%.
Even if this man had long-standing or severe RA, and we
multiplied his risk by 1.5 (on basis of the balance of epide-
miology), his absolute CVD risk would still be well below
the 20% cutoff, and statin therapy would not be warranted.
If the man were 10 year older, had significant hypertension,
or were a smoker, his risk would increase considerably.
Cardiovascular risk calculations need to become common-
place in a rheumatology clinic and in RA patients seen in
primary care. The above-described case example also shows
the need to be comprehensive in addressing CVD risk; CVD
risk factors should be measured and treated aggressively on
the basis of conventional guidelines.57 Finally, in line with
CVD risk screening guidelines in general,57 clinical judg-
ment in CVD risk determination in RA has an important
role. CVD risk calculation in a 50-year-old woman with
mild RA of recent onset perhaps should not be multiplied
by 1.5. Many gray-area cases are to be expected, and each
requires careful consideration.
Surrogate Noninvasive Measures of Vascular Disease
We have previously discussed several noninvasive assess-
ment methods—tests of vascular function (e.g., flow-
mediated vasodilation, pulse wave velocity, laser Doppler
imaging) or tests of subclinical atherosclerosis (e.g., IMT or
coronary artery calcification). There is now excellent and
reasonably consistent evidence of perturbed vascular func-
tion, however measured, and in different vascular beds in
RA patients in parallel with heightened systemic inflamma-
tory levels. There is equally good evidence to show improve-
ment in vascular function with inflammatory suppression in
RA patients. No such measures are in routine clinical use
for risk stratification in the non-RA population, however;
part of the reason is lack of sufficient prospective data, but
equally the variability of techniques does not make stan-
dardization easy. Such techniques remain firmly within the
research domain.
Measurement of carotid IMT is not used for general
CVD risk screening, although, in recognized centers with
robust methodology, it is accepted as a validated measure
to assess efficacy (or otherwise) of novel CVD risk modali-
ties. It also is unclear whether coronary artery calcifica-
tion techniques and carotid IMT are sufficiently robust to
 PART 3  |  effector mechanisms in autoimmunity and inflammation
gauge the extent of excess atherosclerosis risk in RA as
presence of plaques; rather, IMT may be more relevant
to RA vascular events and more informative. Further rel-
evant studies are required. It also is possible that plaque
composition rather than plaque volume may be most rel-
evant in RA, but such assessments are in their infancy and
require more detailed and complex methodologies, such
as cardiac/carotid MRI or positron emission tomography
scanning techniques.
VASCULAR RISK IN OTHER RHEUMATIC
CONDITIONS
Vascular risk is enhanced in other rheumatic conditions,
such as SLE and psoriatic arthritis. Myocardial infarction
risk is increased in SLE—an observation unexplained by
traditional risk factors.58 In addition, compared with healthy
controls, SLE patients exhibit greater carotid atherosclero-
sis, as determined by the presence of carotid plaques, at any
given age.59 In line with factors associated with risk in RA,
independent predictors of plaque in SLE in the latter study
included a longer duration of disease and a higher dam-
age index score.59 Coronary artery calcification scores also
are elevated in patients with SLE.60 Similarly, myocardial
infarction risk has been shown to be significantly elevated
in patients with psoriasis, with greater relative risks associ-
ated with greater disease severity.61
Although a detailed description of risk in other condi-
tions is beyond the scope of this chapter, the principal
mechanisms operating in RA seem to share common fea-
tures across the immunologic diseases. Distinct pathways
could operate particularly in SLE, however, in which altered
underlying signaling and inflammatory cascades that sub-
serve immune-mediated tissue damage also could directly
influence events within the vessel wall. Future intervention
studies are under way in SLE. The outcome of these studies
will be vital in determining how such clinical risk should
be managed beyond the aggressive treatment of underlying
autoimmune disease.
CONCLUSION
Evidence for elevated CHD or CVD risk in RA is abun-
dant and convincing—best estimates indicate that
individuals who have had RA for several years have
approximately a 1.5-fold to 2-fold higher risk for CVD
compared with individuals without RA independent of
most traditional risk factors. This excess CVD risk seems
to be driven mainly by systemic inflammation directly
by its deleterious effects on blood vessels and indirectly
by its accentuation on multiple risk pathways, including
lipids, insulin metabolism, clotting, and oxidative param-
eters. A contribution from existing antirheumatic thera-
pies, in particular, steroids, also may be implicated, but
this requires further direct study. Established therapies
that lessen disease activity and dampen systemic inflam-
mation likely reduce CVD risk, although there remains
scope for larger, robust studies. Emerging evidence indi-
cates that statins may have dual effects in RA with a
modest disease-modifying effect (requiring confirmation)
and a significant lipid-lowering effect, equivalent to the
magnitude of lipid reduction in patients without RA. The
429
latter finding is particularly important because extrapola-
tion of data from all statin end point trials suggests that
the extent of LDL cholesterol reduction accounts for most
statin clinical benefit.
It seems sensible to consider statin therapy in RA
patients with existing vascular disease and to assess CVD
risk in RA patients without prevalent CVD. The latter
would involve minimal additional tests (blood pressure
and nonfasting lipids in most cases) and use of available
risk factor charts. Because most RA patients continue to
exhibit significant systemic inflammation despite potent
therapy, it seems sensible to multiply risk levels derived
from such charts by a factor of around 1.5 to derive the
likely level of risk in RA patients. This conservative
estimate is a balance between excess risk and the lack
of comprehensive prospective CVD studies in RA that
have measured and adjusted properly for the full range
of traditional risk factors. The CHD risk level, and not
the cholesterol level (unless markedly elevated), dictates
whether statin treatment is applicable.
Finally, there is considerable interest in determining
whether novel risk markers or noninvasive vascular tests
can help predict RA patients at greatest subsequent risk.
This work is currently in its infancy, with only limited pro-
spective data linking such measures to future cardiovascular
events in the general population and no such data in RA
patients. Such methods are not proven for risk stratification.
The current focus in clinical practice in RA and vascular
risk should remain on established risk factor algorithms
(e.g., employing smoking, lipids, blood pressure) together
with a multiplying factor in selected patients to determine
absolute CHD risk.
Future Directions
1. Larger comprehensive epidemiologic studies are needed
to determine better the extent to which CVD risk is
or is not explained by traditional risk factors. This goal
would be best achieved by collaboration between several
centers or countries to establish a large cohort with uni-
form baseline phenotyping and ascertainment of future
events. Such a study also could assess whether markers of
current disease severity or disease duration help improve
risk factor stratification beyond traditional CVD risk fac-
tors (i.e., help discriminate individual patients with RA
who will, or will not, experience an incident vascular
event).
2. More detailed noninvasive atherosclerosis assessment
in RA patients is needed using the best current meth-
ods to determine to what extent plaque burden or
plaque composition, or both, is different from patients
without RA and may explain higher CVD risk.
3. Assessment, wherever possible, is needed of the effects
of all new RA therapies on a comprehensive panel of
vascular risk measures.
4. End point statin study is needed to ascertain efficacy
with respect to CVD events and disease severity and
safety in RA patients.
5. More work is needed on body composition changes
and metabolic changes in relation to systemic disease
severity in RA patients.
 430 sattar  |  Atherosclerosis in Rheumatoid Arthritis
REFERENCES
  1. Cobb S, Anderson F, Bauer W: Length of life and cause of death in
rheumatoid arthritis. N Engl J Med 249:553-556, 1953.
  2. Ross R: Atherosclerosis—an inflammatory disease. N Engl J Med
340:115-126, 1999.
  3. Van Doornum S, McColl G, Wicks IP: Accelerated atherosclerosis:
An extraarticular feature of rheumatoid arthritis? Arthritis Rheum
46:862-873, 2002.
  4. Solomon DH, Karlson EW, Rimm EB, et al: Cardiovascular mor-
bidity and mortality in women diagnosed with rheumatoid arthritis.
Circulation 107:1303-1307, 2003.
  5. Turesson C, Jarenros A, Jacobsson L: Increased incidence of cardio-
vascular disease in patients with rheumatoid arthritis: Results from a
community based study. Ann Rheum Dis 63:952-955, 2004.
  6. Maradit-Kremers H, Crowson CS, Nicola PJ, et al: Increased
unrecognized coronary heart disease and sudden deaths in rheuma-
toid arthritis: A population-based cohort study. Arthritis Rheum
52:402-411, 2005.
  7. Van Doornum S, Brand C, King B, et al: Increased case fatality rates
following a first acute cardiovascular event in patients with rheuma-
toid arthritis. Arthritis Rheum 54:2061-2068, 2006.
  8. Solomon DH, Goodson NJ, Katz JN, et al: Patterns of cardiovascular
risk in rheumatoid arthritis. Ann Rheum Dis 65:1608-1612, 2006.
  9. Pasceri V, Yeh ET: A tale of two diseases: Atherosclerosis and rheuma-
toid arthritis. Circulation 100:2124-2126, 1999.
10. Danesh J, Wheeler JG, Hirschfield GM, et al: C-reactive protein and
other circulating markers of inflammation in the prediction of coro-
nary heart disease. N Engl J Med 350:1387-1397, 2004.
11. Palinski W, Napoli C: Unraveling pleiotropic effects of statins on
plaque rupture. Arterioscler Thromb Vasc Biol 22:1745-1750, 2002.
12. Wisse BE: The inflammatory syndrome: The role of adipose tissue
cytokines in metabolic disorders linked to obesity. J Am Soc Nephrol
15:2792-2800, 2004.
13. Wellen KE, Hotamisligil GS: Obesity-induced inflammatory changes
in adipose tissue. J Clin Invest 112:1785-1788, 2003.
14. Sattar N, McInnes IB: Vascular comorbidity in rheumatoid arthri-
tis: Potential mechanisms and solutions. Curr Opin Rheumatol 17:
286-292, 2005.
15. Sattar N, McCarey DW, Capell H, et al: Explaining how “high-
grade” systemic inflammation accelerates vascular risk in rheuma-
toid arthritis. Circulation 108:2957-2963, 2003.
16. Solomon DH, Curhan GC, Rimm EB, et al: Cardiovascular risk factors
in women with and without rheumatoid arthritis. Arthritis Rheum
50:3444-3449, 2004.
17. del Rincon ID, Williams K, Stern MP, et al: High incidence of car-
diovascular events in a rheumatoid arthritis cohort not explained
by traditional cardiac risk factors. Arthritis Rheum 44:2737-2745,
2001.
18. del Rincon I, Williams K, Stern MP, et al: Association between
carotid atherosclerosis and markers of inflammation in rheumatoid
arthritis patients and healthy subjects. Arthritis Rheum 48:1833-
1840, 2003.
19. Nagata-Sakurai M, Inaba M, Goto H, et al: Inflammation and bone
resorption as independent factors of accelerated arterial wall thick-
ening in patients with rheumatoid arthritis. Arthritis Rheum 48:
3061-3067, 2003.
20. Widlansky ME, Gokce N, Keaney JF Jr, et al: The clinical implications
of endothelial dysfunction. J Am Coll Cardiol 42:1149-1160, 2003.
21. Wong M, Toh L, Wilson A, et al: Reduced arterial elasticity in rheu-
matoid arthritis and the relationship to vascular disease risk factors
and inflammation. Arthritis Rheum 48:81-89, 2003.
22. Gonzalez-Juanatey C, Testa A, Garcia-Castelo A, et al: HLA-DRB1
status affects endothelial function in treated patients with rheumatoid
arthritis. Am J Med 114:647-652, 2003.
23. Bergholm R, Leirisalo-Repo M, Vehkavaara S, et al: Impaired respon-
siveness to NO in newly diagnosed patients with rheumatoid arthritis.
Arterioscler Thromb Vasc Biol 22:1637-1641, 2002.
24. Vaudo G, Marchesi S, Gerli R, et al: Endothelial dysfunction in young
patients with rheumatoid arthritis and low disease activity. Ann
Rheum Dis 63:31-35, 2004.
25. Maki-Petaja KM, Hall FC, Booth AD, et al: Rheumatoid arthri-
tis is associated with increased aortic pulse-wave velocity, which is
reduced by anti-tumor necrosis factor-alpha therapy. Circulation 114:
1185-1192, 2006.
26. Datta D, Ferrell WR, Sturrock RD, et al: Inflammatory suppression
rapidly attenuates microvascular dysfunction in rheumatoid arthritis.
Atherosclerosis 192:391-395, 2006.
27. Gonzalez-Juanatey C, Testa A, Garcia-Castelo A, et al: Active
but transient improvement of endothelial function in rheumatoid
arthritis patients undergoing long-term treatment with anti-tumor
necrosis factor alpha antibody. Arthritis Rheum 51:447-450,
2004.
28. Hurlimann D, Forster A, Noll G, et al: Anti-tumor necrosis fac-
tor-alpha treatment improves endothelial function in patients with
rheumatoid arthritis. Circulation 106:2184-2187, 2002.
29. Sattar N, Crompton P, Cherry L, et al: Effects of TNF blockade
on circulating cardiovascular risk parameters in psoriatic arthritis:
Double-blind placebo-controlled study. Arthritis Rheum, 56: 831-
839, 2007.
30. Ershler WB, Keller ET: Age-associated increased interleukin-6 gene
expression, late-life diseases, and frailty. Annu Rev Med 51:245-270,
2000.
31. Banks WA, Phillips-Conroy JE, Jolly CJ, et al: Serum leptin levels
in wild and captive populations of baboons (papio): Implications for
the ancestral role of leptin. J Clin Endocrinol Metab 86:4315-4320,
2001.
32. Kirii H, Niwa T, Yamada Y, et al: Lack of interleukin-1beta decreases
the severity of atherosclerosis in ApoE-deficient mice. Arterioscler
Thromb Vasc Biol 23:656-660, 2003.
33. Whitman SC, Ravisankar P, Daugherty A: Interleukin-18 enhances
atherosclerosis in apolipoprotein E(−/−) mice through release of inter-
feron-gamma. Circ Res 90:E34-E38, 2002.
34. Branen L, Hovgaard L, Nitulescu M, et al: Inhibition of tumor necro-
sis factor-alpha reduces atherosclerosis in apolipoprotein E knockout
mice. Arterioscler Thromb Vasc Biol 24:2137-2142, 2004.
35. Grisar J, Aletaha D, Steiner CW, et al: Depletion of endothelial
progenitor cells in the peripheral blood of patients with rheumatoid
arthritis. Circulation 111:204-211, 2005.
36. Ablin JN, Boguslavski V, Aloush V, et al: Effect of anti-TNFalpha
treatment on circulating endothelial progenitor cells (EPCs) in rheu-
matoid arthritis. Life Sci 79:2364-2369, 2006.
37. Jadhav S, Petrie J, Ferrell W, et al: Insulin resistance as a contribu-
tor to myocardial ischemia independent of obstructive coronary
atheroma: A role for insulin sensitisation? Heart 90:1379-1383,
2004.
38. Raza K, Banks M, Kitas GD: Reversing myocardial microvascular dis-
ease in a patient with rheumatoid arthritis. J Rheumatol 32:754-756,
2005.
39. Chan SY, Mancini GB, Kuramoto L, et al: The prognostic importance
of endothelial dysfunction and carotid atheroma burden in patients
with coronary artery disease. J Am Coll Cardiol 42:1037-1043, 2003.
40. Schoenhagen P, White RD, Nissen SE, et al: Coronary imaging: Angi-
ography shows the stenosis, but IVUS, CT, and MRI show the plaque.
Cleve Clin J Med 70:713-719, 2003.
41. Choi HK, Hernan MA, Seeger JD, et al: Methotrexate and mor-
tality in patients with rheumatoid arthritis: A prospective study.
Lancet 359:1173-1177, 2002.
42. Suissa S, Bernatsky S, Hudson M: Antirheumatic drug use and the risk
of acute myocardial infarction. Arthritis Rheum 55:531-536, 2006.
43. Davis JM 3rd, Maradit-Kremers H, Gabriel SE: Use of low-dose glu-
cocorticoids and the risk of cardiovascular morbidity and mortality in
rheumatoid arthritis: What is the true direction of effect? J Rheumatol
32:1856-1862, 2005.
44. Solomon DH, Avorn J, Katz JN, et al: Immunosuppressive medications
and hospitalization for cardiovascular events in patients with rheuma-
toid arthritis. Arthritis Rheum 54:3790-3798, 2006.
45. del Rincon I, O’Leary DH, Haas RW, et al: Effect of glucocorticoids on
the arteries in rheumatoid arthritis. Arthritis Rheum 50:3813-3822,
2004.
46. Jacobsson LT, Turesson C, Gulfe A, et al: Treatment with tumor
necrosis factor blockers is associated with a lower incidence of first
cardiovascular events in patients with rheumatoid arthritis. J Rheu-
matol 32:1213-1218, 2005.
47. Ferraccioli G, Gremese E: Thrombogenicity of TNF alpha in rheuma-
toid arthritis defined through biological probes: TNF alpha blockers.
Autoimmun Rev 3:261-266, 2004.
48. Wolfe F, Michaud K: Heart failure in rheumatoid arthritis: Rates, pre-
dictors, and the effect of anti-tumor necrosis factor therapy. Am J Med
116:305-311, 2004.
 PART 3  |  effector mechanisms in autoimmunity and inflammation
49. Gaw A, Packard CJ: At what level of coronary heart disease risk should
a statin be prescribed? Curr Opin Lipidol 11:363-367, 2000.
50. McInnes IB, McCarey DW, Sattar N: Do statins offer therapeutic
potential in inflammatory arthritis? Ann Rheum Dis 63:1535-1537,
2004.
51. McCarey DW, McInnes IB, Madhok R, et al: Trial of Atorvastatin
in Rheumatoid Arthritis (TARA): Double-blind, randomised pla-
cebo-controlled trial. Lancet 363:2015-2021, 2004.
52. Leung BP, Sattar N, Crilly A, et al: A novel anti-inflammatory role for
simvastatin in inflammatory arthritis. J Immunol 170:1524-1530, 2003.
53. Palmer G, Chobaz V, Talabot-Ayer D, et al: Assessment of the effi-
cacy of different statins in murine collagen-induced arthritis. Arthritis
Rheum 50:4051-4059, 2004.
54. O’Keefe JH Jr, Cordain L, Harris WH, et al: Optimal low-density lipo-
protein is 50 to 70 mg/dl: Lower is better and physiologically normal.
J Am Coll Cardiol 43:2142-2146, 2004.
55. Van Doornum S, McColl G, Wicks IP: Atorvastatin reduces arte-
rial stiffness in patients with rheumatoid arthritis. Ann Rheum Dis
63:1571-1575, 2004.
431
56. Kakinuma T, Yasuda T, Nakagawa T, et al: Lectin-like oxidized low-
density lipoprotein receptor 1 mediates matrix metalloproteinase 3
synthesis enhanced by oxidized low-density lipoprotein in rheumatoid
arthritis cartilage. Arthritis Rheum 50:3495-3503, 2004.
57. JBS 2: Joint British Societies’ guidelines on prevention of cardio-
vascular disease in clinical practice. Heart 91(Suppl 5):v1-v52,
2005.
58. Manzi S, Meilahn EN, Rairie JE, et al: Age-specific incidence rates of
myocardial infarction and angina in women with systemic lupus erythema-
tosus: Comparison with the Framingham Study. Am J Epidemiol 145:408-
415, 1997.
59. Roman MJ, Shanker BA, Davis A, et al: Prevalence and correlates
of accelerated atherosclerosis in systemic lupus erythematosus.
N Engl J Med 349:2399-2406, 2003.
60. Asanuma Y, Oeser A, Shintani AK, et al: Premature coronary-
artery atherosclerosis in systemic lupus erythematosus. N Engl J Med
349:2407-2415, 2003.
61. Gelfand JM, Neimann AL, Shin DB, et al: Risk of myocardial infarc-
tion in patients with psoriasis. JAMA 296:1735-1741, 2006.
 Part broad issues in the approach
4 to rheumatic diseases
28
KEY POINTS
Epidemiology is the study of the distribution of disease and
its determinants in populations. Epidemiologic methods can
be used to describe the frequency or development of disease
and to determine underlying causes.
Prevalence is the frequency of disease in the population at
a given time, including existing and new cases. ­Incidence
­measures the development of disease over time in a
­population initially free from the disease.
The odds ratio compares the odds of disease in an exposed
population with that in a population without the exposure
or risk factor under study. The relative risk is the risk of
development of disease over time in an exposed population
­compared with an unexposed population. In many instances,
the odds ratio can estimate the relative risk.
Threats to the validity of a study include chance, systematic bias,
and confounding. Confounding occurs when an extraneous
factor, related to the exposure of interest and the disease, but
not part of the causal pathway between exposure and disease, is
superimposed on the true risk factor/disease relationship.
Case-control studies examine exposures in a population that
already has the disease under study and compares them with
exposures in an otherwise comparable population without
the disease. This study design may be subject to recall bias, in
which subjects with a disease report exposure to risk factors
differently than subjects without the disease, but it may be
the design of choice for rare diseases.
Cohort studies follow groups of individuals with and without
an exposure of interest for the development of disease over
time. Because the exposure assessment precedes the disease,
temporality can help determine causation.
Controlled clinical trials most closely resemble formal
­experiments in which the exposure is manipulated by the
­investigator, and the response of disease is compared
­between groups that receive the active intervention and
groups that receive a placebo or other comparator.
Principles
of Epidemiology
in Rheumatic Disease
Joanne M. Jordan
OVERVIEW OF EPIDEMIOLOGIC METHODS
Epidemiology is the study of the distribution of disease and
its determinants in populations.1 The purpose of epidemiol-
ogy is to describe the frequency of disease and to determine
causes responsible for variation in disease occurrence. Com-
parison of the relative strengths of the causes and assessment
of their generalizability can allow “truth” to be inferred. This
chapter explains basic epidemiologic concepts and defini-
tions; describes the major study designs, their strengths and
weaknesses, and their usefulness in inferring causality; and
shows specific applications of these principles to the study
of rheumatic diseases. For the purposes of this chapter, the
term disease is used to represent a disease, death, or other
health outcome of interest, and the term exposure is used to
represent a risk or protective factor examined for its associa-
tion with disease.
MEASURES OF DISEASE OCCURRENCE
Prevalence
Prevalence refers to the frequency of a disease in a population
at any given time. It is measured at one point in time and is
the proportion of individuals with a disease out of the total
population under study. The numerator makes no distinction
between new and established cases of disease. Multiple esti-
mates of prevalence over time are commonly used to deter-
mine trends in disease occurrence or need for health services.
Incidence
To determine the likelihood that disease will develop over
time, repeated observations of the same people are required
to determine who develops disease and who does not. Inci-
dence proportion is the frequency of new cases over a speci-
fied time in a group of people at risk for, but without, the
disease at baseline. During the observation period, a person
may develop the disease in question, die from competing
risks, or be lost to follow-up. All of these situations result in
that individual’s no longer contributing time at risk to the
denominator. The concept of person-time allows the inclu-
sion of the actual time at risk contributed by each individual.
433
 434 JORDAN  |  Principles of Epidemiology in Rheumatic Disease
Year
0 1 2 3 4 5
Subject A I +
Subject B I X
Subject C I LFU
I 4.0
Subject D
¶ X
Subject E
Total years at risk
Figure 28-1  Hypothetical calculation of person-time at risk for a study of the incidence of systemic lupus erythematosus over 10 years. ¶, beginning
of observation period; +, died; X, developed disease; LFU, lost to follow-up. Adapted from Hennekins CH, Buring JE: Epidemiology in Medicine. Boston,
Little, Brown, 1987.
Consider the hypothetical example of incidence of systemic
lupus erythematosus over a 10-year period (Fig. 28-1). Per-
son A may not develop the disease during the observation
period, but may die at year 5 of a competing cause; this per-
son contributes 5-person years to the denominator. Person
B might develop systemic lupus erythematosus 4 years after
the study begins and is no longer at risk of developing the
disease; this person contributes 4 person-years of time at risk
to the denominator. Person C may join the study at year 2,
but be lost to follow-up at year 4, contributing 2 years of
time at risk. Incidence rate is defined by the following:2
New cases developing over
time of observatioon
Incidence rate =
Person-time at risk for each individual
without the diseease at study entry
MEASURES OF EFFECT
More important than just the description of frequency of
disease or its development is the relationship between the
disease and exposures to potentially causative factors. One
way to examine this relationship is to compare the preva-
lence or incidence of disease in groups with a given exposure
compared with groups without that exposure. It is crucial to
the ability to assess potential causality of an exposure/disease
association that the exposed and unexposed groups be com-
parable. Measures to delineate this relationship between dis-
ease occurrence and exposure vary according to study design.
Cross-sectional surveys and case-control studies use the odds
ratio, which is a measure of the odds of disease in the exposed
group compared with the unexposed group. Longitudinal
designs can calculate a relative risk of development of dis-
ease in the exposed compared with the unexposed groups.
STUDY DESIGNS
Study designs include ecologic studies, cross-sectional sur-
veys, case-control studies, cohort studies, and randomized
controlled clinical trials—the last frequently considered the
most rigorous study design and the one most closely repre-
senting a formal experiment. Each study design has its own
Total time
6 7 8 9 10 at risk
5.0
4.0
2.0
LFU
2.5
17.5
inherent strengths and weaknesses (Table 28-1), and the
choice of study design depends on the research question, the
rarity of the disease under study, the availability of appro-
priate study and comparable control populations, resources
available to conduct the study, and logistics.2,3
ECOLOGIC STUDIES
In the ecologic study design, the unit of observation is a
group, rather than an individual.4 Aggregate data on rates of
disease and risk factors are compared to examine associations
between disease frequencies and exposures. The ecologic
study is frequently a design of expediency and can generate
hypotheses for more rigorous testing in studies using indi-
vidual-level data.3 One of the chief drawbacks is its high
susceptibility to confounding. Confounding occurs when an
extraneous factor, not on the causal pathway, masks the true
relationship between exposure and disease, by virtue of its
association with both.5 Associations in the aggregate may
not hold for the individual.3 This concept is termed the eco-
logic fallacy. As a hypothetical example, rates of specific kinds
of cancers may be higher in countries in which cigarette sales
also are high. Whether individuals who are buying, and pre-
sumably smoking, the cigarettes are the same individuals
who develop cancer is unknown from this study design.
CROSS-SECTIONAL SURVEYS
The goal of the cross-sectional survey study design is
usually descriptive, including all individuals, with and
without the disease under study, in the population, or a rep-
resentative sample of them, at one point in time with no
follow-up period. Surveys can estimate prevalence of a par-
ticular ­ disease in the population and determine need for
health services and resource allocation.3 Typically, informa-
tion about risk factors is obtained simultaneously. Such risk
factor data may or may not represent the most relevant time
of exposure, and it cannot be determined whether the expo-
sure preceded or resulted from the disease.2
An example of a cross-sectional survey, conducted approxi-
mately once per decade in the United States, is the National
Health and Nutrition Examination Survey. This survey samples
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES 435

Table 28-1  Common Epidemiologic Study Designs and their Strengths and Weaknesses*
Measure
Study Design Definition of Effect Strengths Weaknesses
Ecologic Aggregate data on exposures   Odds ratio Inexpensive Susceptibility to confounding
and disease; unit of analysis is a Short duration Ecologic fallacy
group, not an individual Hypothesis-generating

Cross-sectional   Data on exposures and disease   Prevalence Can study several   May not be able to determine
survey obtained at one time from all Odds ratio outcomes ­whether disease preceded
individuals in an area (or a sample Short duration ­exposure
thereof) with and without disease Can generate population   Potential survivor bias
­prevalence estimates of disease Not practical for rare diseases
and risk factor ­distributions
Cannot produce incidence or ­relative
risk estimates
Case-control Study of exposure/disease relation- Odds ratio Best for studying rare   Inefficient for rare exposures
ship in cases with a disease and conditions or conditions  
­controls without the disease, who with long latency
are selected from source popula-
tion from which the cases arose
Short duration Potential bias from sampling cases  
and controls separately
Small sample* May not be able to determine whether
exposure preceded disease
Inexpensive* Potential recall bias
Odds ratio can   Potential survivor bias
approximate relative risk
Cannot produce prevalence or inci-
dence estimates
Cohort Individuals without disease are fol- Incidence Can determine   Frequently requires large samples
lowed over time to determine   sequence of events
which characteristics predict who
will get the disease and who will not
Relative   Less susceptibility to survivor bias   Not feasible for rare outcomes
risk and bias in measuring predictors
Can study multiple outcomes More expensive
Can generate population   Long duration
incidence, relative risk
Prospective Study sample selected by   Incidence Investigator control over   Increased expense
investigator and followed forward   selection of participants  
in time for development of disease and measures
Relative risk Long duration
Retrospective Study sample and measurement of ­  Incidence Less expense Less control over selection of  
exposures and disease over time   participants and measures
have already occurred
Relative risk Short duration
Nested   Case-control study within the   Incidence Underlying cohort design May require bank of samples that can
case-­control context of a prospective or   be assayed at a later date until or
retrospective cohort after outcomes occur
Relative   Relatively inexpensive  
risk compared with  
measurement of entire  
cohort
Randomized Exposure (pharmaceutical, nonphar- Relative   Most closely emulates   Costly in time and money
clinical   macologic device, educational risk an experiment
trial ­intervention) manipulated by  
investigator
Hazard   Strongest design to produce   Some research questions unsuitable
ratio evidence for cause and effect because of rare disease or ethical
barriers
Random assignment of interven- May not be generalizable if highly con-
tion minimizes confounding trolled environment does not reflect
“real world” common practice
May be faster and cheaper for   May have narrow scope and study
some study questions than   question
observational studies
*Relative to cohort study design.
Adapted from Hennekins CH, Buring JE: Epidemiology in Medicine. Boston, Little, Brown, 1987; and Hulley SB, Cummings SR: Designing Clinical Research:
An Epidemiologic Approach. Baltimore, Williams & Wilkins, 1988.
 436 JORDAN  |  Principles of Epidemiology in Rheumatic Disease
a proportion of the residents in the contiguous 48 states and
measures various health outcomes and habits, such as blood
pressure, serum lipids, height, weight, smoking, and dietary
intake. These surveys have been used in rheumatology to deter-
mine prevalence of radiographic knee and hip osteoarthritis in
various age, sex, and race/ethnicity subgroups.6
CASE-CONTROL STUDIES
Much maligned by the uninitiated because of its susceptibil-
ity to bias, the case-control study can be the study design of
choice—or sometimes the only appropriate study design—in
certain situations, particularly when the disease under study
is rare. Usually, a case-control study includes fewer indi-
viduals—at much lower cost and higher efficiency—than
would be required for a cohort study because it begins with
­individuals who already have the disease in question, rather
than waiting for a small proportion of a large cohort to
develop the disease over time. Most important in the design
of a case-control study are (1) the choice of the control
group, which must be comparable to the cases, and (2) rec-
ognition of potential biases that may threaten validity.
Strictly defined, the case-control study is a study in which
subjects with the disease (cases) are compared with a control
population without the disease, drawn from the same source
population from which the cases arose.1,5 The source population
may be the residents of a particular geographic area or a hos-
pital’s referral base. The control group serves as an estimate
of the distribution of the exposure in the source population,
and consequently, the control group must be sampled inde-
pendently of exposure status.1,5 If one is interested in examin-
ing the possible association between smoking and progressive
systemic sclerosis, the controls must be from the same source
population that generated the cases, if this can be determined,
and must be sampled without regard to their smoking status.
Selection of Controls for Case-Control Study
If the source of the cases is a well-defined population, the
controls can be sampled directly from that population. If the
source population is too large to allow a complete ­enumeration,
controls may be matched to each case by their residence in
the same neighborhood. Random-digit dialing can be used to
select controls, but this labor-intensive method omits from
selection individuals without telephones and individuals who
cannot be reached.1 If the cases are drawn from a particu-
lar hospital or clinic, the source population should represent
people who would be treated in that hospital or clinic if they
developed the disease under study, but frequently, this source
population can be difficult to identify and is influenced by
referral practices.1 Hospital or clinic controls can be used, but
this method can have particular pitfalls because the controls
might not be selected independently of the exposure in the
source population. In a hospital-based study of smoking in sys-
temic lupus erythematosus, individuals hospitalized for other
diseases, such as myocardial infarction or pneumonia, might
have exposures different from the source population in gen-
eral, especially if the exposure, in this case smoking, causes or
prevents the “control” disease selected. One way to avoid this
is to exclude diseases known to be associated with the expo-
sure under study, but this may create other biases. Another
tactic could be to select hospital controls with diseases that
are believed to be unrelated to the disease or exposures under
study, such as traumatic leg fractures,1 or to use several con-
trol groups selected differently.3 The latter example might
sample controls from hospitalized patients with other diseases
than the disease under study, nonhospitalized patients in the
same medical care system, or nonhospitalized individuals in
the general population, comparing each control group sepa-
rately with the diseased group.
Weaknesses of the Case-Control Design
It is impossible to derive incidence or prevalence estimates
from a case-control study. The greatest threat to validity is
the inherent susceptibility to bias that can exist in this study
design because the cases and controls are sampled separately,
and the assessment of exposure variables is retrospective.3
Matching the cases and controls on factors such as age, sex,
or race/ethnicity, can help ensure comparability of cases and
controls to a degree. As mentioned previously, more than one
control group, selected in different ways, can be used to see
if findings are consistent across control groups with different
sampling biases. A nested case-control design, in which a
case-control study is performed within a larger cohort study,
has the advantage of minimizing sampling bias because the
cases and the controls would have been sampled previously
in identical fashion into the parent cohort study.3
The other chief source of bias in the case-control study is
recall bias, which occurs when exposures predating the dis-
ease may be differentially reported by the controls and the
cases, the latter of whom may have incentive to remember
and report exposures. This bias can be partially prevented by
using exposure data measured before the disease occurred,
if available, and by blinding the observer and the subject
to the exposure under investigation, or, if possible, blinding
them even to the specific disease under study and to case or
control status. In a case-control study examining racial/eth-
nic variation as the exposure variable of interest in systemic
lupus erythematosus, race/ethnicity is immutable and so not
subject to recall bias. In contrast, if study participants know
or suspect that prior exposure to hair dye, for instance, is the
exposure of interest in the same case-control study, partici-
pants with disease may be more prone to “remember” their
exposure than might participants without disease. Investiga-
tors can obtain information about multiple potential expo-
sures or even include several “dummy” exposures to mask
the real hypothesis to try to minimize this type of bias.7
COHORT STUDIES
Cohort studies follow groups of individuals without the dis-
ease in question over time to describe the development or
incidence of disease and to compare the incidence of dis-
ease among groups with different risk factors or exposures.
Cohorts can be prospective or retrospective.1,3
Prospective Cohort Study
Prospective cohorts are characterized by the selection
of the cohort and measurement of risk factors or expo-
sures before the outcome has occurred, establishing time
sequence or temporality, an important factor in deter-
mining causality. This is a distinct advantage over the
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
case-control study, in which exposure and disease are
assessed simultaneously.
The primary disadvantage to the prospective cohort study
is its expense, in that it requires large numbers of individu-
als followed for potentially long periods. Biases can creep
in, particularly if there is significant loss to follow-up. This
study design is highly inefficient and inappropriate to study
rare diseases, but its efficiency increases as the frequency
of the disease in the population increases.3 A prospective
cohort study would be inappropriate to study progressive
systemic sclerosis because of its rarity, but excellent to study
a common condition such as osteoarthritis.8,9
Retrospective Cohort Study
In a retrospective cohort, individuals are followed over time,
but the cohort selection and collection of data have already
occurred, sometimes for a different purpose than the current
disease under study. For example, a cohort of ­individuals with
small vessel vasculitis seen at a particular hospital between
1990 and 1992 could be identified, and data abstracted regard-
ing baseline serologies, physical examination findings, and
biopsy results when the patients were first evaluated. Then,
examination of outcomes such as stroke or development of
dialysis-dependent renal disease could be ascertained in 2000,
by medical record review or by recontact with the individuals
so identified. Because exposure or risk factor assessment pre-
cedes assessment of outcome, this study design can establish
temporality, as in a prospective cohort, and is less subject to
recall bias that can plague case-control studies. By selecting
the cases and controls from the same source population, this
study design also avoids some of the selection biases of case-
control studies in which the cases and controls are sampled
separately. The retrospective cohort design is cheaper and
more efficient than a prospective cohort, but because the
data collection has already occurred, inferences from such
a study are highly dependent on the quality, completeness,
and appropriateness of the original risk factor assessments to
study their association with the disease in question.3
Nested Case-Control Studies
Nested case-control studies are case-control studies that
occur within the context of a prospective or retrospective
cohort and are particularly useful in the assessment of risk
factor variables that would be too expensive to measure
on all members of the cohort.3 In this design, members of
a cohort who have developed a particular outcome during
the observation period are selected and compared with a
sample of individuals within that same cohort who have not
developed the outcome. Then, for example, stored biologic
specimens from baseline may be assayed for an exposure of
interest, such as vitamin D level, and compared between
individuals who developed the disease and individuals who
did not.
CLINICAL TRIALS
The study designs described previously in this chapter all
were observational designs; there was no experimental
manipulation of the exposure or outcome. Experimental
study designs or interventions include clinical trials, field
437
trials, and community intervention trials.4 Inferences from
such trials of treatments assigned randomly to a large enough
sample are much less likely to have biases and other threats
to validity than are observational designs. Randomization
theoretically should eliminate most confounding, although
some variation in risk factors among the intervention groups
may occur by chance and should always be ascertained and
addressed in the analysis if necessary. The validity of conclu-
sions from a clinical trial depends partly on the avoidance of
loss to follow-up or participant dropout.
Clinical trials can be conducted for pharmacologic or
nonpharmacologic interventions, such as dietary, physical
activity, assistive devices, or educational interventions. Trials
can include single or multiple dosages of the study interven-
tion, placebo controls, active comparator controls in which
the intervention of interest is compared with another agent
whose efficacy is known, and combinations of interventions.
The Glucosamine/chondroitin Arthritis Intervention Trial
(GAIT) compared glucosamine hydrochloride alone, chon-
droitin sulfate alone, and the combination of glucosamine
and chondroitin with placebo and with an active compara-
tor, celecoxib, for their effects on symptoms of osteoarthritis
of the knee.10 The Arthritis, Diet, and Activity Promotion
Trial (ADAPT) was a ­ nonpharmacologic intervention
in which diet, exercise, and the combination of diet and
exercise were compared with a control group.11 Such non-
pharmacologic trials may include an “attention-control,”
in which the control group does not get the specific inter-
vention of interest, but does get at least a minimal amount
of attention from the investigator because it is known that
even minimal contact with the participants in a study can
improve outcomes.12
Optimally, to minimize bias, the study should be double-
blind, in which the assignment of treatment is unknown
to the participant and to the data collector evaluating
the participant’s response. A crossover design is a within-
patient design that allows each participant to be his or her
own control and receive the active intervention followed
by a “washout” period, in which no active or inactive treat-
ment is given, and then the control treatment, or vice versa.
This design has some advantages, particularly in sample
size requirements, but can be biased if there is a significant
carryover effect of the active treatment into the “control”
observation period.4 Response to treatment also may differ
depending on whether the active drug is received before or
after the placebo or other comparator.13
Other important considerations in clinical trials are the
selection and means of assessment of primary and second-
ary outcomes, which must be prespecified. Outcomes can
include measures of disease modification, symptom modifi-
cation, and frequency of side effects or other poor outcomes.
Symptom modification trials are frequently of short duration
and less expensive than disease modification trials, which
generally are interested in longer term outcomes. In trials
of biologics for rheumatoid arthritis, effects on symptoms
frequently can be measured in weeks to months, whereas
effects on prevention or healing of radiographic erosions
may require longer follow-up times.14 Similarly, disease
modification trials in osteoarthritis currently require large
numbers of individuals followed for at least 2 years, pre-
dominantly because the metric of change in minimal joint
space on knee radiographs is imprecise and can be fraught
 438 JORDAN  |  Principles of Epidemiology in Rheumatic Disease
with measurement error.15 Outcomes based on magnetic res-
onance imaging, when validated, are expected to be more
sensitive, to be less subject to measurement error, and to
require smaller sample sizes and shorter observation periods
to show an effective response.16
Other trial designs can apply interventions to entire
communities or to health care workers with measurement
of outcome in their patients. An example of the latter
would be an educational intervention designed to increase
physician prescription of physical therapy evaluation of
all patients with knee or hip complaints. The physicians
receive the intervention, but whether the physical therapy
is prescribed, and whether it improves patient symptoms, is
measured by assessing the patient.
Although clinical trials represent the “ultimate” study
design closest to a controlled experiment, there are signifi-
cant potential threats to its validity. One of the most impor-
tant biases can occur when there is large loss to ­follow-up.
To minimize this type of bias, every effort should be made to
continue to obtain outcome information on all participants,
even those who otherwise discontinue study assignment to
therapy. Because all predictors of dropout cannot be known,
and because dropouts may differ from individuals who
remain in a study in ways that cannot be controlled, statis-
tical analytic methods are used to address this. Data always
should be analyzed in an intention-to-treat fashion, in
which all randomized participants are analyzed as members
of the group to which they were initially randomly assigned,
regardless of whether they actually adhered to the group
assignment. Completer, or “according to protocol,” analy-
ses also are often performed, in which only participants who
adhered to their assigned group treatment are included in
the analysis. Prerandomization screening and run-in periods
before randomization can help to avoid randomly assigning
individuals unlikely to adhere to or complete the protocol,
minimizing expense and dilution of effects.17,18 Other issues
to consider in the interpretation of results of clinical trials
deal with generalizability and the difference between effi-
cacy in a controlled environment and effectiveness in the
real world of everyday practice. Postmarketing observations
often can reveal side effects or unintended consequences of
interventions that may not be apparent within the context
of highly regulated trials. More detail regarding design of
clinical trials can be found in Chapter 30.
SUMMARY
Epidemiologic methods can be used to measure frequency or
development of disease and evaluate risk or protective fac-
tors in disease occurrence. Choice of study design depends
on multiple factors, including the research question, disease
under study, availability of appropriate study populations,
and resources available. Each study design has its own set of
advantages and disadvantages, with the clinical trial consid-
ered the most rigorous.
REFERENCES
  1. Rothman KJ: Epidemiology: An Introduction. New York, Oxford Uni-
versity Press, 2002.
  2. Hennekins CH, Buring JE: Epidemiology in Medicine. Boston, Little,
Brown, 1987.
  3. Hulley SB, Cummings SR: Designing Clinical Research. Baltimore,
Williams & Wilkins, 1988.
  4. Rothman KJ, Greenland S: Modern Epidemiology, 2nd ed. Philadel-
phia, Lippincott Williams & Wilkins, 1998.
   5. Rothman KJ: Modern Epidemiology. Boston, Little, Brown, 1986.
  6. Dillon CF, Rasch EK, Gu Q, et al: Prevalence of knee osteoarthri-
tis in the United States: Arthritis data from the Third National
Health and Nutrition Examination Survey 1991-94. J Rheumatol
33:2271-2279, 2006.
  7. Cooper GS, Dooley MA, Treadwell EL, et al: Smoking and use of hair
treatments in relation to risk of developing systemic lupus erythema-
tosus. J Rheumatol 28:2653-2656, 2001.
  8. Felson DT, Zhang Y, Hannan MT, et al: Risk factors for incident
radiographic knee osteoarthritis in the elderly: The Framingham
study. Arthritis Rheum 40:728-733, 1997.
  9. Jordan JM, Helmick CG, Renner JB, et al: Prevalence of knee
symptoms and radiographic and symptomatic knee osteoarthritis in
African Americans and Caucasians: The Johnston County Osteo-
arthritis Project. J Rheumatol 34:172-180, 2007.
10. Clegg DO, Reda DJ, Harris CL, et al: Glucosamine, chondroitin
sulfate, and the two in combination for painful knee osteoarthritis.
N Engl J Med 354:795-808, 2006.
 11. Messier SP, Loeser RF, Miller GD, et al: Exercise and dietary weight
loss in overweight and obese older adults with knee osteoarthritis:
The Arthritis, Diet, and Activity Promotion Trial. Arthritis Rheum
50:1501-1510, 2004.
 12. Rene J, Weinberger M, Mazzuca SA, et al: Reduction of joint pain
in patients with knee osteoarthritis who have received monthly tele-
phone calls from lay personnel and whose medical treatment regimens
have remained stable. Arthritis Rheum 35:511-515, 1992.
 13. Pincus T, Koch GG, Sokka T, et al: A randomized, double-blind, cross-
over clinical trial of diclofenac plus misoprostol versus acetaminophen
in patients with osteoarthritis of the hip or knee. Arthritis Rheum
44:1587-1598, 2001.
14. van der Heijde D, Klareskog L, Rodriguez-Valderde V, et al: Com-
parison of etanercept and methotrexate, alone and combined, in
the treatment of rheumatoid arthritis: Two-year clinical and radio-
graphic results from the TEMPO study, a double-blind, randomized
trial. Arthritis Rheum 54:1063-1074, 2006.
 15. Brandt KD, Mazzuca SA, Conrozier T, et al: Which is the best radio-
graphic protocol for a clinical trial of a structure modifying drug in
patients with knee osteoarthritis? J Rheumatol 29:1308-1320, 2002.
 16. Conaghan PG, Felson D, Gold G, et al: MRI and non-cartilaginous
structures in knee osteoarthritis. Osteoarthritis Cartilage 14(Suppl A):
A87-A94, 2006.
 17. Brandt KD, Mazzuca SA, Katz BP: Effects of doxycycline on progres-
sion of osteoarthritis: Results of a randomized, placebo-controlled,
double-blind trial. Arthritis Rheum 52:2015-2025, 2005.
18. Brandt KD, Mazzuca SA: Lessons learned from nine clinical tri-
als of disease-modifying osteoarthritis drugs. Arthritis Rheum 52:
3349-3359, 2005.
 29
KEY POINTS
Health economics provides information on the equity of care
and informs the debate on resource allocation to improve
health-related quality of life. Economic cost studies also help
identify information gaps and research needs.
In the Netherlands, several national cost-of-illness studies
found that between 5.4% and 12.6% of total health
expenditures was attributable to musculoskeletal conditions,
ranking second after mental retardation.
Musculoskeletal complaints are a major cause of absence
from work in developed countries. They are the most common
medical cause of long-term absence and are second only to
respiratory disorders as a cause of short-term absence
(<2 weeks). Musculoskeletal complaints are also a common
reason for disability pensions.
In people with rheumatoid arthritis, the direct and indirect
costs of illness are twice as high compared with controls,
but two thirds of that difference is related to comorbidities.
The direct costs are initially high and then usually stabilize to
a lower level until joint damage progresses and arthroplasty
becomes necessary, but this may change with the earlier use
of biologics.
The direct costs of osteoarthritis depend on which joint is
affected, disease severity, comorbidities, and access to
arthroplasty. Nonsteroidal anti-inflammatory drug
gastropathy has a significant effect on costs. Work loss is not
as great as with rheumatoid arthritis because of the age of
greatest impact.
The indirect costs of ankylosing spondylitis are considerable,
owing to the long duration of disease throughout working life
and the limitation on activities due to spinal and extraspinal
disease. The direct costs depend on access to rehabilitation
and hospital care and the recent introduction of biologic
therapy.
The direct medical expenditures for osteoporosis are related
predominantly to hospitalization following low trauma
fracture, and the indirect costs are largely related to social
care following hip fracture.
The total cost of low back pain in Organization for Economic
Cooperation and Development countries corresponds to 1%
to 2% of the gross national product; about 10% of the costs
are direct, and 90% are indirect. Most of the costs are related
to work loss. The economic impact is dependent on systems of
social support.
Economic Burden of
Rheumatic Diseases
Anthony D. Woolf
Musculoskeletal conditions are common and are characterized
by pain and physical disability, along with consequential effects
on activities.1 Their prevalence and impact are increasing
worldwide with the aging of the population and changes in risk
factors, such as physical ina­ctivity and ob­esity. Interventions
are available to effectively prevent and control various mus-
culoskeletal conditions.2 H­owever, at present, care is under-
provided in most parts of the world, and priorities do not
reflect the increasing needs and opportunities.3
The burden of musculoskeletal conditions on the indi-
vidual is related to disability and loss of quality of life, as
well as the economic burden on the affected person and
his or her family and caregivers in terms of lost income. In
ad­dition, there are the direct costs of health care and social
support borne by the individual and by society. There is also
a broader cost to society in terms of lost productivity by
affected individuals and their caregivers.
There needs to be a better understanding of the health,
social, and economic burdens of these conditions, as well
as the health and economic benefits of prevention and
tr­eatment. This chapter focuses on the economic issues.
HEALTH ECONOMICS
Health economics is important in a world where there are
constraints on the resources available for health care and
social support. Economic studies can guide health priorities
and decision making about which approach is most benefi-
cial to the greatest number of people. Health economics
provides information on the equity of care and informs the
debate about whether resources should be directed to health
care or other activities that influence health-related quality
of life. Economic cost studies also help identify information
gaps and research needs. These studies should have a soci-
etal perspective—encompassing all costs, no matter who
incurs them, and all benefits, regardless of who receives
them. Health economics can take a macroeconomic per-
spective of the effect of illness on national economies, or
it can take a microeconomic perspective of the effect on
individuals.
ECONOMICS OF ILLNESS
The economic impact of any health condition can be
c­onsidered in terms of the gross loss to the economy. The
cost of illness is the monetary burden on society caused by
the morbidity and premature mortality associated with a
particular illness.4 It can be measured as the societal cost of
pro­viding services related to the delivery of health care and
social support. It can also be considered in terms of the per-
sonal cost to the patient, family, or immediate community.
439
 440 woolf  |  Economic Burden of Rheumatic Diseases
These costs can be estimated within the framework of the
direct, indirect, or intangible costs associated with the condi-
tion. In welfare economics, the total costs are less important
than showing how money spent in one area could be b­etter
spent elsewhere. Resources are limited, but the potential
uses of those resources are unbounded. Health economics is
concerned with how to allocate those resources to generate
maximal health and social benefits. This notion of cost is
based on the value that would be gained from using resources
elsewhere and is referred to as opportunity cost. In practice,
it is usually assumed that the price paid reflects the oppor-
tunity cost; a pragmatic approach is adopted, and ma­rket
prices are used when possible. These opportunity costs may
be within the health care sector or may be considered more
broadly across society as a whole.
DIRECT COSTS
Direct costs are those health care and social costs directly
associated with disease prevention, detection, treatment,
and rehabilitation, including care in the community. The
value of direct costs represents resources that would have
been available for other uses in the health care and social
system, as well as by the individual, if the disease had not
happened. These costs may be disease specific, a direct
result of the condition, or they may be disease associated,
a consequence of the primary disease or its treatment. They
are influenced by comorbidities, and it may be difficult to
a­llocate the costs to a specific diagnosis.
Direct costs include the costs of physician visits, diagnostic
tests, prescription drugs and over-the-counter medications,
hospital stays and procedures, aids and devices, and outpa-
tient procedures. These expenditures may be borne solely or in
combination by the patient, the health insurer, an employer,
or a local or national governmental agency. Direct costs also
include other things generally paid for by the patient, such
as transportation to and from the doctor or other health
care provider, nonphysician services such as complemen-
tary therapy, higher food bills associated with special diets,
or expenditures to adapt the home environment to make it
more functional. In addition, there are direct costs associated
with any change in living status. The direct costs borne by
the individual are called out-of-pocket expenditures, and the
amount varies, depending on what is covered by the health
insurance system. In addition, the costs that are included as
direct costs can vary in economic analyses, depending on the
perspective of the study—who is paying for care and who is
receiving it. For example, in many cases, home care is a direct
cost for the patient but an indirect cost for the funder. Many
direct costs relate to the utilization of available resources and
therefore do not reflect the burden if a need is unmet. Most
direct costs are a consequence of treatment, not prevention.
Direct costs are relatively easy to measure. They can be
estimated by using a “top-down” approach, dividing the total
health expenditure among different diseases, or a “bottom-up”
approach in which the number of health care services for
individuals who fulfill diagnostic criteria are counted and
their costs determined. The certainty of case definition is
better in the bottom-up approach. The total costs of dis-
ease can then be estimated. With comorbidities, it may be
di­fficult to determine which costs to allocate to which con-
dition, especially those costs related to rehabilitation and
changes in living status. However, it is important to avoid
double-counting costs for several conditions.
The attributable costs provide the clearest estimate of
the potential savings, which can be identified by looking
for the incremental costs after the onset of disease or an
event such as a fracture. Alternatively, a case-control study
can compare the total health care costs of individuals with
the disease compared with healthy, matched controls. The
c­a­se-control method has the advantage of allowing for
comorbidities and associated events.
INDIRECT COSTS
Indirect costs result from the consequences of a condition,
such as the limitation of usual activities. The human capital
approach counts these costs as the value of lost production
to society; these costs are less than the assumed loss of gross
earnings, because not all healthy people are economically
active. An alternative approach is to look at indirect costs
as a consequence for individuals, their families, and their
caregivers. These costs include work loss due to treatment,
sick leave, reduced work productivity, early retirement,
and death. Other losses attributable to the condition also
constitute indirect costs; for example, the person may be
prevented from getting a better-paying jobs or suffer from
reduced employment opportunities. The additional costs
related to self-care, maintenance of a home, schooling, or
parenting are also included. Both patients and their caregiv-
ers may incur indirect costs. Chronic physical disability has
a major impact on indirect costs.
These costs are difficult to measure. Some can be given
a monetary value, although this depends on local systems
of social support, sickness benefits, and pensions. Lost work
productivity is important in chronic musculoskeletal condi-
tions but is not often included in economic evaluations.5 It
can be estimated in different ways. With the human capital
approach, the full replacement costs are used, irrespective
of whether the worker is replaced. With the friction cost
approach, only the productivity costs during the period
required to restore the initial production level are consid-
ered, with a replacement worker obtained from the labor
market. This results in lower estimates of lost productivity
costs but is probably more realistic. Other indirect costs
c­annot be given a specific value because reduced productiv-
ity or lost hours at school or household work are difficult to
assign a monetary cost.
Indirect costs are strongly influenced by people who are
working, because employment-related costs are easier to
quantify. The economic impact on children, women, the
elderly, and the unemployed is not adequately captured. In
the case of comorbidities, it can also be difficult to know
which condition to attribute these costs to.
INTANGIBLE COSTS
Intangible costs are those associated with loss of function,
increased pain, and reduced quality of life of patients, fami-
lies, and caregivers. They also include the costs of lost oppor-
tunities. Intangible costs represent a negative benefit in
terms of health economics and are generally not included in
cost-of-illness studies. However, they are very important for
musculoskeletal conditions, because disability is a significant
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES 441

Table 29-1  Health Cost Domains Relevant to Musculoskeletal Conditions

Category Domains How to Identify Costs
Direct Costs
Outpatient costs Visits to physicians (primary care and specialist)
Outpatient surgery Hospital or insurer activity data
Emergency room
Rehabilitation services (e.g., physiotherapist,  
occupational therapist, social worker)
Medication (prescription and nonprescription) Pharmacy records
Diagnostic or therapeutic procedures and tests Radiology activity; laboratory tests
Devices and aids Provision of equipment
Inpatient costs Acute hospital facilities (without surgery) Hospital or insurer activity data on admissions,
Acute hospital facilities (with surgery) length of stay, procedures
Nonacute hospital facilities Rehabilitation activity; nursing home activity
Personal costs Transportation Transportation distance, frequency, methods
Patient time Time spent on health care
Caregiver time Time spent giving care
Other disease-related costs Home health care services Home health care activity
Environmental adaptations Home, work, and transportation adaptations
Medical equipment (nonprescription) Equipment provision
Nonmedical practitioner, alternative therapy Therapist activity
Change of living status Nursing home or residential home Nursing and residential home activity
Home care services Formal and informal home care activity
Indirect Costs
Productivity costs Loss of productivity in employed patients   Sick leave, lost wages, work disability benefits,
or their caregivers no longer working; disabilities leading to
Opportunity costs—reduced employability at   impaired housekeeping or activities of daily
present or higher level living; loss of productivity
Out of pocket Out-of-pocket expenses Survey
Intangible Costs
Deterioration in quality of life of patient, family,   Difficult to quantify
caregivers, friends
Modified from Merkesdal S, Ruof J, Huelsemann JL, et al: Development of a matrix of cost domains in economic evaluation of rheumatoid arthritis. J Rheuma-
tol 28:657-661, 2001.

outcome, including limitations in activities of daily living,

reduction in leisure and community activities, chronic pain,
psychological problems such as depression and an­xiety, and
reduced general health. The inverse of the intangible costs
of arthritis is the benefit a patient receives from effective
treatment. If measuring the costs of care is to have relevance
for clinical decision making, it must also consider the effects
of disease and therapy on the patient’s health and well-
being. Intangible costs are, however, difficult to quantify and
extremely difficult to give a monetary value.
COST ITEMS AND ESTIMATES
Many cost items can be used in the estimation of the cost of
illness. They fall under the categories of direct, ­indirect, and
intangible costs (Table 29-1). Not all items are considered
in every health economic evaluation, and these omissions
need to be recognized. The value of any evaluation is based
on its consideration of all those cost items that might be
influenced by the condition or its treatment. Omissions or
different definitions can affect the interpretation and com-
parability of data. Attempts are being made to standardize
the cost domains for musculoskeletal conditions.6 There also
needs to be some standardization in the way these items are
costed if there is to be any comparability among economic
evaluations. Unfortunately, data collection can be very dif-
ficult, so various assumptions are often made, and modeling
is used.
COST-OF-ILLNESS STUDIES
The economics of health can be looked at in different ways,
depending on the purpose. Cost-of-illness studies and cost
analyses examine the resources used in the provision of
health care related to a certain condition; they do not relate
the costs to the benefits or outcomes of health care.7 These
costs must be related to the effect of the condition and to
its management, including any complications or side effects.
Cost-of-illness studies sometimes include the effect on the
individual’s ability to perform paid or unpaid work and the
costs of social support. It is therefore important to exam-
ine what cost domains are included when examining any
cost-of-illness study. For musculoskeletal diseases, which
have different impacts on individuals during the disease
course, information is also required about the different
health states associated with the particular disease and the
numbers of patients in those states.
Cost-of-illness studies can be performed by a prevalence-
based, top-down approach—looking at the total costs of di­sease
in a population and assigning costs to specific diagnoses, which
enables a comparison between diseases. An alternative is a bot-
tom-up, prevalence approach using patient-derived data from
a cross section of the patient ­population, which allows patient
and disease characteristics to be related to resource use and
costs. An incident approach considers the cost of the illness
from its onset throughout its course, but this is challenging to
do because of the amount of long-term data required.
 442 woolf  |  Economic Burden of Rheumatic Diseases
One weakness of cost-of-illness studies is that they look
only at the supply side and quantify expenditures; there is
no guarantee that the money has been well spent. High
costs may reflect inefficient use of resources, and low costs
may reflect limited availability of health care. Nor do these
studies provide information on the costs of prevention. Low
costs do not mean that a disease should be given low pr­iority,
because it still may be amenable to a very cost-eff­ective
intervention. In addition, increasing expenditures does not
necessarily mean that better outcomes will be achieved,
unless the money is spent efficiently.
Cost analysis is not an economic evaluation because it
does not measure benefits gained by resources consumed;
rather, it provides an evidence base for such analysis. Its
value is in measuring the economic burden and identifying
how it is distributed among the health care system and other
parts of the public sector, the patient, the family, and so­ciety
as a whole. It can facilitate the identification of potential
improvements in the provision of care, showing where
change is likely to have the greatest effect. It can be used
to aid policy-making decisions.8 The value to policy mak-
ers in choosing between alternatives is limited, however,
because this requires a comparison, and the value of bur-
den-of-di­sease and cost-of-illness studies in guiding policy
has been criticized.9,10 Measures of indirect costs are difficult,
because different studies include different cost domains, and
various methods of ascribing cost can be used, which limits
comparability between studies.
HEALTH ECONOMIC EVALUATIONS
Health economic evaluations require methods that enable
costs to be measured against health gains achieved by an
intervention. The concept of cost-effectiveness involves the
achievement of a predetermined goal or the ability to maxi-
mize the benefit from a limited resource. The m­ethods of
health economic evaluation aim to show how well resources
are used to achieve a desired outcome and enable the
s­election of the most cost-effective option from a range of
alternative interventions. Policy makers allocate resources
on the basis of economic efficiency.
Several concepts need to be considered. Efficiency
describes how well resources are used to achieve a desired
outcome. The allocative efficiency measures how resources
can best be allocated to achieve a given outcome. Techni-
cal efficiency measures the extent to which the smallest
amount of resources can be applied to achieve the greatest
outcome.
The benefit is measured as a health outcome, the defini-
tion and measurement of which are central to health eco-
nomic evaluations. A range of outcomes is used relating to
survival and quality of life. They are valued by individuals
expressing their preference for specific outcomes or states of
health—termed a utility value. There are many methods for
placing values on these different states of health. A single
combined measure can be used that considers both quality
and quantity of life—quality-adjusted life-years (QALYs)—
and enables the quality of life experienced by a patient to
be scored: perfect health is equivalent to 1, death is equiva-
lent to 0, and health states worse than death have negative
values. QALYs therefore provide a common currency that
enables the assessment of benefits gained from a variety of
interventions for a variety of conditions in terms of quality
and quantity of life.
Economic costs relate to the consumption of resources
and to the fact that if resources are used in one way, they are
not available to be used in another way, resulting in a loss of
potential benefits. There may not be an actual financial pay-
ment. This loss of potential benefit from an alternative use
of the resources is referred to as the opportunity cost. Health
economics uses this concept of trade-offs.
Economic evaluations provide a systematic and obj­ective
framework for considering costs and benefits. They pre-
dominantly use randomized, controlled trials for evidence
of the effects of any intervention, but what happens in real
life is often different, because many people who have been
treated with an intervention are not eligible to participate
in clinical trials, and other factors such as self-efficacy can
play a role. Because policy decisions are about real people,
some argue that naturalistic studies should be used.11 There
are four main methods of economic evaluation, all of which
measure resources expended in monetary terms. The latter
three differ in how the health outcome is measured.
Cost minimization studies are used to compare alternative
treatments for the same condition, and it is assumed that
the outcomes from the different treatments are the same.
Only the costs of interventions being evaluated are mea-
sured. The most effective treatment is the least costly. The
value of such studies is limited because they do not consider
secondary end points such as side effects. This method is
seldom used today.
Cost-effectiveness analysis is also used to compare treat-
ments for the same condition, but it measures both the health
effects of treatment and the costs. The outcomes measured
are usually clinically relevant and directly quantifiable, such
as life-years gained or morbid events averted. The most effi-
cient therapy is that which has the lowest cost per unit of
effect. These analyses are often used to evaluate pharmaco-
therapies that have single therapeutic goals. They cannot
be used to compare interventions that may have different
effects because they do not consider all the outcomes.
Cost utility analysis uses a measure of outcome that can
be applied to all health care interventions for different con-
ditions, such as QALYs. This measurement should capture
all health effects. It allows a comparison between health
care technologies for different conditions and gives decision
makers information to help them allocate limited resources.
Other methods of measuring utility include time trade-off
and standard gamble.
Cost-benefit analysis brings in the public’s and the
patient’s perspective when using economic information to
set priorities and make choices between treatments for dif-
ferent conditions. Both costs and benefits are expressed in
monetary terms. There are different ways of measuring the
public or patient value of a treatment or condition, such as
willingness to pay. The willingness-to-pay approach values
what individuals are willing to pay (in monetary terms) for
a change that results in improved health, such as a reduced
probability of morbidity. This method allows individuals to
indicate how they value health and death, and calculations
can be made from a societal perspective.
A wide variation in measures of clinical efficacy and whether
and how costs of adverse events are incorporated have been
found in health economic analyses by the OMERACT Health
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
Table 29-2  Methodologic Issues for Economic Evaluation in Rheumatology
Issue
Model horizon
Duration of treatment
Extrapolation beyond trial duration
Modeling beyond trial duration
Synthesis of comparisons when clinical trials do not exist
Outcome measures
Mortality
Valuation of health (e.g., QALYs)
Resource use
Classification and reporting of adverse events
Discontinuation of treatment
Therapeutic strategies
Population risk stratification
QALY, quality-adjusted life-year.
From Gabriel SE, Tugwell P, Drummond M: Progress towards an OMERACT-ILAR guideline for economic evaluations in rheumatology. Ann Rheum Dis
61:370-373, 2002.
Economics Working Group. As a consequence, several issues
have been identified (Table 29-2), and a methodology with a
standardized set of minimum criteria for economic evaluation
in rheumatology has been proposed12; specific recommenda-
tions have been made for rheumatoid arthritis (RA).12
Cost-effectiveness studies, to be valid and meaningful,
must rely on a fair, balanced, and explicit representation of
the disease in a simulation model that includes all relevant
outcomes and treatment consequences.13 One problem is
that effectiveness estimates are usually derived from clinical
trials, particularly for new treatments, but later ob­servational
studies may not demonstrate the same outcomes. This
results in different cost-effectiveness ratios, depending on
the stu­dies considered, and it may affect access to treatment.
These issues have been highlighted by studies of anti–tumor
necrosis factor (TNF) therapy.11
Health economic evaluations for specific interventions
are not discussed in detail in this chapter, because they
must be considered in the context of the condition’s man-
agement. A central issue is the need to extend information
beyond that which is available from clinical trials, such as
the costs and health effects that occur after the clinical
trial, particularly when considering long-term conditions.
M­arkov modeling is frequently used.
This chapter principally considers cost of illness and cost
analysis data.
COST OF MUSCULOSKELETAL
CONDITIONS
Musculoskeletal problems are very common, affecting up
to 20% of adults.1,14 Their overall economic impact can be
measured by the use of health care and social care resources,
443
Question to Ask
How long should costs and benefits be forecast?
What duration of treatment should be included in economic evalua-
tions?
Can extrapolation beyond the duration of clinical trials be performed in a
valid and reliable manner?
Can extrapolation beyond treatment duration be performed in a valid
and reliable manner?
Can comparisons be synthesized in a valid manner when experimental
data are not available?
What are the most meaningful clinical outcomes for economic evalua-
tion?
How should mortality be considered in economic analysis?
What are the optimal sources of health state values?
What are the optimal approaches to estimating resource use?
What are the optimal approaches to measuring, classifying, and report-
ing toxicity?
What are the optimal approaches to determining drug discontinuation
and adherence rates?
Should data on therapeutic strategies (as opposed to individual treat-
ments) be incorporated into economic evaluations?
What are the optimal approaches to incorporating data on the risk status
of the population under study in economic evaluations?
as well as by work disability. There have been several
national cost-of-illness studies in developed countries in
which the costs related to musculoskeletal conditions can
be identified. These reflect the health care expenditures for
the management of these conditions as well as their societal
costs; they measure not only the impact of these diseases
but also the current provision of health and social care for
people with these conditions. They do not measure the
costs of unmet needs due to a lack of adequate services, such
as joint ­arthroplasty in many countries, or due to patients’
unwillingness to seek medical care because of the negative
perception of what can be achieved.15,16
How an individual with a musculoskeletal condition
in a certain country interacts with the health and social
care systems, the normal patterns of care, and local support
systems, such as home care and pensions, are all impor-
tant factors, because these confounders can influence costs.
Expenditures are influenced by the rules of reimbursement
and other factors that influence the management approach
to similar conditions in different countries. For example,
inpatient care is available only for complicated RA in some
countries, whereas in other countries, people commonly
receive inpatient rehabilitation. In addition, the systems
of care are changing in many countries; the trend is away
from inpatient management, which is related to changes
in therapeutic options, better outcomes, and the desire to
reduce health care costs. Social care can be provided in
different ways, from the extended family and caregivers to
agencies prov­iding home care, each with a different cost—
direct, indirect, and intangible. These factors, along with
different methods of costing, make it difficult to compare
findings in different countries or studies done at different
times.
 444 woolf  |  Economic Burden of Rheumatic Diseases

There have been several national cost-of-illness stud-
ies looking at total health expenditures. An examina-
tion of comparable studies shows that between 5.4% and
12.6% of expenditures are attributable to musculoskeletal
conditions.17 These differences can be caused by a range of
methodologic issues that makes comparison difficult, and
interpretation should be based on a set of key questions
(Table 29-3).
In Sweden, musculoskeletal conditions cost SKr 52.7
billion (US$5.3 billion) in 1994.18 The total costs were
attributable to back pain (47%), osteoarthritis (14%), and
RA (5.5%). Most of the costs were indirect and related to
sick leave (31.5%) and early retirement (59%).
Table 29-3  Checklist for the Interpretation   indirect costs ($13.7 billion). Musculoskeletal diseases were
and Comparison of Cost-of-Illness Data
Which types of costs are included? Only health care costs, or also
other costs such as productivity losses and absence from work?
Are all sectors of the health care system included, or are some left out?
What is the definition of the disease, and how is it classified?
Are the costs calculated bottom up or top down?
Are productivity losses based on the human capital or the friction
costs methodology?
What is the year for which data were collected?
Have foreign data been used, and if so, how?
Is the study population representative of the national population,
and is the treatment practice representative of the generally
acknowledged method of treatment?
Are the diagnoses well reported?
How has comorbidity been dealt with?
From Slobbe LCJ, Kommer GJ, Smit JM, et al: Cost of Illness in the Netherlands
2003. http://www.rivm.nl/bibliotheek/rapporten/270751010.pdf.
In the Netherlands, musculoskeletal conditions19 accoun­
ted for 6% of total health care costs in 1994,20 ranking
second behind mental retardation (8.1%). Coronary heart
diseases and other circulatory diseases accounted for 4.8%.
This study considered only medical costs; if the costs of
informal care had been included, this would have greatly
increased the costs related to chronic disabling conditions
such as musculoskeletal diseases. Costs were considerable at
all ages, ranking fifth among those 15 to 44 years old, second
among those 45 to 64 years, and third among those 65 to 84
years (after dementia and stroke).
In Canada in 1998, musculoskeletal diseases ranked sec-
ond and accounted for $16.4 billion of the total costs of ill-
ness (Fig. 29-1).21 Direct costs ($2.6 billion) were less than
the leading cause of disability, accounting for about 39% of
long-term disability costs ($12.6 billion), followed by dis-
eases of the nervous system (12.9%; $4.2 billion), cardio-
vascular diseases (9.8%; $3.2 billion), and mental disorders
(7.0%; $2.2 billion). Musculoskeletal diseases accounted for
10.3% ($1.0 billion) of total short-term disability costs in
1998. In contrast, the expenditure for research on musculo-
skeletal diseases was only 1.3% of the total.
In the United States, the total cost of musculoskeletal
conditions was estimated in 1995 at almost 3% of gross
domestic product,7 compared with estimates of about 0.5%
in 1963 and 1972. This included the spectrum of muscu-
loskeletal diseases and connective tissue disorders, injuries,
neoplasia, and deformities and anomalies. The direct cost
amounted to $88.7 billion, of which 38% ($33.7 billion)
was attributable to hospital admissions, 21% to nursing
home admissions, and 17% to physicians’ visits. Similar to
other established economies, indirect costs were far greater,
amounting to 58% of the total ($126.2 billion). Because

Direct and indirect costs by diagnostic category in Canada*, 1998

Cardiovascular
Musculoskeletal
Cancer
Injuries
Respiratory
Nerv. sys. / Sense org.
Mental disorders
Digestive
Well-patient
Ill-defined
Endocrine and related Direct costs
Genitourinary
Infectious Indirect costs
Pregnancy
Skin and related
Birth defects
Perinatal
Blood
Others (1)
Unattributable (2)
0 10 20 30 40

Figure 29-1  Direct and indirect Cost (Canadian $ billion)

costs of illness in Canada (1998) by
diagnostic category. (From Health
Canada: The Economic Burden of Ill-
ness in Canada 1998. http://www.
hc-sc.gc.ca.)
* Based on total cost of illness of $159.4 billion
(1) Refers to data for which coding is not provided (for diagnostic categories), or data that are grouped
because of small counts.
(2) Refers to data that could not be coded by ICD-9 code.
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
musculoskeletal conditions are rarely fatal, 51% of the cost
attributable to lost wages arose from morbidity. The costs
were greatest in those aged 18 to 44 years and 45 to 64
years; in these groups, indirect costs were greatest due to
work loss. In contrast, in those younger than 18 years and
older than 65 years, most costs were related to medical care
expenditures. Indirect costs relate not only to work loss but
also to lost productivity. More than 7% of almost 30,000
working adults in the United States lost productive time
because of back pain (3.3 hr/wk), arthritis (2.03 hr/wk), or
other musculoskeletal pain (2.02 hr/wk), although absence
was uncommon.22 The total cost of this lost productivity was
estimated at $41.7 billion per year, of which about $31.5
billion occurs while employees are at work but performing
suboptimally. If the costs of arthritis and other rheumatic
conditions alone are considered, the total costs in 1997 were
estimated from the 1997 Medical Expenditure Panel Survey
and the 2002 Behavioral Risk Factor Surveillance System
to be $86.2 billion, including $51.1 billion in incremental
direct costs and $35.1 billion in indirect costs.23,24 This was
approximately 1% of the U.S. gross domestic product, but
the total expenditures for all medical care for any reason
among people with arthritis was estimated in 1996 at $140
billion (in 2000 dollars), representing almost 2.8% of the
gross national product.25 Most recently, based on data from
the Medical Expenditure Panel Study,26 the mean expendi-
ture among persons with arthritis and other rheumatic con-
ditions (excluding injuries, neoplasia, deformities, and most
back pain) was estimated at $6978 in 2003, a 10% increase
over 1997 estimates.27 The proportion of outpatient costs
increased from 29% to 32%, and prescription medication
costs increased from $899 to $1635 in absolute terms and
from 14% to 23% in relative terms from 1997 to 2003. This
reflects changes in clinical practice. The total medical care
expenditures in 2003 incurred by the estimated 46.114
million adult Americans with arthritis or other rheumatic
conditions (excluding injuries, neoplasia, deformities, and
most back pain) were estimated at $321.8 billion, or approx-
imately 3% of gross domestic product.26
The direct costs of musculoskeletal conditions reflect
the fact that such complaints are the second most com-
mon r­eason for consulting a doctor and constitute, in most
cou­ntries, up to 10% to 20% of the primary care practice.28
There were 43.9 million ambulatory physician visits in the
United States in 1997 for a primary diagnosis of arthritis
or other rheumatic conditions.29 Of these, 36.5 million
were arthritis vi­sits unrelated to injury; this constituted
the largest di­agnostic category among chronic conditions.
Most were visits to physicians’ offices; only 11% were visits
to hospital outpatient facilities or emergency rooms. More
than half the visits were to primary care physicians, 19% to
orthopedic surgeons, and 16.5% to rheumatologists. Most
visits involved people older than 45 years, and women had
almost twice the rate of arthritis visits compared with men.
Annually, a person with arthritis reported an average of
eight physician visits for all conditions.25 In Europe, 20.4%
were undergoing long-term treatment for arthritis and rheu-
matism in the ­Eurobarometer Survey.14 Five percent of drug
expenditures in Canada was related to musculoskeletal
­conditions in 1998.30 In the United Kingdom, £363 million
was spent on prescription drugs for musculoskeletal condi-
tions in 2003.31
445
The indirect costs reflect the fact that these conditions
are the most common cause of long-term physical disab­ility.
Musculoskeletal complaints are a major cause of absence
due to sickness in developed countries.32,33 They are second
only to respiratory disorders as a cause of short-term absence
(<2 weeks).34 Musculoskeletal complaints are the most com-
mon medical cause of long-term absence, accounting for
more than half of all illness-related absences longer than
2 weeks in Norway.35 They are also common reasons for dis-
ability pensions, as are mental disorders and cardiovascular
disorders. In Sweden, up to 60% of persons taking early retire-
ment or long-term sick leave claim musculoskeletal problems
as the reason.36 In the United States in 2003, employment
rates were 10.8% lower among those with arthritis or other
rheumatic conditions (excluding injuries, neoplasia, defor-
mities, and most of back pain); however, after controlling
for comorbidities and demographic factors, the employment
gap was 2.3%. In this study, the earnings losses were related
mainly to lost work, but U.S. surveys of working adults have
shown that a larger cost is related to lost productivity while
at work if the full spectrum of musculoskeletal conditions is
considered.22 Many U.S. studies do not include injuries and
back pain, and these are major causes of work loss. Musculo-
skeletal disorders are the most common occupational reason
for work loss.
RHEUMATOID ARTHRITIS
RA can be a devastating condition with a major impact on
the individual and his or her family and friends, although
modern treatment has significantly improved the ­prognosis.
Knowledge of the natural history of the disease and the
typical health interventions and their outcomes enables an
understanding of the associated costs.
The direct costs involve hospital and primary care medi-
cal consultations to assess disease activity and to monitor
treatment safety and efficacy, drug costs, rehabilitation, and
provision of equipment. Arthroplasty may be required. Indi-
rect costs relate to loss of employment by the patient and
often by the caregiver. Importantly, many people with RA or
their caregivers do not have the employment opportunities,
leading to a loss of productivity and income. The intangible
costs of RA are great but difficult to quantify. Various studies
have looked at the considerable health and social costs, but
differences are difficult to interpret and are largely related to
differences in disease severity and disability, different health
and social systems that influence access, and variations in
data sources and types of costs described. As a consequence,
it is impossible to give an average cost.
The direct and indirect costs of illness are twice as high
in people with RA compared with controls,37 but two thirds
of these are related to comorbidities; in part, this could
reflect reimbursement rules for the care of people with RA.
In a systematic review of 15 cost-of-illness studies done pre-
dominantly in the United States,38 it was found that the
average direct annual costs were $5720 per person with
RA. The costs are initially high and then usually stabilize
to a lower level until joint damage progresses and arthro-
plasty becomes ­ necessary. A small proportion of patients
is responsible for the majority of direct costs,38 which were
highest for a younger population with a short duration of
disease and greatest disease severity. Inpatient care strongly
 446 woolf  |  Economic Burden of Rheumatic Diseases
influences these costs, and admission rates varied between
12% and 26%. In the United States, the annual number
of physician visits averages 11 to 12 per patient.25,39,40 In
France, RA patients use health resources more fr­equently,
more intensively, and in a more diverse manner than do
nonarthritic subjects, and 5.1% of all patients had RA-
related surgery in the previous year.41 In a 10-year follow-up
of patients with early RA, 17% had undergone large joint
replacements in Sweden.41a In Germany, the costs of RA
were calculated for 2002 from the German Collaborative
Arthritis Centres database,42 and the median and mean total
direct costs were €2256 and €4727, respectively, attribut-
able mainly to drug costs and inpatient treatments. Out-of-
pocket expenses averaged €559 per year. Major cost drivers
were function, positive rheumatoid factor, and disease activ-
ity. The costs of medication were less than 20% of direct
costs in earlier studies,43 but this was before the widespread
use of anti-TNF. Patients’ time spent on their health care
is a direct cost that is often ignored, but it can be consider-
able in RA. Out-of-pocket expenses vary among countries,
depending on what is covered by health insurance and on
the use of alternative and complementary therapies, which
can be a major contributor.44 Direct costs are high during
the first 2 years, largely related to consultations; they then
fall and gradually increase over subsequent years owing to
the costs of devices and adaptations, which account for 40%
of total costs after 10 years.45 Changes in clinical practice,
such as shared management with primary care physicians
and patients taking greater responsibility, reduce direct
costs. Greater self-efficacy is associated with lower medical
expenditures in Australia.46
The indirect costs are typically between 50% and 75%
of the total in developed countries. They are strongly
influenced by loss of work, either short or long term. Days
lost from work vary in studies from 2.7 to 30 days per
year.38 Employment is 20% lower in men with arthritis and
25% lower in women with arthritis compared with those
without.47 Work capacity is restricted in one third of RA
patients within 1 year48; within 3 years, about 40% will
be considered disabled.49 In the United States, patients
with RA lost their jobs, were unable to get jobs, or retired
early due to their illness.50 Those who do work must often
adjust their work schedules or activities, meaning a loss
of potential income, and work-related disability is greatest
among those doing manual jobs. Loss of activity is substan-
tial, with people reporting 2 to 3 days of restricted activity
within the previous 2 weeks.25 A systematic review found
that work disability rates were similar in the United States
and Europe.51 Age and Health Assessment Questionnaire
(HAQ) disability index are predictors of high indirect
costs, acute and nonacute institutional care,52 and work
disability.53 Disease activity is also associated with higher
costs. The mean indirect costs were $5822 per person in
a syst­ematic review of 15 studies.38 In the recent German
Collaborative Arthritis Centres study,42 the mean indirect
costs due to sick leave and permanent work disability in
2002 were €10,901, applying the human capital approach;
this was reduced to a mean of €3162 by applying the fric-
tion cost approach, which takes into account the fact that
no economy achieves full employment. Costs increased
with disease duration, mainly attributable to costs of per-
manent work disability. Sick leave is the predominant cost
in the initial phase, but as disability increases with disease
duration, long-term disability benefits become an increas-
ing cost. Help in the household is often needed, but in the
United Kingdom, this was provided by family and friends
and was seldom paid for directly.54 This loss of household
productivity, if valued at the market equivalent, is consider-
able and has been estimated to be seven times higher than
the costs from loss of paid productivity estimated by the
friction costs method.45
RA also has a considerable impact on all aspects of qu­ality
of life. Almost two thirds of patients in a cohort study were
restricted in activities of daily living and required help from
family or friends, which in many cases had an adverse effect
on family relations.55
The costs of side effects related to treatment must be
considered, such as fractures subsequent to steroid-induced
osteoporosis or hospitalizations and deaths from nonsteroi-
dal anti-inflammatory drug (NSAID)–related gastropathy.56
OSTEOARTHRITIS
Osteoarthritis is the most common cause of joint pain, and
many cases of self-reported arthritis are related to osteoarthri-
tis. It can affect any joint but occurs most frequently in the
hip, knee, and hands. The impact of osteoarthritis is related
to which joints are affected as well as the presence and sever-
ity of comorbidities that may magnify the disability.
The socioeconomic impact of osteoarthritis has not
been studied extensively. The top-down approach to iden-
tifying costs is difficult because definitive diagnosis requires
a radiograph; joint pain alone is often used, but this results
in the inclusion of other causes, including some cases of
inflammatory arthritis. It is also difficult to estimate the
costs attributable to osteoarthritis because of the presence of
comorbidities.57
National studies have largely identified the costs of
arthritis as a whole. However, in Sweden, osteoarthritis
was estimated to incur costs of SKr 7.4 billion (US$749.4
million) in 1994, of which SKr 739 million (US$75 million)
was for inpatient care and SKr 6.4 billion for productivity
losses.18 Based on national data from France, the costs of
osteoarthritis were estimated at 0.1% of 1991 gross national
product,58 which was equivalent to US$51.4 billion (in 2000
dollars). Almost two thirds of the total was attributable to
direct costs of medical care. The total medical costs for
those with osteoarthritis younger than 65 years are double
compared with the similar individuals without osteoarthri-
tis; they are 50% higher in those older than 65 years.59 In a
case-control study of a large national managed care organi-
zation in the United States, of those with an International
Classification of Diseases (ICD) diagnosis of osteoarthritis
(ICDD-9), the attributable costs were $2827 per patient
year in those younger than 65 years and $1963 per patient
year in those 65 years and older (in 1993 dollars). Two thirds
of these costs were related to health care facilities; attribut-
able drug costs were 7% ($199) in those younger than 65
years but only 2% ($30) in those 65 years and older. In the
United States, annual physician visits average 9 per person,
and noninstitutionalized people with osteoarthritis have an
average of 0.3 hospitalization lasting 8 to 9 days.25
Health resource utilization also relates to the complica-
tions of treatment, most commonly NSAID gastropathy,
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
which results in increased outpatient care, hospitaliza-
tion, and the prescribing of drugs to prevent gastropathy.
It is estimated that the United Kingdom’s National Health
Service spends an average of £251 million per year (range,
£166 million to £367 million) on NSAID-induced gastric
side effects.60
Complementary therapy is often used by those with
osteoarthritis. One study found that nearly half the patients
used at least one type of alternative therapy during a 20-
week period. The cost was estimated at $1127 per year on
alternative care providers, compared with $1148 on ambu-
latory medical care services.61
End-stage osteoarthritis is the major indication for hip
and knee replacement surgery, which is responsible for sig-
nificant health care resource utilization. These costs reflect
supply, not need, and there is a gap between these two fac-
tors in all countries. The estimated prevalence of primary
total knee replacement was 8.4 joints per 1000 people aged
35 years and older in England; in contrast, the estimated
requirement was 27.4 joints per 1000 people aged 35 years
and older, based on the severity of knee disease.62 In Sweden,
the rate of total knee replacement is less. The arthroplasty
rate more closely meets needs in the United States.62a
Total hip replacement rates in Organization for Eco-
nomic Cooperation and Development (OECD) countries
vary between 50 and 140 procedures per 100,000.63 The
primary reason for such surgery is osteoarthritis, except in
the very elderly, when it is more often done after hip frac-
ture.64 The number of hip replacements performed depends
on the resources available and on clinical criteria used to
determine when total hip replacement is appropriate; there
is no consensus on such criteria among physicians and
­orthopedic surgeons.65 In the United Kingdom, 47,932 total
hip replacements were performed in 2000, 20% of which
were revisions; this number is predicted to increase, particu-
larly revisions.66 Forty-nine percent of primary hip replace-
ments were done in patients aged 60 to 74 years; 33% were
in those older than 75 years. This more closely meets the
predicted requirement.66a The estimated cost was £4076 at
2000 National Health Service prices, compared with £641.
53 annual cost per patient for watchful waiting.67 Those
with knee disease were more likely than those with hip dis-
ease to seek treatment from their primary care physicians
but less likely to be referred to specialists or to be awaiting
arthroplasty.62 Patient preferences influence resource utiliza-
tion, and 30% to 55% of those with severe large joint osteo-
arthritis are unwilling to undergo arthroplasty.15,62
Indirect costs are difficult to estimate for previously stated
reasons. National surveys in the United States estimated that
between 0.1 and 0.3 day was lost from employment in the
previous 2 weeks by those with osteoarthritis,39,40 More than
half of those with symptomatic osteoarthritis reported work
disability.68 Those with osteoarthritis are more likely to report
a reduction in working hours or an inability to get a job due
to their illness.50 However, work loss is not as great as with
RA, because many patients are no longer of working age.
In Australia, out-of-pocket expenses by those with osteo-
arthritis for medications, special equipment, and assistance
from family and friends increased, despite a government-
funded health care system.69
Loss of mobility is common in those with arthritis, espe-
cially those with osteoarthritis; 18% report a significant loss.
447
Limitation of activities, defined as the inability to perform
one’s major activity, was reported by 25% of people with
osteoarthritis.39 These limitations increase with age.
ANKYLOSING SPONDYLITIS
Ankylosing spondylitis commonly starts in early adulthood
and has a chronic, progressive course leading to increased
disability, which results in considerable costs related to both
health care and inability to perform paid work. Only recently
have studies been performed to measure this cost of illness,
stimulated by the introduction of effective but expensive ther-
apies and the need to justify their use and cost-effectiveness.
There is a long delay in the diagnosis and specialist
management of ankylosing spondylitis, with a mean disease
duration of 5.9 years before the first visit to a rheumatolo-
gist in the German Collaborative Arthritis Centres study.70
Following diagnosis, management varies by country; for
example, there are differences in the frequency of routine
clinical review, the use of physiotherapy and hydrotherapy,
and inpatient care. The greatest differences are between
Europe and the United States.71 In a study of patients in
France, Belgium, and the Netherlands with ankylosing
spondylitis of around 10 years’ duration, the average annual
direct costs were €2640 (median, €1242) per patient.72
Direct health care costs accounted for 82% of this total;
the rest was attributable to direct non–health care costs,
such as exercise classes, household help, or transportation.
The greatest direct costs were related to inpatient care
(27%), combined formal and informal care (22%), physio-
therapy (13%), and drugs (13%). Cost ­differences between
­countries were related to clinical practice and availability
of or access to resources. In Germany, where costs were ana-
lyzed only among those of working age, the annual direct
costs were €3676 (median, €1705); these costs were more
similar to those in other European countries when all ages
were considered.42 The difference in health care utilization
is greater between Europe and the United States, where,
for example, physiotherapy is not covered by health insur-
ance and inpatient care is uncommon. As a consequence
of this fact and the differences in clinical practice, the dis-
tribution of costs is considerably different.71,73 The direct
health costs were only 26% of the total annual costs in the
United States, with average direct costs of $1775. Of these
direct costs, 42% were related to drugs, 16% to inpatient
care, and 16% to ambulatory care; however, the majority
of ambulatory care was related to medical visits, not phys-
iotherapy.73 Higher costs are related to lower educational
levels, higher depression scores, more pain and stiffness,
longer disease duration, higher disease activity, and greater
functional disability.71,73
The indirect costs of ankylosing spondylitis are consider-
able owing to the long duration of disease (throughout most
of working life) and the limitation of activities due to spinal
and extraspinal disease. In the German Collaborative Arthri-
tis Centres study, indirect costs for those of working age due
to sick leave, early retirement, and lost productivity were
estimated as a mean of €7204 by the frictional costs method
and €13,513 by the human capital approach,42 accounting
for 49% or 72.8%, respectively, of the total cost of illness.
Workforce participation was 11% lower, and ankylosing
spondylitis–related work disability was 15% higher, than in
 448 woolf  |  Economic Burden of Rheumatic Diseases
the general Dutch population.74 In a 2-year follow-up study
in three European countries among people of working age,
lower employment was found only in the Netherlands, but
there was increased work disability in all three countries, with
an adjusted work disability rate of 41% in the Netherlands,
23% in France, and 9% in Belgium.75 It is clear that factors
other than ankylosing spondylitis play a role in determining
work status and productivity costs, and these factors, such as
patient characteristics and differences in employment and
social security systems, must be considered when interpret-
ing data and making comparisons between populations.71
Patient costs and intangible costs can be substantial, but
the data are limited. Out-of-pocket expenses can include a
wide spectrum of additional costs, such as transportation,
paid help in the home, home modifications, heating, and
private physiotherapy. Such out-of-pocket expenses were a
mean of €517 in the German study.42 It is more difficult to
determine the impact on quality of life, the influence on
career choices by the patient or caregiver, and many other
consequences that ankylosing spondylitis can have.
OSTEOPOROSIS
Osteoporosis manifests clinically as fragility fractures, and
its socioeconomic impact relates to these fractures. They
occur most commonly at the distal forearm, hip, and spine.
The prevalence of osteoporosis and the incidence of fragility
increase with age. Their impact increases with comorbidity.
Therefore, costs are dependent on the age of the population
being considered. The costs incurred, and whether they are
attributed to health or social care, depend on clinical man-
agement practices and existing systems of long-term care
and support.
The direct medical expenditure is attributable predomi-
nantly to hospitalization. In Sweden in 1996, there were
54,000 admissions for fractures in men and women aged
50 years or older, accounting for 600,000 hospital-bed days
in a population of just over 9 million, 30% of whom were
50 years or older.76 Duration of hospital stay increased with
age. Hip fractures accounted for 69% of hospital-bed days in
men and 73% in women.
The total direct medical expenditure for osteoporotic
fractures in the United States in 1995 was estimated at
$13.8 billion.77 In the United Kingdom, the total costs
were estimated at £942 million in 1998 for all osteoporotic
fractures, of which only 45% was estimated to be for direct
costs,78 although the cost of antifracture therapy was not
fully included. These estimates were revised to £1.7 b­illion
in 200178a and are predicted to increase further with the
aging population. In Sweden, hospital days for osteoporotic
fr­actures accounted for higher costs than for myocardial
infarction, breast cancer, and prostate cancer combined.76
Across Europe, it has been estimated that osteoporotic frac-
tures cost €36,248 million—€24,353 million for hip fracture,
€719 million for vertebral fracture, and €11,177 million for
other osteoporotic fractures.79 The total costs for women are
estimated to be threefold those for men.
In Canada, the cost of hip fracture has been estimated
at US$22,292 during the first year; 58% was related to
the in­itial hospitalization and inpatient rehabilitation,
but there was wide variation, depending on the patient’s
age and the care setting before and after the fracture.80
Estimates of the total cost of hip fractures in the United
States range from $24,000 to $33,500 (1995 dollars).81 In
the United Kin­gdom, hip fractures are most expensive, esti-
mated to account for 87% of the total costs of osteoporotic
fractures in women; 60% of their cost is indirect owing to
the need for long-term hospital and community care.78 The
acute hospital cost of a hip fracture was estimated at £4808
in 1998, with a total cost per fracture of £12,124.78 A more
recent bottom-up costing of hip fractures in the United
Kingdom was ca­lculated at a mean of £12,163 for acute
hospitalization costs per patient in 2003; ward costs con-
tributed 84%, operative costs 9%, and investigations 7%.82
The direct costs of hip fracture in Sweden over 12 months,
excluding community care, were estimated at SKr 63,420
(approximately €6000).83 The mean length of stay in Swe-
den is much shorter than in the United Kingdom—12 to13
days76 compared with 23 days.82
Direct costs of distal forearm fractures are less, but one in
five men and women were admitted to a hospital in a U.K.
study84; the proportion requiring admission increased with
age, from 14.5% at age 50 to 59 years to 26% at age 80 years
and older. Wrist fracture has been estimated to cost £468
(£368 for acute costs).78
Vertebral fractures present acutely in only about one
third of cases, but they were responsible for almost 70,000
hospital admissions in 1997 in the U.S. Nationwide Inpa-
tient Sample, about one fourth the number of admissions
resulting primarily from hip fracture. Hospitalization rates
increased with age and comorbidity. Hospital stays aver-
aged almost 6 days each and generated charges of $8000 to
$10,000.84a More than half the patients required posthos-
pital discharge care. Hospitalization for vertebral fracture
resulted in almost 400,000 total hospital days that gener-
ated charges in excess of $500 million a year. In the United
Kingdom, vertebral fracture has been estimated to cost £479
(£96 for acute costs).78
In the age group that sustains hip fracture and the asso-
ciated comorbidities, it is more meaningful to consider the
attributable costs. In the United States, the attributable
costs were $3300 for a hip fracture and $1300 for other
nonvertebral fractures, based on Medicaid data.85 In a case-
control study of osteoporotic fractures in Olmsted County,
Minnesota, the incremental direct medical costs for the
year following an osteoporotic fracture were found to be
greatest following distal femur fracture ($11,756) and hip
fracture ($11,241).86 Similar figures have been reported in
Europe.87
The costs of fracture prevention need to be considered.
This includes both testing, such as bone densitometry, and
treatment, such as with bisphosphonates. There has been a
10-fold increase in bone mineral density testing in Ontario,
Canada—from about 37,000 tests in 1992 to more than
404,000 in 2001; in addition, there has been an increase in
the number of people filling at least one prescription for an
antiresorptive agent—from about 12,000 in 1996 to nearly
226,000 in 2003.88
The indirect costs related to social care are a major com-
ponent of the cost of hip fracture: only about 45% of patients
are discharged home, and 20% require long-term residential
care after discharge from the hospital.78 However, some were
already in long-term care facilities before the fracture,76 so
there would be little additional cost. Productivity loss is
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
not important because of the age at which fractures occur,
although caregivers may be affected.
With the increased prevalence of fracture with age and
the global increase in the aged population, it is estimated
that the burden and associated costs will increase signifi-
cantly—in Europe, from €36.3 million in 2000 to €76.8
million by 205079—unless effective preventive strategies are
implemented.
BACK PAIN
Back pain is most often nonspecific in cause and transient,
although it may be recurrent. A small number of patients
have chronic back pain with continuous symptoms for more
than 3 months, and they account for the largest percentage
of costs.89 In the United States, only 4.6% to 8.8% of cases
lasted longer than 1 year, but they accounted for 64.9%
to 84.7% of the costs.89 In Jersey in the United Kingdom,
only 3% of patients were absent from work for more than 6
months, but they accounted for 33% of the benefits paid.90
The total cost in OECD countries corresponds to 1% to
2% of the gross national product; about 10% of the costs
are direct and 90% indirect.91 Most of the costs are related
to work loss. The economic impact is dependent on the
country’s system of social support, and the methods used to
identify and estimate the costs vary, making comparisons
between studies and countries difficult.
A cost-of-illness study of low back pain in the United
Kingdom estimated direct costs in 1998 at £1.7 billion; total
costs were between £6.6 billion and £12.3 billion, depend-
ing on the method used.92 In Sweden in 1994 and 1995,
back pain was responsible for almost half the economic
burden related to musculoskeletal conditions, costing SKr
25 billion to 29 billion (US$2.5 billion to $3.0 billion), of
which SKr 24 billion to 27 billion was spent on productivity
losses.18 These societal costs of back pain equate to US$290
per inhabitant per year (1991 dollars) in Sweden and $323
in the Netherlands and about 80% to 90% of these amounts
in the United Kingdom.
In Sweden, the total costs of low back pain to society
were estimated at €1860 million in 2001. The indirect costs
due to lost productivity accounted for 84% of this total
cost.93 This represented 11% of the total costs of short-term
sick leave; about 13% of all early retirement pensions were
granted for back problems. Although substantial, these costs
have not changed greatly over 10 to 15 years.
Health resource utilization varies between countries
and is dependent on clinical practice and the use of inpa-
tient treatment, including investigations such as mag-
netic resonance imaging and the frequency of surgery. In
general, expenditures are related predominantly to ambu-
latory care and are distributed between physicians and ther­
apists (including osteopaths, chiropractors, and alternative
the­rapists), depending on clinical practice. Physician visits
vary from 1.1 per year in the Netherlands to 3.71 in the
United Kingdom.91 Surgery is more common in the United
States than in most European countries.94 Based on the
1998 Medical Expenditure Panel Survey, the total health
care expenses incurred by individuals with back pain in the
United States was estimated at $90.7 billion.95 The total
incremental expenditure attributable to back pain was esti-
mated at $26.3 billion; the health costs of individuals with
449
back pain were about 60% higher than those without back
pain.95 In those with back pain, 75% of the total service
expenditure was attributable to the 25% of patients with
the highest costs.
Number of sick days varies between countries and says
more about a country’s system of workers’ compensation
than about disease severity. In the United States, the back
was the body part most frequently affected in injuries
involving days away from work in a worker health review.96
In the 1998 U.S. National Health Interview Survey,97 the
prevalence of back pain lasting more than a week was
17.6%, and 4.6% had low back pain associated with lost
work.98 This amounted to 149 million lost days annually.
The annual cost of lost work time associated with chronic
low back pain has been estimated at $1230 for males
and $773 for females, based on data from the 1987 U.S.
National Medical Care Expenditure Survey; this translates
into annual productivity losses of about $28 billion.99 The
annual compensation cost of work loss due to medically
certified low back pain in the working population of Jersey,
United Kingdom, was £3.16 million per 100,000 in 1994—
10.5% of the total paid for all claimed absences.90 Low back
disorders are the most co­mmon reason for disability pen-
sions in Norway.100
SUMMARY
The economic impact of musculoskeletal conditions
is great. They have a major effect on the indirect costs
of health care. The direct costs reflect the supply rather
than the demand and do not indicate what health gain
is achieved. The documented expenditure is clearly an
underestimate of need, as there is little priority for these
conditions and not much investment in preventive
approaches. Many patients do not present for treatment
or do not comply for fear of side effects. Highlighting this
significant economic impact can be a means of gaining pri-
ority for these conditions, but it can also help identify the
spectrum of cost items that need to be considered in any
economic evaluation of strategies to reduce the burden of
musculoskeletal conditions.
REFERENCES
  1. Woolf AD, Pfleger B: Burden of major musculoskeletal conditions.
Bull World Health Organ 81:646-656, 2003.
  2. European Action towards Better Musculoskeletal Health. Lund, Sweden,
Bone and Joint Decade, 2004. http://europa.eu.int/comm/health/ph_
­projects/2000/promotion/fp_promotion_2000_exs_15_en.pdf.
  3. Woolf AD, Akesson K: Understanding the burden of musculoskeletal
conditions: The burden is huge and not reflected in national health
priorities. BMJ 322:1079-1080, 2001.
  4. Rice DP, Hodgson TA, Kopstein AN: The economic costs of illness:
A replication and update. Health Care Financ Rev 7:61-80, 1985.
  5. Pritchard C, Sculpher M: Productivity Costs: Principles and Prac-
tice in Economic Evaluation. London, Office of Health Economics,
2000.
  6. Merkesdal S, Ruof J, Huelsemann JL, et al: Development of a matrix
of cost domains in economic evaluation of rheumatoid arthritis.
J Rheumatol 28:657-661, 2001.
  7. Rice D: The economic burden of musculoskeletal conditions, 1995.
In Praemar A, Furner S, Rice DP (eds): Musculoskeletal Conditions
in the United States. Rosemont, Ill, American Academy of Orthopedic
Surgeons, 1999, pp 27-33.
 450 woolf  |  Economic Burden of Rheumatic Diseases
  8. Murray CJL, Lopez AD (eds): The Global Burden of Disease:
A Comprehensive Assessment of Mortality and Disability from
Diseases, Injuries, and Risk Factors in 1990 and Projected to
2020. Cambridge, Mass, Harvard University Press, 1996.
  9. Byford S, Torgerson DJ, Raftery J: Economic note: Cost of illness
studies. BMJ 320:1335, 2000.
10. Mooney G, Wiseman V: Burden of disease and priority setting.
Health Econ 9:369-372, 2000.
11. Maetzel A: Cost-effectiveness analysis: Out of touch with clinical
reality? Arthritis Rheum 53:3-4, 2005.
12. Gabriel SE, Tugwell P, Drummond M: Progress towards an
OMERACT-ILAR guideline for economic evaluations in rheuma-
tology. Ann Rheum Dis 61:370-373, 2002.
13. Weinstein MC, O’Brien B, Hornberger J, et al: Principles of good
practice for decision analytic modeling in health-care evaluation:
Report of the ISPOR Task Force on Good Research Practices—Mod-
eling Studies. Value Health 6:9-17, 2003.
14. European Opinion Research Group EEIG: Health, Food and Alcohol
and Safety: Special Eurobarometer 186. Brussels, European Commis-
sion, Directorate General, Communication Public Opinion Analysis
Sector, 2003.
15. Hawker GA, Wright JG, Coyte PC, et al: Differences between men
and women in the rate of use of hip and knee arthroplasty. N Engl J
Med 342:1016-1022, 2000.
16. Woolf AD, Zeidler H, Haglund U, et al: Musculoskeletal pain in
Europe: Its impact and a comparison of population and medical per-
ceptions of treatment in eight European countries. Ann Rheum Dis
63:342-347, 2004.
17. Slobbe LCJ, Kommer GJ, Smit JM, et al: Cost of Illness in the Netherlands
2003. http://www.rivm.nl/bibliotheek/rapporten/270751010.pdf.
18. Jonsson D, Husberg M: Socioeconomic costs of rheumatic dis-
eases: Implications for technology assessment. Int J Technol
Assess Health Care 16:1193-1200, 2000.
19. World Health Organization: International Classification of Diseases.
9th ed. Geneva, WHO, 1977.
20. Meerding WJ, Bonneux L, Polder JJ, et al: Demographic and epide-
miological determinants of healthcare costs in Netherlands: Cost of
illness study. BMJ 317:111-115, 1998.
21. Health Canada: The Economic Burden of Illness in Canada 1998.
http://www.hc-sc.gc.ca.
22. Stewart WF, Ricci JA, Chee E, et al: Lost productive time and
cost due to common pain conditions in the US workforce. JAMA
290:2443-2454, 2003.
23. Update: Direct and indirect costs of arthritis and other rheumatic
conditions—United States, 1997. MMWR 53:388–389, 2004.
24. Direct and indirect costs of arthritis and other rheumatic condi-
tions—United States, 1997. MMWR 52:1124–1127, 2003.
25. Dunlop DD, Manheim LM, Yelin EH, et al: The costs of arthritis.
Arthritis Rheum 49:101-113, 2003.
26. Yelin E, Murphy L, Cisternas MG, et al: Medical care expenditures
and earnings losses among persons with arthritis and other rheu-
matic conditions in 2003, and comparisons with 1997. Arthritis
Rheum 56:1397-1407, 2007.
27. Yelin E, Cisternas MG, Pasta DJ, et al: Medical care expenditures and
earnings losses of persons with arthritis and other rheumatic condi-
tions in the United States in 1997: Total and incremental estimates.
Arthritis Rheum 50:2317-2326, 2004.
28. Rasker JJ: Rheumatology in general practice. Br J Rheumatol 34:
494-497, 1995.
29. Hootman JM, Helmick CG, Schappert SM: Magnitude and character-
istics of arthritis and other rheumatic conditions on ambulatory medical
care visits, United States, 1997. Arthritis Rheum 47:571-581, 2002.
30. Coyte PC, Asche CV, Croxford R, Chan B: The economic cost of mus-
culoskeletal disorders in Canada. Arthritis Care Res 11:315-325, 1998.
31. The Office of Health Economics Compendium, 18th ed. Abingdon
UK, Radcliffe Publishing, 2007.
32. Szubert Z, Sobala W, Zycinska Z: [The effect of system restructuring
on absenteeism due to sickness in the work place. I. Sickness absen-
teeism during the period 19989-1994]. Med Pr 48:543-551, 1997.
33. Tellnes G, Bjerkedal T: Epidemiology of sickness certification—a
methodological approach based on a study from Buskerud County in
Norway. Scand J Soc Med 17:245-251, 1989.
34. Stansfeld S, Feeney A, Head J, et al: Sickness absence for psychiatric
illness: The Whitehall II study. Soc Sci Med 40:189-197, 1995.
35. Brage S, Nygard JF, Tellnes G: The gender gap in musculoskeletal-
related long-term sickness absence in Norway. Scand J Soc Med
26:34-43, 1998.
36. National Board of Health and Welfare (Sweden): Yearbook of Health
and Medical Care 2001. Stockholm, Socialstyrelsen.
37. Birnbaum HG, Barton M, Greenberg PE, et al: Direct and indirect
costs of rheumatoid arthritis to an employer. J Occup Environ Med
42:588-596, 2000.
38. Cooper NJ: Economic burden of rheumatoid arthritis: A systematic
review. Rheumatology (Oxford) 39:28-33, 2000.
39. Felts W, Yelin E: The economic impact of the rheumatic diseases in
the United States. J Rheumatol 16:867-884, 1989.
40. Kramer JS, Yelin EH, Epstein WV: Social and economic impacts of
four musculoskeletal conditions: A study using national community-
based data. Arthritis Rheum 26:901-907, 1983.
41. Girard F, Guillemin F, Novella JL, et al: Health-care use by rheuma-
toid arthritis patients compared with non-arthritic subjects. Rheuma-
tology (Oxford) 41:167-175, 2002.
41a. Kuper IH, Prevoo ML, van Leeuwen MA, et al: Disease-associated
time consumption in early rheumatoid arthritis. J Rheumatol 2000;
27(5):1183-1189.
42. Huscher D, Merkesdal S, Thiele K, et al: Cost of illness in rheumatoid
arthritis, ankylosing spondylitis, psoriatic arthritis and systemic lupus
erythematosus in Germany. Ann Rheum Dis 65:1175-1183, 2006.
43. Fautrel B, Guillemin F: Cost of illness studies in rheumatic diseases.
Curr Opin Rheumatol 14:121-126, 2002.
44. Hulsemann JL, Mittendorf T, Merkesdal S, et al: Direct costs related
to rheumatoid arthritis: The patient perspective. Ann Rheum Dis
64:1456-1461, 2005.
45. Verstappen SM, Boonen A, Verkleij H, et al: Productivity costs among
patients with rheumatoid arthritis: The influence of methods and sources
to value loss of productivity. Ann Rheum Dis 64:1754-1760, 2005.
46. Cross MJ, March LM, Lapsley HM, et al: Patient self-efficacy and
health locus of control: Relationships with health status and arthri-
tis-related expenditure. Rheumatology (Oxford) 45:92-96, 2006.
47. Yelin E: The earnings, income, and assets of persons aged 51-61 with and
without musculoskeletal conditions. J Rheumatol 24:2024-2030, 1997.
48. Jantti J, Aho K, Kaarela K, Kautiainen H: Work disability in an
inception cohort of patients with seropositive rheumatoid arthritis:
A 20 year study. Rheumatology (Oxford) 38:1138-1141, 1999.
49. Albers JM, Kuper HH, van Riel PL, et al: Socio-economic conse-
quences of rheumatoid arthritis in the first years of the disease. Rheu-
matology (Oxford) 38:423-430, 1999.
50. Gabriel SE, Crowson CS, Campion ME, O’Fallon WM: Indirect and non-
medical costs among people with rheumatoid arthritis and osteoarthritis
compared with nonarthritic controls. J Rheumatol 24:43-48, 1997.
51. Burton W, Morrison A, Maclean R, Ruderman E: Systematic review
of studies of productivity loss due to rheumatoid arthritis. Occup Med
56:18-27, 2006.
52. Clarke AE, Zowall H, Levinton C, et al: Direct and indirect medical
costs incurred by Canadian patients with rheumatoid arthritis: A 12
year study. J Rheumatol 24:1051-1060, 1997.
53. Eberhardt K, Larsson BM, Nived K, Lindqvist E: Work disability in
rheumatoid arthritis—development over 15 years and evaluation of
predictive factors over time. J Rheumatol 34:481-487, 2007.
54. Cooper NJ, Mugford M, Symmons DP, et al: Total costs and pre-
dictors of costs in individuals with early inflammatory polyarthritis:
A community-based prospective study. Rheumatology (Oxford) 41:
767-774, 2002.
55. Hawley DJ, Wolfe F, Pincus T: Use of combination therapy in the
routine care of patients with rheumatoid arthritis: Physician and
patient surveys. Clin Exp Rheumatol 17(6 Suppl 18):S78-S82, 1999.
56. Henry D, Lim LL, Garcia Rodriguez LA, et al: Variability in risk of
gastrointestinal complications with individual non-steroidal anti-
inflammatory drugs: Results of a collaborative meta-analysis. BMJ
312:1563-1566, 1996.
57. March LM, Bachmeier CJ: Economics of osteoarthritis: A global per-
spective. Baillieres Clin Rheumatol 11:817-834, 1997.
58. Levy E, Ferme A, Perocheau D, Bono I: [Socioeconomic costs of
osteoarthritis in France]. Rev Rhum Fr Ed 60:63S-67S, 1993.
59. MacLean CH, Knight K, Paulus H, et al: Costs attributable to
osteoarthritis. J Rheumatol 25:2213-2218, 1998.
60. Moore RA: The hidden costs of arthritis treatment and the cost of
new therapy—the burden of non-steroidal anti-inflammatory drug
gastropathy. Rheumatology (Oxford) 41(Suppl 1):7-15, 2002.
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
61. Ramsey SD, Spencer AC, Topolski TD, et al: Use of alternative
therapies by older adults with osteoarthritis. Arthritis Rheum 45:
222-227, 2001.
62. Juni P, Dieppe P, Donovan J, et al: Population requirement for pri-
mary knee replacement surgery: A cross-sectional study. Rheumatol-
ogy (Oxford) 42:516-521, 2003.
62a. Holland R, Harvey I: Population needs assessment and knee replace-
ment surgery. Rheumatology (Oxford) 2003; 42(4):503-506.
63. Merx H, Dreinhofer K, Schrader P, et al: International variation in
hip replacement rates. Ann Rheum Dis 62:222-226, 2003.
64. Malchau H, Herberts P, Eisler T, et al: The Swedish Total Hip Replace-
ment Register. J Bone Joint Surg Am 84(Suppl 2):2-20, 2002.
65. Dreinhofer KE, Feron JM, Herrera A, et al: Orthopaedic surgeons
and fragility fractures: A survey by the Bone and Joint Decade and
the International Osteoporosis Foundation. J Bone Joint Surg Br 86:
958-961, 2004.
66. Dixon T, Shaw M, Ebrahim S, Dieppe P: Trends in hip and knee joint
replacement: Socioeconomic inequalities and projections of need.
Ann Rheum Dis 63:825-830, 2004.
66a. Frankel S, Eachus J, Pearson N, et al: Population requirement for pri-
mary hip-replacement surgery: A cross-sectional study. Lancet 1999;
353(9161): 1304-1309.
67. Vale L, Wyness L, McCormack K, et al: A systematic review of the effec-
tiveness and cost-effectiveness of metal-on-metal hip resurfacing arthro-
plasty for treatment of hip disease. Health Technol Assess 6:1-109, 2002.
68. Pincus T, Mitchell JM, Burkhauser RV: Substantial work disability and
earnings losses in individuals less than age 65 with osteoarthritis: Com-
parisons with rheumatoid arthritis. J Clin Epidemiol 42:449-457, 1989.
69. Lapsley HM, March LM, Tribe KL, et al: Living with osteoarthri-
tis: Patient expenditures, health status, and social impact. Arthritis
Rheum 45:301-306, 2001.
70. Zink A, Thiele K, Huscher D, et al: Healthcare and burden of disease
in psoriatic arthritis: A comparison with rheumatoid arthritis and
ankylosing spondylitis. J Rheumatol 33:86-90, 2006.
71. Boonen A, Severens JL: Ankylosing spondylitis: What is the cost
to society, and can it be reduced? Best Pract Res Clin Rheumatol
16:691-705, 2002.
72. Boonen A, van der Heijde D, et al: Direct costs of ankylosing spon-
dylitis and its determinants: An analysis among three European
countries. Ann Rheum Dis 62:732-740, 2003.
73. Ward MM: Functional disability predicts total costs in patients with
ankylosing spondylitis. Arthritis Rheum 46:223-231, 2002.
74. Boonen A, Chorus A, Miedema H, et al: Employment, work disability,
and work days lost in patients with ankylosing spondylitis: A cross
sectional study of Dutch patients. Ann Rheum Dis 60:353-358, 2001.
75. Boonen A, van der Heijde D, et al: Work status and productivity
costs due to ankylosing spondylitis: Comparison of three European
countries. Ann Rheum Dis 61:429-437, 2002.
76. Johnell O, Kanis JA, Jonsson B, et al: The burden of hospitalised
fractures in Sweden. Osteoporos Int 16:222-228, 2005.
77. Ray NF, Chan JK, Thamer M, Melton LJ III: Medical expenditures
for the treatment of osteoporotic fractures in the United States in
1995: Report from the National Osteoporosis Foundation. J Bone
Miner Res 12:24-35, 1997.
78. Dolan P, Torgerson DJ: The cost of treating osteoporotic fractures in the
United Kingdom female population. Osteoporos Int 8:611-617, 1998.
78a. Torgerson DJ, Iglesias CP, Reid DM: The economics of fracture preven-
tion. In Barlow DH, Francis RM, Miles A (eds): The Effective Man-
agement of Osteoporosis. UK Advances in Clinical Practice Series.
London: Aesculapius Medical Press, 2001, pp 111-121.
79. Kanis JA, Johnell O: Requirements for DXA for the management of
osteoporosis in Europe. Osteoporos Int 16:229-238, 2005.
451
80. Wiktorowicz ME, Goeree R, Papaioannou A, et al: Economic impli-
cations of hip fracture: Health service use, institutional care and cost
in Canada. Osteoporos Int 12:271-278, 2001.
81. Tosteson AN: Economic impact of fractures. In Orwoll E (ed): The
Effects of Gender on Skeletal Health. San Diego, Calif, Academic
Press, 1999, pp 15-27.
82. Lawrence TM, White CT, Wenn R, Moran CG: The current hospital
costs of treating hip fractures. Injury 36:88-91, 2005.
83. Zethraeus N, Borgstrom F, Johnell O, et al: Costs and quality of
life associated with osteoporosis related fractures: Results from
a Swedish survey. SSE/EFI Working Paper Series in Economics
and Finance No. 512, 2002. Stockholm School of Economics,
­Sweden.
84. O’Neill TW, Cooper C, Finn JD, et al: Incidence of distal forearm frac-
ture in British men and women. Osteoporos Int 12:555-558, 2001.
84a. Gehlbach SH, Burge RT, Puleo E, Klar J: Hospital Care of osteoporo-
sis-related vertebral fractures. Osteoporos Int 2003; 14(1):53-60.
85. Martin BC, Chisholm MA, Kotzan JA: Isolating the cost of osteopo-
rosis-related fracture for postmenopausal women: A population-based
study. Gerontology 47:21-29, 2001.
86. Gabriel SE, Tosteson AN, Leibson CL, et al: Direct medical costs attrib-
utable to osteoporotic fractures. Osteoporos Int 13:323-330, 2002.
87. Autier P, Haentjens P, Bentin J, et al: Costs induced by hip fractures:
A prospective controlled study in Belgium. Belgian Hip Fracture
Study Group. Osteoporos Int 11:373-380, 2000.
88. Jaglal SB, Weller I, Mamdani M, et al: Population trends in BMD
testing, treatment, and hip and wrist fracture rates: Are the hip frac-
ture projections wrong? J Bone Miner Res 20:898-905, 2005.
89. Hashemi L, Webster BS, Clancy EA: Trends in disability duration
and cost of workers’ compensation low back pain claims (1988-1996).
J Occup Environ Med 40:1110-1119, 1998.
90. Watson PJ, Main CJ, Waddell G, et al: Medically certified work loss, recur-
rence and costs of wage compensation for back pain: A follow-up study of
the working population of Jersey. Br J Rheumatol 37:82-86, 1998.
91. Norlund AI, Waddell G: Cost of back pain in some OECD coun-
tries. In Nachemson A, Jonsson E (eds): Neck and Back Pain:
The Scientific Evidence of Causes, Diagnosis, and Treatment.
Philadelphia, Lippincott Williams & Wilkins, 2000, pp 421-425.
92. Maniadakis N, Gray A: The economic burden of back pain in the
UK. Pain 84:95-103, 2000.
93. Ekman M, Johnell O, Lidgren L: The economic cost of low back pain
in Sweden in 2001. Acta Orthop 76:275-284, 2005.
94. Deyo RA, Cherkin D, Conrad D, Volinn E: Cost, controversy, crisis:
Low back pain and the health of the public. Annu Rev Public Health
12:141-156, 1991.
95. Luo X, Pietrobon R, Sun SX, et al: Estimates and patterns of direct
health care expenditures among individuals with back pain in the
United States. Spine 29:79-86, 2004.
96. Worker Health Chartbook 2004. Cincinnati, Ohio, U.S. Depart-
ment of Health and Human Services, CDC (NIOSH), 2004. www.
cdc.gov/niosh/docs/chartbook/pdfs/Chartbook_2004_NIOSH.pdf.
97. Guo HR, Tanaka S, Cameron LL, et al: Back pain among workers in
the United States: National estimates and workers at high risk. Am J
Ind Med 28:591-602, 1995.
98. Guo HR, Tanaka S, Halperin WE, Cameron LL: Back pain preva-
lence in US industry and estimates of lost workdays. Am J Public
Health 89:1029-1035, 1999.
99. Rizzo JA, Abbott TA III, Berger ML: The labor productivity effects of
chronic backache in the United States. Med Care 36:1471-1488, 1998.
100. National Insurance Administration, Norway. 1998.
 30
KEY POINTS
Clinical trials play an important role in new drug
development, but also can serve to explore existing drugs or
interventions further or refine their use.
Pragmatic trials have a lower level of internal validity
(methodologic robustness) compared with explanatory trials,
but a higher level of external validity (generalizability).
A fundamental choice in the consideration of the design of
the study is to decide about a superiority design or a noninfe-
riority design.
An appropriate trial design is of decisive importance to
optimize the likelihood that the trial would provide results
that are interpretable, robust, and applicable. The decisions
made during the design phase are a reflection of the continu-
ous balance between internal validity and external validity.
Disturbing factors, such as missing data, dropout, and
confounding, may jeopardize the interpretation of the trial
results. The consumers of trial results (investigators,
pharmaceutical industry workers, readers of medical journals)
should be challenged to interpret the results of a trial in the
light of these potentially disturbing factors.
Clinical trials are studies designed to assess the efficacy and
toxicity of drugs or other interventions. Although the term
clinical trial often refers to randomized clinical trial (RCT),
every study in which patients are exposed to an interven-
tion and in which data are systematically collected can be
considered as a clinical trial. Clinical trials play an impor-
tant role in new drug development, but also can serve to
explore existing drugs or interventions further or refine
their use, such as the determination of predictive factors for
treatment efficacy, or to test treatment strategies. This chap-
ter is broadly confined to RCTs. It discusses methodologic
principles of clinical trials and RCT analysis in the context
of rheumatology, and it unveils limitations of RCTs, while
briefly discussing alternative solutions in design and analy-
sis. This chapter is intended as an introduction for clini-
cians and researchers working in rheumatology.
TRIAL DESIGN
RANDOMIZED CLINICAL TRIALS
The classic template of an RCT includes two (or more) trial
arms, comparing the drug or intervention of interest (e.g., the
new drug) with a control intervention. The latter may include
a placebo or sham intervention, or an intervention that is
considered to represent standard care. By definition, the treat-
ment arms are created by the process of randomization, which
is pivotal and is outlined subsequently in more detail.
Clinical Trial Design
and Analysis
ROBERT B. M. LANDEWÉ •
DÉSIRÉE M. F. M. VAN DER HEIJDE
To understand differences in trial design better, it is often
helpful to distinguish explanatory RCTs and pragmatic
RCTs.1-3 Trials of new drugs, such as trials designed for drug
registration, aiming at showing efficacy and short-term
safety, belong to the group of explanatory RCTs. Generally,
all elements of trial design, such as the selection of patients,
the sample size, the choice of the comparative intervention,
and the duration of the trial, are chosen in such a manner
that the trial can optimally show a treatment effect (i.e., a
difference in efficacy between the new drug and the con-
trol intervention). The methodologic robustness of a trial,
which depends on these elements of trial design, is referred
to as internal validity. Explanatory trials do not always resem-
ble clinical practice. For methodologic reasons, they often
include patients with a high level of disease activity that
form only a minority in clinical practice.
The extent to which clinical trial results can be extrap-
olated to common clinical practice is referred to as exter-
nal validity. As a rule of thumb, explanatory trials have a
high level of internal validity, which may be at the cost
of external validity. Pragmatic trials more closely resemble
the clinical situation. Such trials aim at optimizing treat-
ment by exploring existing drugs or treatment strategies
further. Pragmatic trials incorporate fundamental principles
of RCTs, such as randomization, but include a more realis-
tic representation of patients, may have a longer duration,
and may allow cointerventions. Generally, pragmatic tri-
als have a lower level of internal validity compared with
explanatory trials, but a higher level of external validity.
Often, explanatory trials are initiated and sponsored by the
pharmaceutical industry, whereas most pragmatic trials are
investigator-driven initiatives sponsored by the academic
community.
Randomization
Randomization, the process by which patients are assigned
to treatment by chance, is the most important methodo-
logic characteristic of an RCT and warrants some explana-
tion. Randomization makes treatment arms similar for all
variables except treatment, or, in other words, randomiza-
tion divides all known and unknown variables that may or
may not be of prognostic importance equally across treat-
ment groups, reducing the probability that factors other
than treatment may influence the results. Randomization
does not completely preclude imbalances. Differences in
variables that are of prognostic importance may occur sim-
ply by chance. Randomization precludes intentional imbal-
ances, however (e.g., dissimilarities created by physicians
who consider a particular treatment more appropriate for a
particular patient [selection]).
453
 454 LANDEWÉ  |  Clinical Trial Design and Analysis
From a statistical perspective, chance differences occur
more frequently in small study samples than in larger ones
and may be of higher magnitude in small trials. It is neces-
sary to compare treatment groups at baseline with respect
to important prognostic variables, and to adjust for differ-
ences in the statistical analysis in case of doubt. Usually,
computer-generated randomization lists are used to random-
ize patients in a RCT. Technically, randomization is often
performed in “blocks” so that in every block of 4 or 10, there
is an equal number of patients in the treatment and control
groups. Randomizing in blocks ensures that if the sample
size is less than expected, there is an equal proportion of
patients in each treatment group. Often, in multicenter tri-
als, one center is assigned one or more blocks, ensuring that
the number of patients receiving the new drug and the con-
trol drug are distributed evenly per center.
Some trials randomly enroll patients in strata (stratifi-
cation) of equal or unequal size. Stratification (a better
wording is stratified randomization) makes sense only if the
variable subject for stratification represents a prognostically
important feature. An appropriate example is a situation
in which there is circumstantial evidence that the efficacy
of a treatment is different in men compared with women.
Stratified randomization with “men” and “women” as strata
implies that randomization to treatment groups occurs after
the assignment of the appropriate stratum. It allows a justifi-
able comparison between the treatment groups within each
stratum because there is prognostic similarity at baseline.
Appropriate stratified randomization requires a trial design
and a sample size that allows such a comparison (see later
discussion of statistical power).
Stratified randomization should be distinguished from
post-hoc subgroup analysis, in which the “strata” are
determined during the analysis of the trial. In such post-
hoc comparisons, prognostic similarity cannot be ensured,
and statistical adjustments can account only rarely for
this.
Design Considerations
A fundamental choice in the consideration of the design
of the study is to decide about a superiority design versus a
nonsuperiority design. The latter theoretically can be cat-
egorized further as a noninferiority design and an equiva-
lence design. The basis supporting this choice is the null
hypothesis underlying the study. The consequences of the
choice for the design are important. If a new treatment is
tested against placebo, the a priori hypothesis is that this
new drug is more effective than placebo, and a superiority
design is a rational choice. If treatments are already avail-
able for a particular disease or condition, it is ethically often
not justifiable to subject patients to a placebo treatment for
longer periods. It is not always rational to assume that a new
treatment would be better than the best available treatment
at that moment, and a superiority design would have a high
likelihood of failure. In such situations, one can opt for a
nonsuperiority design. These designs have the underlying
hypothesis that the treatment to test is at least not worse
than (or equivalent to) the comparative treatment, which
can be the standard of care or, alternatively, the best cur-
rently available treatment.
In a superiority design, the question is whether the new
treatment is more efficacious than the control intervention
(e.g., placebo). Formally, such a study tests whether the null
hypothesis of no difference between both treatment groups
can be rejected. To do so, the investigators agree on a mini-
mally clinically important difference (MCID) between the
intervention of interest and the control intervention that
such a study should be able to show, and they design the
study in such a way that this difference can be shown with
high likelihood (statistical power) when it really exists (see
later).
In a noninferiority design, the reasoning is opposite.
The null hypothesis is that the new treatment is less effi-
cacious than the control intervention.4,5 Even if the new
intervention and the control intervention are truly similarly
effective, a trial almost never would yield a result with a
treatment effect of exactly zero (no difference). There would
be variation around zero, and it is the task of the investiga-
tors to decide in the design phase of the study which devia-
tion from a treatment effect of zero they would accept to
conclude that the interventions are equivalent—termed the
noninferiority margin.
The determination of the MCID in a superiority design
and the noninferiority margin in a noninferiority design is
a subjective decision with important consequences for the
sample size. When it is important in a superiority design
to be able to show very small treatment effects with a high
likelihood, large sample sizes are needed, and the same is
true with a very narrow noninferiority margin in a nonin-
feriority design. Especially in a noninferiority design, other
considerations than efficacy alone may give guidance to the
level of the noninferiority margin. If a new drug is less toxic
or less costly than the existing drugs on the market, and
as such may have additional benefits, one could be more
lenient with regard to determining the noninferiority mar-
gin. Generally, noninferiority designs require (far) more
patients than superiority designs.
Subject Selection
Subjects who are entered into clinical studies should meet
accepted criteria for the disease or disorder under study.
Most rheumatologic conditions lack single and unequivo-
cal diagnostic tests, and classification criteria have been
developed to identify patients with similar characteristics.6
The classification criteria serve as eligibility criteria in an
RCT. To homogenize patient populations for scientific
purposes, classification criteria are designed to be highly
specific. Consequently, sensitivity may fall short, and clas-
sification criteria are often of limited use in diagnosis. The
high specificity of classification criteria has implications for
the composition of the trial population. Generally, there is
an overrepresentation of patients with classic, often severe
disease and an underrepresentation of patients with early,
less typical disease.
In many trials in rheumatology, patients also must meet
certain criteria for disease activity or duration. Some trials
require that the patient experiences a flare after withdrawal
of medication as evidence of active disease. Other studies
define disease activity before withdrawal of medication as
evidence of lack of response to current treatment. Disease
severity can be defined by accepted clinical criteria or by
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
lack of response to previous treatments. Rheumatoid arthri-
tis (RA) studies may be limited to patients who have not yet
received methotrexate (who presumably have early disease
or mild disease) or to patients who have failed treatment
with at least one other disease-modifying antirheumatic drug
(more disease severity). There are ethical and methodologic
reasons for the use of such activity or severity criteria. Ethi-
cal arguments may proscribe that a novel intervention is first
tested in patients with severe, sometimes intractable disease
in which common alternatives have failed. Methodologic
arguments are that a treatment effect can be shown best in
a population of patients prone to change. Most inflamma-
tory rheumatic diseases have a cyclic course characterized
by exacerbations and remissions. Patients with a high level
of disease activity tend to improve over time, even without
an intervention, a phenomenon known as regression toward
the mean, and the additional effect of a new intervention
compared with a control treatment can be shown more eas-
ily in such a context.
Exclusion criteria usually include conditions such as
cancer, cardiac, hepatic, or renal disease; abnormalities in
hematologic parameters; medication allergies; and preg-
nancy. Exclusion criteria decrease the background noise or
variability resulting from differences in patient characteris-
tics. Generally, inclusion and exclusion criteria homogenize
the trial patient population and contribute to an environ-
ment that is most optimal to show a treatment effect. Inclu-
sion and exclusion criteria also prevent entry of patients in
whom an adverse response is more likely to occur or patients
for whom the experimental treatment could be dangerous a
priori. Inclusion criteria and exclusion criteria contribute to
a high level of internal validity, but this jeopardizes external
validity. Explanatory trials usually have a comprehensive set
of inclusion and exclusion criteria. Pragmatic trials are more
lenient in this regard because they should reflect the com-
mon clinical practice better.
Informed Consent
Ethical considerations determine whether eligible subjects
participate in a clinical trial. Governmental agencies of
most countries require that institutions involved in human
research have a local institutional review board. The insti-
tutional review board reviews all protocols before imple-
mentation and monitors ongoing studies at an institution.
A crucial element in the review of a trial is the informed
consent process.7 The consent form should explain to the
study participant the purpose of the study, all potential
benefits and risks (including risks to pregnant mothers and
fetuses), alternatives to participation, and who is responsi-
ble for conducting the study. Patient confidentiality should
be ensured. The consent form should state clearly that par-
ticipation is completely voluntary, and that refusal to par-
ticipate or withdrawal from the study would not affect future
care. If compensation is provided, it must be documented
in the consent form. Participants should be given contact
information for questions or in case of injury and a state-
ment about whether any medical treatment would be given
if injury occurs. Investigators are responsible for ensuring
that the risk to subjects is minimized and appropriate for the
anticipated benefits.
455
Follow-Up Considerations
The optimal duration of the trial is a compromise between
economical, ethical, and methodologic considerations. A
trial should not be too short because an intervention needs
time to exert its potentially advantageous (but also deleteri-
ous) effects; in particular, a short trial does not reflect clini-
cal reality of most rheumatologic conditions. Equally, a trial
should not be too long because RCTs are expensive, patients
should not be subjected to experimental interventions with
uncertain adverse events for an excessive length of time,
and too much longitudinal bias should be avoided. Longitu-
dinal bias may occur if during the trial the treatment groups
increasingly become dissimilar owing to selective dropout,
cointerventions, and behavior of other patients or the treat-
ing physician.
Selective dropout may occur if patients with a particu-
lar profile preferably withdraw from one of the treatment
groups, creating prognostic imbalance. A common example
is that of an RA trial comparing an effective drug with pla-
cebo. Patients with severe and active disease in the placebo
group may preferably discontinue trial medication and drop
out because they do not experience benefit, whereas the
patients with less severe and less active disease remain in
the trial. Cointerventions—allowed or not allowed—may
similarly jeopardize prognostic similarity if they occur in
an unbalanced (i.e., unequal) manner across the treatment
groups. A common example is a trial that tests a nonsteroidal
anti-inflammatory drug versus placebo with respect to pain
relief. Simple analgesics that are preferentially used in the
placebo group may inadvertently influence the pain scores
and lead to incorrect conclusions. The treating physician
also may contribute to prognostic imbalance by prescribing
cointerventions, or in general terms treating patients differ-
ently according to their clinical response or the occurrence
of adverse events.
A short follow-up would decrease the likelihood that
unintended events occur and as such contributes to main-
taining prognostic similarity and increasing internal valid-
ity. Explanatory trials usually have a follow-up duration that
is as short as possible, and cointerventions are prohibited.
The most important limitation of short-term trials in life-
long rheumatologic diseases is that they do not appropri-
ately reflect the course of the disease encountered in clinical
practice. Sometimes, RCTs, especially pragmatic trials, have
a long trial duration that better reflects the clinical reality.
In such a trial, the internal validity is deliberately sacrificed
to some extent in favor of an increased external validity
(generalizability) and the yield of long-term information.
Blinding
In double-blind studies, neither the patient nor the investi-
gator is aware of the treatment group assignment. In ­single-
blind studies, the investigator is aware of the treatment
allocation, but the patient is not. In open-label studies,
the patient and the investigator are aware of the treatment
assignment. The most important reason to blind treatment
allocation is to avoid the possibility that any expectation
about the kind of treatment could influence the measured
outcome (expectation bias), especially (but not exclusively)
if the measured outcome includes subjective components.
 456 LANDEWÉ  |  Clinical Trial Design and Analysis
Subjective refers to the patient (e.g., pain scores) and to the
investigator (e.g., joint scores). To avoid the latter, many
drug trials make use of independent (joint) assessors who are
not responsible for decisions regarding patient care and are
blinded to the treatment. Another common example of a
blinded independent assessor in rheumatology is the reader
of radiographs in imaging studies in RA, psoriatic arthritis,
or ankylosing spondylitis.
Regardless of any precaution, unblinding may inadver-
tently occur because of identifiable adverse reactions or minor
side effects, lack of efficacy, or changes in laboratory param-
eters. A disturbing effect of such a type of unblinding is not
easy to prove, and it cannot be adjusted for in the analysis.
CHOICE OF THE OUTCOME VARIABLES
Any clinical trial has one or more outcome variables of
interest. Outcome is broadly defined and refers to a clinical
situation or a change in a clinical situation that is quanti-
fiable by using assessment instruments. Outcome variables
can measure real outcome, which directly affects the patient
(e.g., vertebral fracture in osteoporosis), or alternatively can
reflect a situation that is associated with real outcome, but
does not affect the patient per se (e.g., low bone mineral
density in osteoporosis). The latter type of outcome is often
termed surrogate outcome. Reasons to use surrogate outcome
measures rather than real outcome measures are that the
former occur (far) earlier and more frequently and often
can be assessed on a continuous scale (which is a statistical
advantage), whereas the latter often describe an event (the
presence or absence of a clinical situation) with negative
implications for statistical power.
The Outcome and Measurement in Rheumatoid Arthri-
tis Clinical Trials (OMERACT) initiative was started to
bring unanimity in the multitude of outcome measures in
rheumatology on the basis of expert consensus.8 It was initi-
ated in RA and expanded to include most other rheumato-
logic diseases. The OMERACT framework is the so-called
OMERACT filter, which describes the methodologic pre-
requisites that an appropriate outcome measure should ful-
fill to be considered valid for clinical trials.
The OMERACT filter prescribes three validation require-
ments: An outcome measure should be truthful, discrimi-
natory, and feasible. Truthful refers to whether or not an
outcome measure truly measures what it is intended to
measure, and approximates the concepts of face, content,
and construct validity. It means that the disease activ-
ity score (DAS) in RA should truly measure what is con-
sidered important in RA (e.g., swelling and tenderness of
joints) (content validity) and what is a relevant construct to
describe the process of RA (e.g., the DAS is associated with
radiographic progression and limited physical function)
(construct validity). Discriminatory refers to whether or not
an outcome measure can be measured reliably (intra­
observer variation and interobserver variation), whether
it can distinguish between two stages of the disease (e.g.,
RA with high disease activity versus RA with low disease
activity), whether it can be applied to groups of patients or
an individual patient, whether the measure is sensitive to
change (e.g., whether the DAS measurably decreases if the
disease improves), and whether the measure can discrimi-
nate between groups of patients on effective therapy versus
placebo or less effective therapy. Feasible refers to whether
or not an outcome measure is easily applicable and inexpen-
sive in the setting in which it is intended to use.
Eight biannual OMERACT conferences have now resulted
in sets of outcome measures for almost all inflammatory
rheumatologic diseases and for many noninflammatory dis-
orders. These so-called core sets have importantly improved
the homogeneity across different clinical trials, favoring
comparability. In the design phase of a clinical trial, it is
highly recommended to choose a primary outcome measure
from these core sets, and to measure all components of the
core set as secondary outcome measures. The reporting of
all core set measures prevents the selective reporting of only
positive results with respect to a few variables.
Increasingly, indices replace single outcome variables in
rheumatology. An index is a weighted or unweighted com-
bination of single variables that together reflect a particular
domain of outcome.9 A general rule is that indices perform
better than single-item variables only if they consist of vari-
ables that correlate moderately with each other. If variables
correlate at too high a level, there is redundancy of informa-
tion (colinearity). If variables do not correlate, they reflect
different domains, which complicates interpretability, and
it is better to describe them separately. Important examples
of useful indices in rheumatology are the already mentioned
DAS10 and the American College of Rheumatology (ACR)
response criteria in RA.11
MEASURING EFFECT
After the choice of the outcome measures, it is important
to consider how the change in outcome measures is assessed
in the clinical trial. One could simply calculate a before/
after difference in a continuous variable (a change score),
but this is statistically not the best. The ACR has devel-
oped the ACR20 response criteria as a tool to determine
in clinical trials whether one drug is more efficacious than
another or placebo.11 The ACR20 is an index, containing
several outcome measures from the World Health Organiza-
tion/International League against Rheumatism core set for
RA,12 which is based on expert consensus about different
measures to assess disease activity. The ACR20 response cri-
teria require a 20% improvement and have been thoroughly
validated in several validation steps, have been shown to
perform better in trials than individual core set measures,
and currently are the standard for measuring drug efficacy
in clinical trials.
The ACR50 and ACR70 response criteria have been
derived from the ACR20 response criteria in that they
require a higher level of improvement. These derivatives
have never been appropriately validated, but have proved to
be useful in drug research, despite an inferior discriminatory
potential compared with the ACR20 response criteria.13
A specific limitation of the ACR70 is that the baseline
disease activity needs to have a certain (high) level to allow
a 70% improvement.
The European League against Rheumatism (EULAR)
has endorsed its own response criteria in RA, which are
based on the DAS.14 The DAS is a continuous index mea-
sure that includes four core set measures in a statistically
weighted manner. The advantage of the DAS, which also
underwent numerous validation steps, is that it describes a
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
state of disease activity, rather than a response, or a change.
The EULAR response criteria are categorical response cri-
teria (good response, moderate response, and no response)
that are entirely based on the DAS and require an absolute
change in the DAS and a certain state level of the DAS.14
Another disease in which response criteria have been
developed for use in clinical trials is ankylosing spondyli-
tis. The ASsessment in Ankylosing Spondylitis (ASAS)
international working group has developed the ASAS20
response criteria, which includes four patient-oriented dis-
ease activity measures, for use in clinical trials of ankylosing
spondylitis.15
In general, the consensus-based response criteria have
contributed to better trial design, better trial conduct, and
better comparability across trials. An important drawback
is that the interpretability of the different indices (“what
does it mean that a patient has experienced an ACR20
response?”) remains difficult.
SAMPLE SIZE AND STATISTICAL POWER
Statistical power is the likelihood that if a treatment effect
truly exists (or drug A is truly better than drug B), the trial
will show this with statistical significance. A power of 0.80
(80%) means that if there is a true difference between the
treatment group and the control group, the likelihood that
this RCT will confirm that difference is 80%. At the basis of
this definition is the reasoning that truth (difference or not)
is unknown—that we never will know the truth with abso-
lute certainty, and that we can only approximate the truth
by performing RCTs. Intuitively, an RCT does not always
give the right answer. An RCT may conclude that there is
a difference, whereas in truth there is not. Alternatively, a
true difference would not be supported by the results of the
trial. The former is termed a type I error; the latter is termed
a type II error (Fig. 30-1).
The theoretical problem is that—by definition—we do
not know which RCT gives the right answer and which does
not. Although we do not know the true answer, we try to
design the trial in such a manner that type I error and type II
error are minimal. The likelihood of type I and type II error
is less with larger sample sizes. Consequently, the likelihood
of correctly drawing a conclusion from the experiment of
one RCT increases by increasing sample size. Apart from
sample size, the likelihood that a study can detect a differ-
ence between treatment groups also depends on effect size
(if the treatment has a larger effect, it is easier to detect with
smaller sample sizes) and reliability of the outcome measure
(if the outcome measure is more precise, or less influenced
by measurement error, it is easier to detect with smaller
sample size).
The probability of a type I error (false positive) is referred
to as alpha and is better known as the level of statistical sig-
nificance. The probability of a type II error (false negative) is
referred to as beta. Statistical power is defined as (1 − beta). If
beta is 10%, there is a 90% likelihood of finding a difference
between groups of the expected effect, or greater when this
difference truly exists. Usually, alpha, or the P value, is set at
0.05, and beta is set between 0.2 and 0.1 (power of 80% to
90%). It is generally considered for ethical reasons that type
I error should be avoided because it may directly and nega-
tively affect patient care (a new drug is falsely ­ considered
457
Truth
Drug A is better Drug A is not better
than drug B than drug B
True-positive trial result False-positive trial result
(Type I error)
Drug A is better
than drug B
Correct rejection of Erroneous rejection of
null-hypothesis the null-hypothesis
Trial result
False-negative
True-negative trial result
trial result
Drug A is not (Type II error)
better than
drug B Erroneous acceptance Correct acceptance of
of null-hypothesis null-hypothesis
Figure 30-1  The interpretation of the trial result in the context of the
unknown truth of a trial challenging the null hypothesis that drug A is not
better than drug B.
to be better than an existing drug). Type II error is subtler,
but should be avoided for more than one reason. First, new
effective treatments would not reach the market for false
reasons, but more importantly, trials with a high probability
of type II error are truly inconclusive. Because new drugs
may cause harm, a trial that a priori does not allow a firm
scientific conclusion is ethically unjustifiable and spends a
lot of money without any yield.
Sample size is one of the most important determinants
of the power of a study to find a treatment difference. To
determine the appropriate sample size for a study, the inves-
tigator needs to have an estimation of the effect size of the
intervention (i.e., difference in outcome between treatment
groups, or MCID), the variability of the data (e.g., standard
deviation), the statistical test to be used, and the alpha (P
value) and beta (i.e., false-negative rate) level. It is impor-
tant to consider nonspecific effects (placebo effect, regres-
sion toward the means), such as the proportion of subjects
who meet criteria for improvement in the placebo group
(reported 20% to 40% in studies of patients with RA). The
variability or standard deviation of the outcome measure
can be estimated from pilot data or from other published
clinical trials.
Often, the sample size in each treatment arm is the same,
but unequal but proportional treatment groups also can be
used (2:1 ratio of treated subjects to control). RCTs with
equal-size treatment groups have greater statistical power,
but unequal groups are sometimes used to maximize the
number of patients who receive treatment, especially if
information on safety is the most important reason for the
trial. Also, patients are sometimes more willing to enter a
trial in which they have a greater likelihood of receiving an
active treatment.
DECLARATION OF HELSINKI
The World Medical Association’s Declaration of Helsinki
(1964) is a document that spells out a set of ethical guidelines
for physicians and other participants in medical research.16
It is considered the most widely recognized source of ethical
guidance for biomedical research. The fifth and last revision
(Edinburgh, October 2000)17 contains 32 paragraphs that
 458 LANDEWÉ  |  Clinical Trial Design and Analysis
aim to find a balance between the physician’s duty to “pro-
mote and safeguard the health of the people” (paragraph 2),
implying that “the well-being of the human subject should
take preference over the interests of science and society”
(paragraph 5), and the scientific and societal appreciation
that “medical progress is based on research which ultimately
must rest in part on experimentation involving human sub-
jects” (paragraph 4), inevitably involving “risks and burden”
(paragraph 7).
The Declaration is not a static document, and the cur-
rent debate focuses on the place of placebo-controlled trials
(paragraph 29).18 Although this paragraph—which raised a
lot of argument—justifies placebo-controlled trials only in
circumstances in which “no proven prophylactic, diagnostic
or therapeutic method exists,” a Note of Clarification by the
World Medical Association outlines a more liberal interpre-
tation of this paragraph, providing circumstances in which
placebo-controlled trials are allowed even if proven therapy
is available. It is generally accepted and required by govern-
mental institutions and institutional review boards that trial
design, trial conduct, and trial report are in accordance with
the stipulations of the Declaration of Helsinki.
PLACE OF NONINFERIORITY DESIGNS
IN RHEUMATOLOGY
Paragraph 29 of the Declaration, issuing the place of placebo-
controlled trials, has generated interest in designing noninfe-
riority trials in rheumatology. The more recently developed
biological therapies have had an important impact on the
treatment of chronic inflammatory diseases. If paragraph
29 of the Declaration of Helsinki is interpreted conserva-
tively, this means that placebo-controlled trials are ethically
not justifiable anymore in RA, ankylosing spondylitis, and
psoriatic arthritis. It immediately follows from paragraph 30
of the Declaration (“every patient entering into the study
should be assured of access to the best proven … therapeutic
methods identified by the study”) that future trials investi-
gating new treatments in these diseases should include the
best available treatments in the control arm.
The introduction of these ethical principles are expected
to have methodologic consequences because RCTs with
a superiority design become virtually impossible because
of the high level of efficacy in the control group. The only
acceptable alternative is the noninferiority trial, which
was discussed briefly earlier. An illustrative example of a
noninferiority design, emerging from the more recent con-
troversy about cardiovascular safety of nonsteroidal anti-
­inflammatory drugs and cyclooxygenase-2 inhibitors, is the
Multinational Etoricoxib and Diclofenac Arthritis Long-
term (MEDAL) study program,19 which we briefly discuss
here. Although this program was focused on cardiovascular
safety rather than on efficacy, the noninferiority principles
underlying the study are applicable in different settings.
The primary hypothesis of the MEDAL study was that
treatment with etoricoxib was noninferior to treatment
with diclofenac. As a noninferiority margin, a relative risk
of 1.30 was chosen. It was defined before the start of the
trial that the upper limit of the 95% confidence interval for
the hazard ratio (etoricoxib versus diclofenac) should not
exceed the noninferiority margin to justify the conclusion
that etoricoxib was noninferior to diclofenac with respect to
causing cardiovascular events; this implies that the relative
risk itself should be far less than 1.30 to justify a conclusion
of noninferiority. It was assumed based on previous experi-
ence that diclofenac would give a cardiovascular event rate
of 1.3%. With approximately 40,000 patient-years of expo-
sure, it was possible to calculate that the maximal absolute
event rate in the etoricoxib group that would still meet the
noninferiority criterion would be 1.46%, or, in other words,
greater than 1.6 cases per 1000 patient-years of treatment.
This example illustrates that noninferiority trials always
imply a certain expense (in this example, numerous excess
cardiovascular events). It is possible to limit this expense
by narrowing the bound of noninferiority, but this occurs at
the cost of increased sample size in an exponential manner.
The determination of the noninferiority margin is the result
of careful considerations about drug safety (which require a
margin very close to 1) on the one hand, and the statistical
appreciation that the demonstration of true equivalence is
not possible on the other hand.
CONCLUSIONS
An appropriate trial design is important to optimize the
likelihood that the trial would provide results that are inter-
pretable, robust, and applicable. Usually, the decisions made
during the design phase are a reflection of the continuous
balance between internal validity and external validity.
Which of both characteristics prevails in the ultimate design
depends on the aims and the context of the trial. The ideal
trial design does not exist. It is the challenge of the investi-
gators to find the most appropriate design for their goal.
TRIAL ANALYSIS
HYPOTHESIS TESTING
Suppose a scenario with a particular disease for which an
effective treatment A exists, and a new treatment B is pro-
posed. It is unknown whether the new treatment B would
be more effective than the established treatment A, and a
clinical trial should provide resolution. It is useful to realize
that this trial provides only an approximation of the truth
that we—by definition—do not know. If you could repeat
this trial many times, you might find a mean treatment
effect across trials that is the best possible approximation
of the truth, and most trials would give results that are close
to this mean treatment effect. A few trials would give more
deviant results, however, simply by chance.
Because you would not be able to carry out this trial
numerous times, you would have to interpret the results
of your particular trial in the context of numerous illusory
trials with the same design. To do so, a null hypothesis of
the truth should be carefully formulated. The character of
the null hypothesis depends on the type of trial. In a clas-
sic superiority design in which a new drug is tested against
an established drug, the null hypothesis of the truth states
that the new treatment is as effective as the established (con-
trol) treatment. In a noninferiority design, however, the
null hypothesis of the truth states that the new treatment
is less effective than the established treatment. The statistical
analysis would challenge the actually observed trial results
against the null hypothesis of the truth.
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
Essentially, the statistical test provides the probabil-
ity that a treatment effect (e.g., the difference in propor-
tion of ACR20 responders between the treatment groups,
or the difference in mean decrease of DAS) can be found
that is as large as what was shown in this trial—or even
larger—whereas in truth such a difference does not exist. In
the paragraph about statistical power, we referred to this sce-
nario as type I error or alpha, and its likelihood is called the
P value. Usually, we consider a probability of type I error of
less than 5% (P < .05) sufficiently low to assume safely that
the null hypothesis of the truth is not a valid hypothesis,
and that it can be rejected. We now decide that the new
drug is not as effective as the established drug. Note that not
as effective may imply better than as well as worse than. Sta-
tistical testing does not differentiate between both scenarios
(two-sided testing). The crude data should provide the cor-
rect interpretation.
The advantage of thinking in probabilistic terms (this
trial is only one example of many possible trials with similar
design) is that you immediately accept that there is always a
chance that—even with very convincing results—the inter-
pretation of this trial may be false. The lower this chance is,
the more convincing is the interpretation of the trial results.
We also have alluded to the scenario that a trial does not
show a treatment difference, whereas in truth there is—type
II error or beta. Such a trial lacks (statistical) power. Sample
size is most often of pivotal importance, and a classic exam-
ple of potential type II error is the small clinical trial with
a superiority design that tests a new drug against an estab-
lished drug. Such a trial may give a treatment effect that is
of potential clinical interest, but the P value exceeds 0.05
(not statistically significant).
It is important to make numerous restrictions here. In
analogy with type I error, you can never be certain whether
type II error is truly responsible for not statistically show-
ing a potentially important difference. You can only suspect
type II error and approximate the probability that it occurs
(power calculation). A priori power calculations during
the design phase inform you about the probability that the
designed trial would statistically “help you” (rightfully reject
the null hypothesis) if a particular treatment effect is shown.
Post-hoc power calculations can be made after the comple-
tion of the trial, using the actually shown treatment effect.
Post-hoc power calculations inform you about the power of
this trial to support statistically the observed difference if
this difference would have been considered clinically important
before the trial. Insufficient post-hoc power (e.g., 0.30) is
an indication that type II error is operative, but it does not
prove type II error.
INTENTION TO TREAT
A classic clinical trial with a superiority design is analyzed
on the basis of intention to treat (ITT) by default. ITT
means that every patient is analyzed as belonging to the
group to which he or she was allocated by randomization,
regardless of what happened with this patient afterward. In
the extreme scenario, a patient who is randomly assigned
to group A may drop out even before the first drug dose is
administered and may be treated outside the clinical trial by
his or her physician with the drug that is actually adminis-
tered in group B (crossover of treatment). True ITT requires
459
that this patient—regardless of the data that are present or
missing—is analyzed in group A, even if he or she has expe-
rienced the effects of the drug in group B. ITT is considered
the only means of analysis that preserves prognostic similar-
ity that was created at baseline. Many trial reports mention
an ITT analysis, but such an analysis is often not rigorously
performed. Randomized patients who did not receive treat-
ment are often excluded from the analysis.
Alternatively, a trial analysis could be limited to only the
patients who completed the study (completers analysis), or
to the patients who completed the study while complying
to the study protocol (per-protocol analysis). Both types of
analysis may introduce bias, and consequently prognostic
similarity should not be assumed. Dropout can occur because
of lack of efficacy, and completers may be a less severe rep-
resentation of the entire trial population (see later). Often,
a completers analysis tends to enlarge the treatment effect,
whereas an ITT analysis is more conservative. In a noninfe-
riority trial, the situation is just the reverse: The ITT analysis
tends to favor noninferiority, whereas the completers or the
per-protocol analysis is more conservative in this regard.
PROBLEM OF INCOMPLETE DATA
A major threat for the interpretation of the results of clini-
cal trials is early withdrawal or dropout. Early withdrawal
can be due to a variety of reasons. Some patients withdraw
consent immediately after randomization, even before the
first drug dose, because they simply changed their mind.
Patients may drop out because of actually occurring or feared
adverse events, because of lack of efficacy (no response,
disease progression, unrealistic expectations), because of
a combination of both, or because they have died or have
relocated. Usually, dropout results in missing data because
most patients do not come back for outcome measurement.
Often, patients who formally withdraw are encouraged to
continue trial assessment, but it is difficult to decide how
to handle the data of these patients because they are often
treated with different drugs outside the trial.
The trial analysis is negatively affected by missing data
in several aspects. First, the individual response or disease
course cannot be calculated anymore, but leaving out the
entire patient jeopardizes statistical power, especially if the
number of withdrawn patients is high (e.g., >20%). Second,
early trial withdrawal is not a random process. It is easy to
imagine that the most severe patients are at risk for effi-
cacy failure, which may lead to selective dropout in a trial
with high treatment contrast, or that certain subgroups of
patients are at risk for particular adverse events that occur
preferentially in one of the trial arms.
The net result of these inadvertent selection processes
can be that the treatment groups that were entirely similar
at baseline lose their prognostic similarity during the trial.
Indicators for selective dropout include a difference in the
rate of dropouts between treatment groups, differences in
the reasons for dropout between treatment groups, and a
high rate of dropout for a specific reason that is reducible to
a specific treatment. Usually, missing data are handled in the
database by data imputation, to keep the patient in the trial
analysis. Data imputation means that a missing data point
is supplied by an imaginary value. There are several means
of imputation, and there is no consensus about the best way
 460 LANDEWÉ  |  Clinical Trial Design and Analysis
to impute. Most likely, the best imputation method depends
on the characteristics of the outcome measure, such as the
natural course of this outcome measure.
Last observation carried forward imputation is a fre-
quently applied method of imputation, in which the last
truly measured value is imputed (carried forward) in the
subsequent (missing) data points. Other imputation rules
are the imputation of a mean group score of the same or the
comparator group, the imputation of the 95th percentile, or
linear intrapolation or extrapolation. It is not easy to pre-
dict how different imputation rules affect the study results.
Increasingly, investigators perform sensitivity analyses with
different imputation rules to challenge the robustness of
their trial results. A trial result that is robust to various
means of imputation has more credibility than a trial result
that depends on the means of missing data imputation.
PRESENTATION OF TRIAL RESULTS
An increasing number of journals require the presentation of
trial results following consensus guidelines, such as the Con-
solidated Standards of Reporting Trials (CONSORT) guide-
lines,20 to increase the comprehensibility and to maximize
information. Such guidelines require the exact presentation
of the randomization process, a description of blinding,
and eligibility criteria (about inclusion and exclusion). An
appropriate trial report should include exact information
about the fate of all patients after randomization, includ-
ing the major reasons for withdrawal. It is essential that the
total numbers of patients per group can be reconstructed.
Increasingly, such information is provided in a flow chart.
The initial part of data analysis is to examine the baseline
characteristics of the trial groups, including demographics,
previous and current treatments, and disease characteristics
(e.g., severity scales, duration, extent of organ involvement)
with descriptive statistics. Occasionally, baseline charac-
teristics per group are statistically compared, and baseline
similarity is assumed if no statistically significant differences
between groups could be shown. Such an approach is useless
and sometimes overtly false. Groups are similar by definition
because they were formed by randomization. Randomiza-
tion is a probabilistic procedure, and groups may statistically
differ in one or more variables at baseline just by chance.
Often, such differences are small in relation to the treatment
effect, or the particular baseline characteristic is not associ-
ated with the measured outcome and is not contributory.
Rarely, baseline differences are not negligible, and the
trial result should be adjusted for these differences by mul-
tivariate analysis (e.g., regression analysis or analysis of
covariance). A particular problem may occur in small tri-
als, if even clinically important differences at baseline are
not statistically significant, whereas their impact on the
treatment effect may be substantial. A general rule is to
“eyeball” baseline differences, and to adjust in the statisti-
cal analysis for variables that show potentially important
differences.
Descriptive Analysis
Simple descriptive analysis includes the presentation of
means and standard deviations in the case of a normal
(parametric) distribution of continuous data (e.g., DAS),
or medians and key percentiles in the case of an abnor-
mal distribution of continuous data (e.g., Sharp scores).
Dichotomous data (e.g., ACR20 responses) are presented
as proportions or rates, and ordinal/nominal or categorized
data are presented as percentages per category. Graphic
representations (e.g., line or bar graphs) are preferred
because they give the clearest representation of the treat-
ment effect.
An illustrative example of visual presentation of data
is the use of probability plots for radiographic data.21 The
emphasis should be on the primary outcome variable, but
all measured outcome variables should be presented. It is
recommended to present data such that they can be used
post-hoc for systematic reviews or meta-analysis. Status
scores plus a measure of variability (e.g., mean DAS at
baseline and at follow-up and standard deviations) and
change scores plus a measure of variability (e.g., mean
change score and standard deviation of change score)
should be presented for primary and secondary outcome
variables. Dichotomous outcome variables (e.g., ACR20
response criteria) should be accompanied by extensive
information about the status values of (separate) vari-
ables. It is relevant to present not only response measures
(e.g., ACR20), but also state measures (e.g., absolute
DAS) because the combination of both gives additional
information and increases the interpretability of trial
results.
A useful extension of state measures is the concept of
patient-acceptable symptom state. The patient-acceptable
symptom state reflects the level of symptom severity that
best discriminates an acceptable situation from an unac-
ceptable situation from the perspective of the patient.22
Patient-acceptable symptom state levels can be determined
by different methodologies and are presented as proportions
or rates.
Statistical Analysis
Because the process of randomization provides prognos-
tic similarity at baseline, the statistical analysis of a clini-
cal trial is simple, as long as one assumes that prognostic
similarity is preserved during the trial. The statistical test
of choice is a test for binomial data (e.g., χ2 test) if the
outcome measure of choice is dichotomous (e.g., ACR20,
ASAS20 response criteria), and a test for continuous
data (e.g., Student t test or Mann-Whitney U test) if the
outcome measure is continuous. An extension of the sta-
tistical test that provides useful information to increase
interpretability is the 95% confidence interval of the
treatment effect. The 95% confidence interval can be cal-
culated for the treatment effect measured by continuous
and dichotomous outcome variables. It provides the range
within which the estimate of the treatment effect would
lie in 95% of the cases in the imaginary situation that this
trial would be repeated numerous times. If the lower limit
of the 95% confidence interval of the mean treatment
effect in a trial comparing drug A and drug B does not
cross zero, this means that 95 out of 100 times that this
trial is repeated, drug A is better than drug B, but 5 out of
100 times, drug A is not better than drug B. The formula-
tion of the 95% confidence interval closely resembles that
of the P value.
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
STATISTICAL SIGNIFICANCE AND CLINICAL
RELEVANCE
Clinical trials can be designed in such a way that even
very small treatment effects can be statistically shown.
Explanatory trials in which new drugs are tested are often
performed in a highly selected population of patients with
strong adherence to the protocol, without comorbidity, and
a high probability of responding. Sometimes, sample sizes
are huge (“overpowered”). Such trials aim at a high level of
internal validity and a low probability of type II error, but
it is difficult to interpret the results in the clinical situation
immediately because the mean treatment effect can be so
small that it is considered irrelevant in the context of clini-
cal practice, and patients in the trial often do not resemble
the individual patients in practice (external validity).
There are several guides to evaluate the quality of a clini-
cal trial.23 A trial may show a small improvement in a pri-
mary outcome criterion that is statistically significant, but
the effect may not have clinical importance because it is
insufficient to affect quality of life or survival, or it does not
outweigh the risk or cost of treatment. A result may be sta-
tistically and clinically significant, but have little medical
relevance because the benefit does not outweigh the risk or
cost of treatment, or because the benefit is seen only in a
very small subgroup of patients.
CONFOUNDING
Confounding is a type of bias (systematic error) that can
occur in a trial when the trial groups differ with respect to
a particular factor other than trial treatment (prognostic
dissimilarity). If this factor also is related to the outcome
measure of interest, a fake treatment effect may emerge that
would erroneously be attributed to the difference in treat-
ment (confounding). As long as confounding factors are
known, measurable and measured, one can adjust for them
in the statistical analysis. If such variables that are referred
to as prognostically important are not measured and even
unknown, adjustment is impossible, however.
The likelihood of confounding is far higher in obser-
vational studies than in randomized trials, and we use an
example from an observational study to clarify. If in an
observational database the efficacy of the disease-modifying
antirheumatic drug sulfasalazine in the treatment of RA is
compared with the efficacy of methotrexate, and it is com-
mon practice to give sulfasalazine treatment primarily to
patients with less severe disease (rheumatoid factor–negative
RA) and methotrexate to patients with more severe disease
(rheumatoid factor–positive RA), radiographic progression
may be less in patients treated with sulfasalazine than with
methotrexate. It is difficult to determine whether this differ-
ence is due to differences in efficacy between sulfasalazine
and methotrexate or to differences in severity of the RA
that also may drive radiographic progression (prognostic
dissimilarity). Variables such as rheumatoid factor in this
example may confound the relationship between treatment
and outcome (radiographic progression). In this example,
with a well-known confounder, the analysis may adjust for
differences in rheumatoid factor positivity. In theory, many
unknown variables, or known but unmeasured variables,
also can cause prognostic dissimilarity.
461
We have mentioned previously that treatment groups in
an RCT are prognostically similar only at baseline, and that
prognostic similarity can be lost during follow-up. Conse-
quently, confounding is possible in RCTs, and trial results
should be judged in light of this possibility.
INTERPRETATION OF SAFETY ANALYSES
Safety is crucial in the consideration of whether or not a new
drug or treatment should be approved. A detailed descrip-
tion of the process of drug approval is beyond the scope of
this chapter, but there are a few methodologically impor-
tant issues with regard to the interpretation of safety data in
clinical trials. Usually, clinical trials aim at showing efficacy
of a drug or treatment. Many relevant adverse events occur
in a low frequency or (far) beyond the duration of the trial.
Consequently, the probability that such a relevant adverse
event would occur within the context and time frame of the
clinical trial is low, and the interpretation of safety results of
a clinical trial does not exclude important adverse events.
Such information should be obtained from long-term obser-
vational studies or drug registries.
CONCLUSIONS
The descriptive and statistical analysis of clinical trial data
is not an extremely challenging task and should follow the
straightforward protocol imposed by the trial design. Uni-
versal guidelines are published to guide the investigator,
and these guidelines are increasingly warranted by medical
journals. The interpretation of the trial results is not always
easy and straightforward, however. We have mentioned
numerous disturbing factors that may jeopardize the inter-
pretation of the trial results, such as missing data, dropout,
and confounding, and the investigators and the readers
of medical journals should be challenged to interpret the
results of the trial in the light of these potentially disturbing
factors.
SUMMARY
Clinical trials are used to test the efficacy of new drugs and
devices and to compare efficacy and safety of combinations
of drugs. New drug development is a long and expensive
process. The choice of clinical trial design and implementa-
tion is crucial for safe, efficient, and successful drug devel-
opment. Because the cost of clinical trials for new drug
development has increased substantially, research leading to
regulatory approval of new treatments is done primarily by
the pharmaceutical industry in multicenter clinical trials.
Such trials usually are explanatory in nature, with internal
validity prevailing over external validity. It is widely recog-
nized in medical science that investigator-initiated clinical
trials are crucial in studies addressing the effects of combi-
nations of standard drugs, or standard drugs in combination
with new treatments, and in the initial studies of the effects
of newly approved drugs for other indications. Such trials
are often more pragmatic in nature and have a higher level
of external validity (generalizability). Important contribu-
tions in the field of investigator-initiated pragmatic RCTs
have been published in the rheumatologic literature.24-26 It
will be a considerable challenge to fund such trials in the
 462 LANDEWÉ  |  Clinical Trial Design and Analysis
future, and this may be an opportunity for collaborations
between academic clinical scientists and pharmaceutical
industry workers.
The clinical trial is not the only type of clinical research
study. An important drawback of clinical trials is that the
duration is short, and as a result they have to rely on inter-
mediate or process measures for outcomes, rather than the
“hard” outcomes themselves. Longitudinal practice-based
observational studies may fill in a gap in that they provide
important information about the effects of a new treatment
when used in diverse groups of patients; about drug toxicity;
and about the long-term effects of a treatment on functional
status, morbidity, and mortality. They may provide a wealth
of information about the relationship between process or
surrogate measures and “true” outcome measures. They also
may provide useful information about prognostic factors for
a certain outcome. A general judgment about the effective-
ness of a particular treatment is in the end a compilation
of impressions obtained from various sources, including
explanatory drug registration trials, pragmatic trials better
meeting the need of clinicians, and observational studies
with a focus on true outcome and long-term safety.
REFERENCES
  1. Armitage P: Attitudes in clinical trials. Stat Med 17:2675-2683,
1998.
  2. MacRae KD: Pragmatic versus explanatory trials. Int J Technol
Assess Health Care 5:333-339, 1989.
  3. McMahon AD: Study control, violators, inclusion criteria and defin-
ing explanatory and pragmatic trials. Stat Med 21:1365-1376, 2002.
  4. D’Agostino RB Sr, Campbell M, Greenhouse J: Non-inferiority tri-
als: Continued advancements in concepts and methodology (special
papers for the 25th Anniversary of Statistics in Medicine 25[7]). Stat
Med 25:1097-1099, 2006.
  5. D’Agostino RB Sr, Massaro JM, Sullivan LM: Non-inferiority tri-
als: Design concepts and issues—the encounters of academic con-
sultants in statistics. Stat Med 22:169-186, 2003.
  6. Fries JF, Hochberg MC, Medsger TA Jr, et al: Criteria for rheu-
matic disease: Different types and different functions. The Ameri-
can College of Rheumatology Diagnostic and Therapeutic Criteria
Committee. Arthritis Rheum 37:454-462, 1994.
  7. Prout TE: The ethics of informed consent. Control Clin Trials 1:
429-434, 1981.
  8. Boers M, Brooks P, Strand CV, et al: The OMERACT filter for Out-
come Measures in Rheumatology. J Rheumatol 25:198-199, 1998
(editorial).
  9. Bombardier C, Tugwell P: A methodological framework to develop and
select indices for clinical trials: Statistical and judgmental approaches.
J Rheumatol 9:753-757, 1982.
10. van der Heijde DM, van’t Hof M, van Riel PL, et al: Development
of a disease activity score based on judgment in clinical practice by
rheumatologists. J Rheumatol 20:579-581, 1993.
11. Felson DT, Anderson JJ, Boers M, et al: Preliminary definition of
improvement in rheumatoid arthritis. American College of Rheuma-
tology. Arthritis Rheum 38:727-735, 1995.
12. Felson DT, Anderson JJ, Boers M, et al: The American College
of Rheumatology preliminary core set of disease activity measures
for rheumatoid arthritis clinical trials. The Committee on Outcome
Measures in Rheumatoid Arthritis Clinical Trials. Arthritis Rheum
36:729-740, 1993.
13. Felson DT, Anderson JJ, Lange ML, et al: Should improvement in
rheumatoid arthritis clinical trials be defined as fifty percent or sev-
enty percent improvement in core set measures, rather than twenty
percent? Arthritis Rheum 41:1564-1570, 1998.
14. van Riel PL, van Gestel AM, van de Putte LB: Development and
validation of response criteria in rheumatoid arthritis: Steps towards
an international consensus on prognostic markers. Br J Rheumatol
35(Suppl 2):4-7, 1996.
15. Anderson JJ, Baron G, van der Heijde D, et al: Ankylosing spondylitis
assessment group preliminary definition of short-term improvement in
ankylosing spondylitis. Arthritis Rheum 44:1876-1886, 2001.
16. World Medical Association: World Medical Association Declaration
of Helsinki: Ethical principles for medical research involving human
subjects. Available at: www.wma.net/e/policy/b3.htm. Accessed Octo-
ber 8, 2007.
17. Macklin R: After Helsinki: Unresolved issues in international research.
Kennedy Inst Ethics J 11:17-36, 2001.
18. Carlson RV, Boyd KM, Webb DJ: The revision of the Declaration of
Helsinki: Past, present and future. Br J Clin Pharmacol 57:695-713,
2004.
19. Cannon CP, Curtis SP, Bolognese JA, et al: Clinical trial design and
patient demographics of the Multinational Etoricoxib and Diclofenac
Arthritis Long-term (MEDAL) Study Program: Cardiovascular out-
comes with etoricoxib versus diclofenac in patients with osteoarthritis
and rheumatoid arthritis. Am Heart J 152:237-245, 2006.
20. Begg C, Cho M, Eastwood S, et al: Improving the quality of re-
porting of randomized controlled trials—the CONSORT statement.
JAMA 276:637-639, 1996.
21. Landewe R, van der Heijde D: Radiographic progression depicted by
probability plots: Presenting data with optimal use of individual val-
ues. Arthritis Rheum 50:699-706, 2004.
22. Tubach F, Ravaud P, Baron G, et al: Evaluation of clinically relevant
states in patient reported outcomes in knee and hip osteoarthritis: The
patient acceptable symptom state. Ann Rheum Dis 64:34-37, 2005.
23. DerSimonian R, Charette LJ, McPeek B, et al: Reporting on meth-
ods in clinical trials. N Engl J Med 306:1332-1337, 1982.
24. Boers M, Verhoeven AC, Markusse HM, et al: Randomised com-
parison of combined step-down prednisolone, methotrexate and sul-
phasalazine with sulphasalazine alone in early rheumatoid arthritis.
Lancet 350:309-318, 1997.
25. Goekoop-Ruiterman YP, de Vries-Bouwstra JK, Allaart CF, et al:
Clinical and radiographic outcomes of four different treatment strat-
egies in patients with early rheumatoid arthritis (the BeSt study):
A randomized, controlled trial. Arthritis Rheum 52:3381-3390,
2005.
26. Grigor C, Capell H, Stirling A, et al: Effect of a treatment strat-
egy of tight control for rheumatoid arthritis (the TICORA study):
A single-blind randomised controlled trial. Lancet 364:263-269,
2004.
 31
KEY POINTS
Any single health outcome can give only a partial view of the
impact of a disease on a patient.
Core sets are minimal, but not exclusive, domains of
outcomes agreed on by professional groups as important to
include in studies; they are available for several rheumato-
logic conditions.
Defining measurement need is key to the choice of the right
instrument.
Choosing an instrument follows a step-by-step
process—looking for evidence of practical aspects
of using the instruments and statistical properties.
If an instrument lacks evidence of a certain property, a study
can be conducted to create the evidence, rather than
abandon the instrument.
In an era of rising health care costs, increased choice, and
greater provider accountability,1 health outcome ­ measures
have become essential tools for researchers, ­clinicians, and
funding bodies. By definition, outcomes refer to “all possible
effects of a disease or intervention.”2 Outcomes cover a spec-
trum of the burden of arthritis on a patient from biomark-
ers of disease to subjective appraisals of overall well-being.
Other chapters refer to some of the most common measures
of health and disease encountered in rheumatology, includ-
ing the Disease Activity Scale (DAS, DAS28),3 the Health
Assessment Questionnaire (HAQ),4 and the SF-36 (short
form, 36 items).5 Using any one of these health outcome
assessments is like looking out a window in a house, with
the burden of arthritis the landscape outside. Each window
in the house provides a view of the outside world, but it is a
specific view defined by the size of the window and the side
of the house it is on. Another window may offer a slightly
better angle on what you would like to see. Different health
outcome assessments can have a degree of overlap in their
views, in which case an informed choice needs to be made
between them, whereas others can hold quite distinct views.
One assessment might be useful to compare the burden of
arthritis against the general population, another might be
useful to assess differences in the prevalence in different
subgroups, and yet another might be useful to measure the
specific benefits of an arthritis intervention.
This chapter focuses on describing the different windows
clinicians have on the burden of arthritis and how they
relate to each other. A framework is provided for ensuring
that a selected measure is the right one for a given need. This
chapter addresses four questions: (1) What health outcome
assessment tools are available generally and ­specifically for
Assessment of Health
Outcomes
Dorcas Eleanor Beaton  •  Maarten
Boers  •  Peter Tugwell
use in rheumatology? (2) How do the different assessment
tools relate to one another? (3) How does one characterize
what one needs to measure? (4) How does one find a mea-
sure that can meet that need?
WHAT HEALTH OUTCOME ASSESSMENT
TOOLS ARE AVAILABLE?
In reading the rheumatology literature, and in monitor-
ing the clinical care of patients, certain highly relevant
outcomes emerge. A group of these often emerge and are
termed core sets of outcomes.
DISEASE-SPECIFIC MEASURES—THE CORE SETS
Core sets are the minimal, but not exclusive, set of domains
to be measured in a study of arthritis. Historically, they fol-
low the Ds of outcome measurement in arthritis: disability,
disease activity, damage, discomfort, dissatisfaction, and
death.6,7 They are usually recommended by groups such as
OMERACT, EULAR, ILAR, or ACR or groups formed
around specific diseases, such as ASAS for ankylosing spon-
dylitis or GRAPPA for psoriatic arthritis. All core sets have
a great interest in agreeing on a common set of relevant and
psychometrically sound outcomes that would allow them to
compare findings across studies and of clinical care.
Table 31-1 presents the list of core sets for clinical trials
in six types of arthritis7-16; it also shows what each group rec-
ommends as additional domains or as needing more research
before they become core set members. The first column on the
left of Table 31-1 is the core set for longitudinal observational
studies in rheumatology by Wolfe and colleagues7 with some
minor additions. Wolfe’s core set is broader than the core sets
presented in other columns because observational studies are
often looking for a broader range of outcomes that are rel-
evant for studies outside of treatment trials. It also is designed
to be used across different forms of arthritis. The table also
uses this broader list of outcomes as an axis against which the
other core sets can be described. The remaining columns show
that across different types of arthritis, the core sets have many
common elements; most contain or recommend pain, physi-
cal function, patient and clinician global assessments, and
markers of inflammation. Many also include disease activity
indices, which are often an aggregation across other clini-
cal findings (e.g., joint count, acute-phase reactants, global
ratings of severity) into a score reflecting the activity of the
disease at that point in time. Some core sets contain domains
reflecting the unique aspects of the disease (e.g., spinal mobil-
ity in ankylosing spondylitis)17 or the unique target of the
study using the outcomes (fractures are core outcomes only in
osteoporosis studies focused on fracture prevention).18
463
 464

Table 31-1  Core Sets for Six Rheumatologic Conditions and for Longitudinal Observational Studies*
BEAToN 

Clinical Trial Core Sets of Domains by Disease Group

| 

Longitudinal Study Core Rheumatoid Systemic Lupus Ankylosing

Set of Domains7 Arthritis15,16 Osteoporosis11 Osteoarthritis13 Erythematosus14,19 Spondylitis8 Psoriatic Arthritis10,12
Health status/quality of life ✓
Quality of life R (utility) R (BL)(†) R ✓ ✓
Symptoms ✓ Pain R (back)(†) ✓ Pain R (fatigue) ✓Pain ✓ Pain
✓ Fatigue
Physical function ✓ Disability R(†) ✓ R ✓ ✓
Psychosocial R R

Disease process ✓
Aggregate index ✓ (EULAR DAS) ✓ DAI, R = severity Pending ✓ DAS
Biomarkers ✓ Biochemical O
Joint tenderness ✓ 28 or 68 joints ✓ Peripheral (44 joints) ✓
Assessment of Health Outcomes

Enthesitis Enthesitis‡
Joint swelling ✓
Joint stiffness O ✓ Spinal stiffness
Global ✓ Spinal mobility
Patient ✓ ✓ ✓ ✓
Physician ✓ R ✓
Acute-phase reactants ✓ O R ✓‡ ✓
Damage ✓
Radiography or imaging ✓ >1 yr ✓ Bone mineral density ✓ >1 yr ✓ Spine and hip§ ✓ Structural
Deformity
Surgery
Organ damage R (BL)/ ✓ (†) fractures ✓ Damage index ✓Skin disease
R (BL)/ ✓ (†) Change height
Disadvantages
Toxicity effects ✓ R ✓ ✓
Death ✓

Dollar costs [R] R R(†) R ✓

Work disability [R] R
*The left-hand column is the broader set of measures recommended by Wolfe and colleagues7 for longitudinal studies. It serves here as a broader range of outcomes, and an axis for the organization of core outcomes
in the other conditions (columns).
✓, core domain; R, recommended for further research and possible inclusion in core set; O, optional outcome (osteoarthritis category only).
Osteoporosis: Core set depends on focus: BL, bone loss studies; †, studies aiming to reduce fracture rates.
Ankylosing spondylitis: Core set elements vary depending on focus of study: ‡, clinical records and symptom modifying; §, disease modifying only; others = all.
DAI, disease activity index; EULAR DAS, European League Against Rheumatism Disease Activity Scale (revised = DAS-28, 28-joint count).
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
Table 31-1 focuses on the core domains that should be
measured; the next step is deciding on the instrument that
is able to provide that well in a reproducible, accurate man-
ner. In some cases, an instrument choice has been suggested
(e.g., the HAQ for disability in rheumatoid arthritis). Other
times several options are provided. Strand and cowork-
ers19 reviewed six disease activity indices in systemic lupus
erythematosus and found they gave comparable results. In
some cases, the domains are shared, but the measurement
technique varies within or by disease; in rheumatoid arthri-
tis, the DAS28 uses 28 joints,3 and in ankylosing spondy-
litis, 44 joints are counted.17 Some of the more commonly
encountered instruments in arthritis are briefly reviewed.
Health Status/Quality of Life
General Health Status. Generic health outcomes provide
information on an aspect of health across many conditions
so that theoretically comparisons can be made to compare
the burden of low back pain with that of arthritis or diabe-
tes. This comparison depends on the ability of that measure
to capture the burden in a disease group well. Generic mea-
sures have advantages of allowing comparisons across dis-
eases and covering a broader range of health issues—things
that may have been overlooked in a core set (e.g., mental
health issues). Often because of their breadth, however, the
generic measures tend not to delve as well into the depth of
experience in any one disease. Arthritis-related fatigue is not
detected well in many generic measures because a generic
measure asks about being tired or not sleeping well. Because
of this, a generic measure is usually weaker in its ability to
detect specific changes and their sensitivity to different lev-
els of disease activity and should usually be supplemented
with disease-specific measures (described previously).20
Two commonly used generic measures are the Sickness
Impact Profile (SIP)21 and the SF-36.22 The SIP is a 136-
item list of illness behaviors that provides a weighted score
for the impact of a disease across 12 categories, such as bodily
pain, work and role functioning, and dressing,21 which lead
to global scores (physical, psychosocial, and overall). The
SIP has been shown to measure illness across a wide vari-
ety of health conditions.20 The SF-36 is a 36-item question-
naire of which 35 items are used to obtain eight domain
scores (including physical functioning, mental health, role
functioning, and pain) scored on a 0-to-100 scale (with
100 = better health)22 and two summary scores (mental and
physical); the questionnaire is scored with normal of 50 and
standard deviation of 10. The SF-36 and briefer SF-12 are
well supported on the website (www.qualitymetric.com) and
through manuals that supply age and disease group distri-
butions of scores.23 Direct comparisons of generic measures
have shown differences in scores and health states attribut-
able to the choice of measure.24-26 Studies or clinical results
may not be comparable to each other if they are using differ-
ent health status scales.
Utilities—Value of Health State. Utility scales offer an
overall score for the value of a health state, setting death
at 0 and full health at 1. The emphasis is not on describing
the state, but on assigning a value, worth, or preference to
that state.27,28 Utilities are needed for economic appraisals
and form the health assessment for cost per quality-adjusted
465
life-years estimations. Utility states can be obtained by di-
rect or indirect methods. Direct methods, such as standard
gamble and time tradeoff, involve the respondent work-
ing through exercises to elicit the value for his or her own
health state against things such as time or more or less fa-
vorable health situations.27 Indirect methods capture the
state with standardized questions and apply predetermined
weights.28 Examples include the EQ-5D which is five items
(three response categories) combined to describe a health
state. Similarly, the Health Utility Index gathers informa-
tion on six or seven dimensions of health (depending on the
version) on five-item to six-item response scales to define a
health state.28 Both these scales then use weights determined
in different populations to assign the value to these health
states—hence the “indirect” weighting. The absolute value
obtained across these different approaches varies.27
Generic measures of health and utility scores are very
broad. They often do not perform as well as the more specific
measures described in the following sections because they are
designed to allow comparisons across populations and need to
include items that might not be relevant in arthritis. In some
areas, there are measures of quality of life that are designed
for that disease, such as rheumatoid arthritis or osteoporosis,
which offer a measure of the broad concept of quality of life
in a disease-specific manner. Such measures would not allow
comparisons across diseases. At the time of this writing, these
were not yet recommended as core instruments.
Symptoms. Pain is usually measured using a 10-cm visual
analog scale or a 0-to-10 numeric rating scale of the intensity
of the symptoms.29 This simple measure has been well tested
and is easily understood by patients. Fatigue is another im-
portant symptom and quite distinct from being “tired.” The
ankylosing spondylitis modified core set contains fatigue,
and it is a recommended area of further research in rheuma-
toid arthritis and lupus. Measures are being tested or devel-
oped at present. Ankylosing spondylitis is currently using the
10-cm visual analog scale of fatigue from the Bath Ankylos-
ing Spondylitis Disease Activity Index (BASDAI).30
Disability Scales. Physical disability in rheumatoid arthritis
and osteoarthritis is often measured using the Health Assess-
ment Questionnaire–Disability Index (HAQ-DI),31 which
covers 20 items looking at different aspects of daily func-
tioning. Patients score each item on a 0-to-3 scale, where
3 represents the greatest disability. Scores are obtained for
each domain and summed into a total score expressed on
the same 0-to-3 scale. Scores are adjusted to a 2/3 if an aid
is used to ­complete a task. More details on the HAQ-DI are
widely available in print and on the Internet.
There are other scales asking about physical function such
as the Arthritis Impact Measurement Scale (AIMS)32 and the
AIMS233 and measures with even more specific foci, such as the
Western Ontario McMaster (WOMAC) osteoarthritis index,
which is commonly used in hip and knee osteoarthritis,34 and
the AUSCAN osteoarthritis index for hand osteoarthritis.35
Disease Process (Activity, Severity)
Core sets often include indices of disease process. Disease
process can be divided into activity (inflammatory activity)
and severity (overall severity of disease). There are several
 466 BEAToN  |  Assessment of Health Outcomes
disease activity indices, the most commonly known being
the DAS36 and DAS283 in rheumatoid arthritis. Using a
subset of the core outcomes (i.e., acute-phase reactants,
joint counts, global ratings), the EULAR group formulated
a weighted index that provides a score of 2 to 10 (DAS)
or 0 to 9 (DAS28). From this, cutoffs were established to
define high, moderate, and low disease states. The low dis-
ease state (DAS28 <2.6) is considered an indicator of remis-
sion of arthritis and is touched on again later. More recently,
the concept of “stable remission” has been proposed; it is
defined as an initial DAS28 of less than 3.2 and a lack of
change in DAS over time of 1.2 (2 standard errors).37 Dis-
ease activity indices track the level of inflammatory activity.
There are others, such as the BASDAI30 or the six available
in systemic lupus erythematosus.19 When more than one is
available, look for direct comparisons such as Strand’s to see
if similar information is provided.19,38
Damage Indices
A great deal of work has gone into measures of joint damage
in arthritis. van der Heijde and Landewe17 provide a succinct
summary of the Sharp, van der Heijde, and Larsen/Scott tech-
niques. Guidelines should be followed closely, focusing on the
hands and feet. Changes in radiographic progression of joint
damage often are measured by the smallest detectable differ-
ence, the boundary between measurement error (day-to-day
variability) and change discernible from error.39,40
Disadvantages—Toxicity/Adverse Events
Medical and nonmedical management of many rheumatic
conditions carries a risk of toxicity and adverse events,41
many of them unexpected. A comprehensive documenta-
tion of a range of adverse events is important in outcome
assessments separate from the treatment benefits.42 An
OMERACT group is currently working on standardizing
reporting of toxicities in rheumatologic trials.43
Death
Arthritis is associated with increased mortality, and arthritis-
specific mortality should be monitored. Death is not specifically
mentioned in the disease-specific core sets for clinical trials,
but would be important to monitor in observational studies.
Attributing death to arthritis is challenging given its depen-
dence on documentation at time of occurrence, which may or
may not be linked to underlying arthritis in the coding.
Dollar Costs
The economic burden of arthritis and the cost of care are core
outcomes for psoratic arthritis and recommended for consid-
eration in observational studies and rheumatoid arthritis,
osteoporosis, and lupus disease groups. Standards for what
should be included in a cost analysis are under development.
Disease Self-Management
Lorig’s work in self-management programs often have
focused on improving self-efficacy, which has been found to
reduce pain and health care use.25 Self-efficacy is a health
outcome. Similarly, Tugwell’s “effective consumer” captures
the degree to which the patient is effectively managing
his or her own health care decisions, interactions with the
health care team, and disease monitoring.44 This may be a
reasonable target for self-help interventions.
EMERGING DOMAINS
Work Productivity and Disability
With the shift toward more aggressive management of ear-
lier disease, more people with arthritis are working, and
outcomes need to shift to track work disability (absentee-
ism and at-work productivity loss).7 Work is hard to mea-
sure because it depends on the job and the organization the
individual works in. Absenteeism can mean many things,
and measures should articulate how this was operational-
ized—full days off work, days on insurance payments, or full
and part days off work. More challenging still is measuring
the difficulty someone is having at work (presenteeism).
There are several measures—16 were found in a more recent
review45—but there are few direct comparisons of these con-
ceptually diverse instruments. The most commonly used is
the work limitations questionnaire (amount of time expe-
riencing difficulty).46 Two scales developed in arthritis are
promising—Gignac’s Work Activity Limitations Scale
(amount of difficulty experienced)47 and Gillworth’s Work
Instability Scale (risk of future work loss).48 Direct compari-
sons of these measures are under way.
Nonpaid Work
Participation in valued nonpaid roles, such as parenting,
volunteer work, or leisure activities, can be an impor-
tant aspect of the burden of disease.49 Outcome measures
reflecting this are needed to capture fully the concept of
participation.
Patient-Specific Domains
Patient-specific scales, including the MACTAR or PET in
arthritis,50,51 allow the patient to nominate his or her own
scale content, within a guided framework. Most patients
report three to five items that are particularly salient to them.
A surprising number of these scales have been developed.52
Each taps relevant content for that patient, and because of
this they also are responsive to change.53 The challenge is
in the mathematics and how to analyze the numeric score
when the items vary across patients. Analysis that focuses
on individual level quantification is likely best.
Satisfaction with Health Outcomes
Satisfaction scales are often linked with the goals of a health
care organization and focus on the attributes of the structure
and process of care. Instruments developed to look at satis-
faction with a specific health end point (e.g., how satisfied
are you with the results of your surgery)54 become a health
outcome. Satisfaction with outcome is complex, and Hudak
and colleagues55 point out the complex balance of experi-
ences and ability to “live with” ongoing limitations that
influence a patient’s response.
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
Sleep
The OMERACT patient group has identified sleep qual-
ity as an important domain for outcome assessment.
The concept and measurement of it are expected to be
addressed at the upcoming OMERACT 9 meeting in
2008.
HOW DO OUTCOME MEASURES RELATE
TO ONE ANOTHER?
With an array of potential measures, or windows to view
the burden of disease, how does one organize them to
get an accurate picture of the whole? Conceptual frame-
works help one to understand how domains, such as those
described previously, theoretically relate to one another
when applied in research or clinical practice. They also
usually provide an operational definition of each of
their domains, which becomes essential when choos-
ing between instruments or deciding how to model the
outcome of an intervention and its modifying factors.56,57
Conceptual frameworks facilitate accurate communica-
tion and the generation of hypotheses of understanding
of a disease process and impact. In the past, the most
common conceptual framework was the main (sometimes
causal) pathway of a biomedical model: Pathology leads
to organ/system changes leads to pain/symptoms leads to
functional loss leads to diminished quality of life. This is
similar to a Wilson and Cleary model58 and might help
in understanding that function and quality of life might
relate more closely than sedimentation rate and quality of
life—based on their proximity and distance in the model.
Expanding on this type of model are conceptual frame-
works such as Nagi59 or Verbrugge’s60 disablement process.
In these models, there is a main pathway, with slightly
different definitions, but there also are boxes of influence
from outside this pathway—the patient’s personal factors
and environmental factors. In 2001, the World Health
Organization ratified the International Classification of
Functioning (ICF),61 which offers an even more expanded
framework based on a biopsychosocial understanding of
disease. It is receiving wide endorsement in arthritis.56,62
In this model (Fig. 31-1), there are three main elements of
burden. A disease can affect an individual by impairment
(body part), activity limitations (individual’s ability to
do a task), or participation restriction (restrictions in the
execution of the individual’s roles in life situations). The
boxes in Figure 31-1 are joined by bidirectional arrows
because this model suggests that the right-side boxes also
could influence the left with secondary problems (e.g.,
joint contractures or wounds secondary to prolonged bed
rest). For the ICF, disablement is not a linear progression;
it is a dynamic interaction influenced by several factors.57
The ICF strongly emphasizes the influence of personal
factors and environmental factors on all three domains.
Several initiatives are under way to examine the fit and
usefulness of the ICF framework in various forms of arthri-
tis.57,63,64 Readers are referred to Jette and Keysor65 for an
excellent comparison of the ICF and Verbrugge models in
the context of arthritis.
In addition to providing the conceptual framework, the
ICF undertook the task of a classification system listing
467
Health condition
(disorder/disease)
Body function
and structure
Activities Participation
Environmental Personal
factors factors
Figure 31-1  The International Classification of Functioning conceptual
framework showing the hypothesized relationships between domains of
impairment, activity limitations, and participation restrictions.
all possible categories of impact falling under each of the
five main headings. Many groups, particularly in arthritis,
have reviewed the categories and developed a list of the
categories relevant to that form of arthritis. This list sug-
gests which of these categories should be reflected in core
set measures in arthritis, offering a form of standard for the
content of scales.63
Frameworks define the realm of outcomes that should
be considered, and the hypothetical relationships between
them. They form the basis for understanding observa-
tions, testing hypotheses, or planning and executing an
analysis. Shifting frameworks is challenging because dif-
ferent tools may vary in how they define certain aspects
of health or disability. Shifts can prompt fruitful rethink-
ing of concepts, however, and how they relate to each
other.64
HOW DOES ONE CHARACTERIZE WHAT
ONE NEEDS TO MEASURE?
Just as investigators define a research question before
embarking on a clinical trial, so should users of health out-
comes define a measurement question before choosing an
instrument.
WHY MEASURE?
Clarity about a measure’s intended purpose helps ensure the
right one is selected. Measures can be used in three ways: to
describe people at one point in time, to predict a future
state, and to measure change over time.66,67 This chapter
focuses on the purposes used for health outcome assessment:
describing an end point state in a trial, which is descriptive,
and evaluating change over time.
WHAT TO MEASURE
An understanding of concepts and definitions is important.
It is not good enough to decide to measure physical func-
tion, for example; a better outcome would be physical func-
tion at the level of disability according to Verbrugge and
Jette.60 Similarly, when measuring pain, is intensity more
important than frequency? What about the degree to which
 468 BEAToN  |  Assessment of Health Outcomes

pain interferes with daily activities? Questions such as these
should be addressed before any instruments or core sets are
reviewed. The instrument should meet the need, and not
the reverse.
WHO CONSTITUTES THE TARGET POPULATION
The target population is crucial, but often overlooked. A
given instrument may work well in severe osteoarthritis of
the hip, but not be sensitive to the early symptoms of the
disease. Equally important is to consider if one wants to
measure for an individual patient or for describing a group of
patients as a whole. The former demands much higher lev-
els of measurement properties (e.g., reliability coefficients
>0.90 as opposed to 0.75 to 0.80 being adequate for group
descriptions).66
SELECTING THE OUTCOME THAT CAN
MEET THE MEASUREMENT NEED
The selection of an outcome measure depends entirely
on a clear understanding of measurement need. Often a
well-used instrument is used, rather than looking for one
that matches the concept, population, and purpose. Many
guidelines that offer more detail than can be described
here are available, particularly for the acceptable levels
of reliability and validity.66-72 This section describes a
decision-making process for fit between a given instru-
ment and the clinician’s need (Fig. 31-2). This process
builds on the work of Law72 and the OMERACT filter69
and highlights key understandings in each area from the
published guidelines.
This decision-making process emphasizes three things.
First, it begins by stating the measurement need (why,
what, and in whom), which reinforces that a candidate
measure meet one need, but not the next. Second, it
emphasizes that a lot of the appraisal can be done with-
out statistics. It is done by appraising the questionnaire
or instrument itself and knowledge of its administration.
Third, the inability to affirm each stage suggests that
there is no need to continue. At the later data-based
stages, the clinician may choose to run a small study to
create the evidence (the “do-it” loops) in patients, rather
than abandoning the instrument that seems like a good
candidate. Given these three key features, the process
from left to right is reviewed next.

Need: Concept Population Intended purpose: describe evaluate change

Candidate measure:

1. Matches
target concept? Boxes marked with “do it loop” are
those where you can create the
No evidence and continue on
2. Feasable Blue boxes = pre-data evaluation
to use? Yellow boxes = data-based evaluation
No

a. One point in time (descriptive)

3. Does it measure what Reliability
it says it does? (truth) -internal consistency
No
-inter-rater reliability
Face Construct validity
Content Construct
-differentiates high/low levels
-acts as expected with other indicator

4. Does it have b. Change over time (evaluative)

purpose-specific -test-retest reliability
properties in your -inter-rater reliability
No -responsive to change that is
population?
similar to target situation

Benchmarking
Choose scores (states)
another 5. Is it interpretable?
tool No Change
thresholds

Combinations:
change and state
Good fit for your needs!

Figure 31-2  Algorithm showing decision-making process for the fit of a candidate measure with your measurement need. The left-hand side of the
page is done by appraising the instrument and its instructions. The right-hand side requires numeric evidence of the relevant measurement properties.
Many instruments are weeded out as a poor fit in steps 1, 2, and 3. The “do-end” loop denotes stages at which you can pause to create evidence if it is
missing and not have to abandon the instrument.
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
STEP 1: IS THERE A MATCH BETWEEN
THE INSTRUMENT’S CONCEPT AND THE
MEASUREMENT NEED (CONCEPT,
POPULATION, PURPOSE)?
An operational definition of the target concept, the appli-
cable populations (patients or general population), and
intended purpose should be articulated by the developer and
match your current need.42,68,71,73 If this is not the case, or if
it is not a good match, start with another candidate measure
because this one would not work.68
STEP 2: IS IT FEASIBLE TO USE?
Feasibility covers the practical aspects of using this scale in
the intended setting.42,69,72,73 Does it take too much time? Are
the licensing costs too high? Does it require special equip-
ment? Is it too burdensome for your patients (language,
literacy, acceptability of questions)? Is it formatted well on
the page, and do the responses make sense given the tar-
get and the question? Are the questions phrased in a clear
and simple manner? Are the necessary scoring instructions
available? A negative response to any of these questions
could direct you to go to another, more feasible, instrument.
Feasibility often makes or breaks a decision about a candi-
date measure.69
STEP 3: IS IT MEASURING WHAT IT SAYS
IT IS MEASURING? (TRUTH)
Does the instrument measure what it says it will measure?
We divide this into three areas: content, face, and construct
validity. Content validity appraises the items and domains
of a scale. Have the authors covered what McHorney and
Tarlov66 call the breadth and the depth of the concept, that
is, all the important areas, but also enough depth to capture
the range of experience of the patients? Face validity is an
appraisal of the general direction of the scale—will it hit
the target? Are the response options organized in a logical
direction for high and low levels of this attribute? Does the
scoring make sense?
The stage of construct validity is the dividing point
in the decision process between data-free and data-based
appraisal. Up until now, the appraisal is done by look-
ing at the instrument and its manuals. In construct valid-
ity, we begin to explore data to see if the numeric scores
arising from the instrument make sense. Basic construct
validity should be established regardless of the purpose.
Sometimes it is de-emphasized in evaluative instruments;
however, responsiveness without knowing if the instru-
ment is measuring the target seems misplaced. We place
it before the purpose-specific properties to emphasize its
need as a basis. Construct validity is generally measured
by comparisons with other similar scales or related con-
structs (i.e., high and low levels of pain and function) to
see if the numeric scores are behaving in the way they
should if this were a valid measure of the target con-
cept. Theoretic situations are set up before analysis, the
direction and magnitude of the expected relationship are
declared, and then the relationship is tested.68,73 Com-
parisons also should be made between groups known to
differ (high versus low severity) or with scales where no
469
relationship is found—again based on an a priori the-
ory—to see if the candidate measure behaves according
to the theory. These add to the evidence that the instru-
ment is measuring what it is supposed to measure.68 If the
evidence is unavailable or is not in the intended popula-
tion, you have a choice of abandoning the measure or
doing a study to create that evidence and then continu-
ing to advance.
STEP 4: PURPOSE-SPECIFIC EVIDENCE IN YOUR
POPULATION
After getting a general sense of the construct validity of the
instrument, the next step is to address the specific attributes
needed for the instrument to function to meet your measure-
ment need (discrimination component of the OMERACT
filter).69 More attention is now paid to the intended purpose
and the intended population and the properties of consis-
tency in measurement (reliability—obtaining the same score
in different settings) and additional validity (cross-sectional
again or responsiveness or sensitivity to change).
Descriptive Purpose
Descriptive outcomes often are used to classify individuals
as to severity of condition or to identify them by a prog-
nostic group. In health outcome assessment, descriptive
instruments are needed to classify individuals as respond-
ers or as being in a low disease activity state or remission.
An instrument needs to be precise, that is, the observed
score is very close to the true score with low error. This is
estimated by the internal consistency of a multi-item scale
or questionnaire and Cronbach alpha coefficients or Kuder
Richardson 20 if the scale is dichotomous (yes/no). Internal
consistency is a feature of a scale with many items measur-
ing the same thing—are the responses similar across items
within the instrument? It is not a feature of a scale con-
taining weighted sums of different attributes, such as disease
activity measures.37
If more than one person will be gathering the data, inter-
rater/observer reliability should be measured and quantified
with an intraclass correlation coefficient (ICC) for continu-
ous measures or a weighted kappa for ordered categories.74
There are different types of ICC depending on the model
used for the variance estimates; the type of ICC should be
named.74 The ICC and weighted kappa measure the com-
parability of actual numeric scores and are preferred over
correlation coefficients that look only for trends and not
a direct match in number values. Cutoffs are always chal-
lenging, but in general, reliability (including test-retest)
should be at minimum 0.7566,73 for group level analyses,
and for describing an individual patient, it should be 0.90
to 0.95.66,73 The internal consistency reliability can be con-
verted back into the scale score by calculating the precision
limits—using 95% limits, the true score lists somewhere
within 1.96 × s[1 − r]1/2 where r = internal consistency and
s = standard deviation. This calculation tells us the range
within which the true score for an individual can be found.
If it is too wide (reliability too low), it is impractical to use
that instrument.
Construct validity is revisited for the descriptive instru-
ment, but with more attention to looking for evidence close
 470 BEAToN  |  Assessment of Health Outcomes
to the intended application. If the goal is to measure high
versus low health, the sample should be divided into known
groups with high and low health according to another
accepted opinion, and then this scale is tested against it.
The image of a window can help here in selecting compara-
tors to use for testing. What else gives a bit (or more) of
overlap with the target view? How much correspondence is
expected between scores? For good construct validity, this
a priori hypothesized relationship should be recreated with
data, whether that be a strong correlation or no correlation
at all. An instrument or measure is never universally valid
and requires ongoing testing to improve understanding of
the scores in different situations.
Evaluative Purpose
In evaluative measures, the intent of the study is to focus on
the amount of change over time. Many clinical trials are doing
this and comparing results between treatment and control
groups. Interobserver reliability is important if more than one
measurer is to be involved. The hallmark of a good evaluative
measure relates, however, to time: First, do the scores remain
the same when the target concept has not changed over time
(test-retest reliability)? Second, when the concept changes,
does the score on the instrument/measure change as well?
Test-retest reliability requires two administrations of
the measure over a time when no change has occurred.
This may be easier said than done sometimes, but the
authors should justify their design and how they ensured
no change had occurred. Similar to interobserver reliabil-
ity, the ICC is the preferred statistic for continuous scores,
and weighted kappa, its equivalent, is preferred for cate-
gorical scores. The cutoffs are the same, and a coefficient
can be converted into a “minimal detectable change”75
as 1.96 × s(2[1 − r])1/2, where s = standard deviation and
r = test-retest reliability (ICC).66,75 Ninety-five percent of
subjects who are stable have change scores less than this
value; a change greater than this is not likely to occur in a
stable patient, only in a changing one. It becomes a lower
boundary of meaningful change—anything below that
could be day-to-day fluctuations in scores.
Responsiveness—the accurate detection of change when
it has occurred—is sometimes best thought of as longitudinal
construct validity. Similar to construct validity, responsive-
ness depends on an a priori theoretic relationship—one in
which the attribute is changing over time. Often the focus
is on the amount of change picked up, rather than the type
or amount of change that had occurred. A large change is
not useful if we were expecting a small one; rather it suggests
noise. The construct embedded in a study of responsiveness
should be described carefully and should be a clear match with
the intended application (measurement need). If the goal is
to detect change in a clinical trial, it is important to assess the
instrument’s ability to detect the difference in change between
treatment and control groups. If the goal is to detect change
in a cohort, it might be more useful to examine change in a
single group perhaps in a treatment of known efficacy (hip
replacement) or in subjects who rated themselves as improved
on an external anchor (global index of change).
Responsiveness is summarized with statistics of sig-
nal (change) over noise (error), such as the standardized
response mean (mean change/standard deviation of change),
t ­statistic (mean change/standard error), and effect size (mean
change over standard deviation of baseline)74; each can be
adapted to quantify the relative change between treatment
and control groups.53,76 Deyo and Centor77 also described
the correlational approach (correlate change and another
indicator of change) and the receiver operator curve approach
(various change scores against external “gold standard” that
the person has changed) where the area under the curve is
a summary statistic.77 The numeric summaries of responsive-
ness, such as effect sizes or areas under the curve, should cor-
respond to the type of change expected (a priori theory). A
large effect size or area under the curve does not mean an
instrument is “responsive.” It should correspond with the
change anticipated in the study—small or large. Comparisons
of the effect sizes are helpful if different instruments are being
compared in the same study, as done by Buchbinder and col-
leagues53 or by Verhoeven and coworkers,76 who focused on
responsiveness in early rheumatoid arthritis. Responsiveness
is a highly contextualized property, and the same instrument
may not be responsive in another situation (e.g., early versus
late disease, osteoarthritis versus rheumatoid arthritis).73
STEP 5: INTERPRETABILITY OF SCORES
The final step, often deemed the most elusive,78 is the inter-
pretability of the scores.
Benchmarking States
What is the meaning of a score of 2/10 on a pain score? Is it
a good outcome? The meaning of different scores on an out-
come assessment is used for classifying subjects at the begin-
ning of a trial and at the end point. To do this, comparisons
are made to other known health states—­severity indices,
ability to work, self-rating as mild.79 Gradually, enough
trends might be seen across different scenarios to gain confi-
dence in the meaning of “good” or “mild.”80,81 In rheumatol-
ogy, we see the emergence of low disease activity states82,83
or patient acceptable symptom states84 or remission criteria
with the DAS2838 as thresholds below which subjects are
considered to be in an acceptable state (either tolerable
symptoms or disease activity where it does not require medi-
cation changes). At this point, these thresholds are being
established, and similar to change thresholds, we may find
variability in the values38 that need to be sorted out with
methodologic work and application in clinical practice.
Changes in State
The second type of interpretability concerns change scores.
American College of Rheumatology Response Criteria.
The American College of Rheumatology took the core
set measures and determined that if one observed an X%
change in joint count and in swollen joint count and in at
least three other areas—erythrocyte sedimentation rate or
C-reactive protein, physician global, patient global, pain,
or physical disability—one had a clinical response, and the
individual would be classified as a responder. The percent
is usually 20%, but 50% and 70% have been considered.
The ACR20 is widely used, catches responses across a
wide variety of domains, and discriminates well in clinical
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
trials76; however, it is currently being revalidated owing to
the changing nature of rheumatoid arthritis and its care.85
Minimal Clinically Important Differences and Improve-
ments. Defining the threshold of change above which an
individual has had an “important” shift in outcome is what
Kirwan78 has described as the “elusive crock of gold at the
end of the rainbow.” Nevertheless, important advances have
been made. There are many sources of variation in score,
including the method used, the baseline severity, and the
type of change to be sought.86,87 In 2000, Wells and cowork-
ers88 described nine different methods for deriving minimal
clinically important differences from the literature. Some
use distributional cutoffs (½ standard deviation, or effect
size of 0.2 or 0.5),89 which have been criticized as lacking
any meaningful anchor. Other methods depend on some
external anchor that important improvement has occurred,
but are sometimes challenged by the dependence on that
anchor and the perspective of the individual who deter-
mines it (patient, physician, third-party payer). Minimal
clinically important differences repeatedly have been shown
to vary with baseline state90,91 and with improvement versus
deterioration.92 Tubach and associates93 changed the term
to minimal clinically important improvement and looked only
at improvement. Minimal clinically important differences
vary depending on the context of measurement. You need
to plan on working with a range of values,42,86,87 to make sure
the measurement situation is similar to your own (severity,
timing, type of intervention), and to build confidence with
congruence in minimal clinically important differences from
across methods if you can achieve that.
Combined Approaches: Change and State
An attractive, although often overlooked, option is com-
bining change and state. In 1996, EULAR defined clini-
cal response as a change in DAS28 score of more than 1.2
(change) plus a final DAS28 score of less than 2.4 (final
state).36 Jacobson and colleagues94 did the same in defining
response to psychotherapy; change greater than error was
used (minimal detectable change mentioned earlier) plus a
final “normal” state. Studies from the patient’s perspective
have often reflected the same thing.80,81,95 Treatment needs to
induce a change, but perhaps it also needs to land patients
in a healthy state to make them feel better.
The approaches described previously focus on interpreta-
tion at the level of the individual, perhaps for use in clinical
practice or in a response-type analysis of a clinical trial or eco-
nomic appraisal (% responder). Verhoeven and ­coworkers76
showed that the same instrument may not perform equally
well in a responder type of analysis and a group level change.
At each stage of this appraisal, there is an element of
judgment. It is likely there will never be perfect evidence
across all stages. The user needs to assess the potential risk
of accepting less than ideal evidence or abandon the scale.
Users also may create the evidence, however, by doing it
themselves. An instrument that makes it through this
appraisal is likely a good fit with the measurement need.
Anything short of that could lead to error and be prone to
misinterpretation. By working from left to right, scales that
are not targeting the right concept or are impractical to use
in the intended setting can be eliminated quickly before
471
reviewing the literature extensively for the measurement
properties.
SECTION 6: AREAS OF GROWTH IN HEALTH
OUTCOME ASSESSMENT
Item Response Theory
Users of outcomes in arthritis come across item response
theory (IRT) and computer adaptive testing (CAT). These
terms relate to newer methods of ordering and calibrating
items on a scale so that there is equal meaning in score incre-
ments across the scale. Most of our outcomes were developed
in “classic test theory” (internal consistency, summed scores
without weights). There are two schools within IRT: Rasch,
which fixes the parameters and assesses if items in a scale fit
or do not fit that model, and IRT itself, which fits a model
to the data, rather than the reverse. IRT and Rasch are often
presented as conflicting schools, but they are both working
toward an item calibration that allows more accuracy and
precision. In the future, direct comparisons may reveal their
similarities and differences in practical ways. The weights
are cumbersome to apply for the clinician, but can be eas-
ily integrated into a computer-based scoring system for easy
data entry and CAT. CAT chooses items based on the previ-
ous set of responses and uses the fewest number of items to
reach a precise score skipping easier items if confident the
subject can do the harder ones. This streamlined scoring is
quite attractive, but there are some limitations. It depends
on technology that may not at this time be available in
every setting. It also may be influenced by differential item
functioning, which means an item might change weight, or
order, in certain subgroups and necessitates more complex
weights. An example of differential item functioning would
be putting on a pullover sweater. It is a hard task if you have
shoulder pain, but pretty easy if you have a hand problem
and would require a different weight.
The National Institutes of Health is currently funding
PROMIS (Patient Reported Outcomes Measurement Infor-
mation System; available at http://www.nihpromis.org/) to
develop a CAT system (currently based on a two-parameter
graded response model IRT) for common chronic diseases,
including arthritis.96 Several measures have been pooled
into a large database and are being refined and rescaled at
the time of this writing. Well-known measures, such as the
HAQ, also will be used to allow for cross-calibration with
the newer items. All findings will be reported on the PRO-
MIS website, as will access to the scoring algorithms.
Use of Technology in Health Outcomes Assessment
In addition to enabling efforts such as PROMIS to develop
CAT systems, information technology has changed many
aspects of health outcome assessments. Streamlined, cus-
tomized assessments can be set up on the Internet or on a
stand-alone computer with interfaces such as touch screen,
light pens, or “point and click.” Patients can complete the
questionnaires at home, at the clinic, on their PDA, or
on a tablet. Language and literacy issues can be overcome
with talking screens. Scoring becomes instantaneous,
and reports can be printed immediately summarizing the
scored results in time for the clinical visit.97,98 ­Comparisons
 472 BEAToN  |  Assessment of Health Outcomes
between touch screens and traditional paper and pen-  
cils are promising, and the acceptability by patients with
arthritis is good.97-99 New technology means that health  
outcome assessment can become part of the patient-
­clinician ­experience and facilitate the ability of the clini-
cian to monitor the patient’s health.97  
 
Adaptation to an Ongoing Disease
This chapter has focused on the measurement of health states  
and their interpretation over time. Individuals with chronic
diseases adapt to ongoing disease with behavioral strategies or
cognitive reframing of their situation.100 In some circles, this  
is adjustment95; in others, it is response shift.101 The challenge
in health outcomes assessment is to tell when a state is chang-
ing only because of adaptation and not the intervention. In
many situations, we try to induce adaptation, or cognitive
reframing, and it can be constructive. It does create a bias
in measurement,101 however, and a challenge to the health
outcome assessor. Numerous groups are researching how to
incorporate adaptation into health outcome assessments.
SUMMARY
There is considerable room for improvement in health out-
come assessment in rheumatology, despite the work done to
date. A battery of instruments have been developed, many of
which exhibit the measurement properties described in this
chapter and meet the challenge of a changing arthritis tar-
get (less severe, earlier disease), and several more measures
are being considered for membership in core sets to capture a
comprehensive view of the burden of arthritis. We are on the
brink of deciding on the role to be played by IRT and CAT
in widespread care settings. Despite progress in assigning a
numeric value to a complex health state, however, we are now
struggling with the back-translation—what does the numeric
score mean in the real patient world. It is not always a simple
translation from questionnaire score to clinical meaning.
Health outcome assessment is well advanced in arthritis care,
and we should recognize the years of work and commitment of
many professional and patient/consumer groups. Advances will
continue in the use of technology, the breadth and depth of
outcomes, and the quality of measurement to keep pace with
the needs of patients, clinicians, and researchers.
Acknowledgments
Dorcas Beaton is supported by a New Investigators Award through the
Canadian Institutes of Health Research. Peter Tugwell holds a Canada
Research Chair.
The authors would like to thank Ms. Taucha Inrig, Dr. Claire
­Bombardier, Dr. Fred and Mrs. Janet Krieger, Mr. William Francis, and
the OMERACT executive for their help with this manuscript, and
Dr. M. Ward, whose chapter in the seventh edition of Kelley’s Textbook of
Rheumatology was a helpful guide.
REFERENCES
  1. Relman AS: Assessment and accountability: The third revolution in
medical care. N Engl J Med 319:1220-1222, 1988.
  2. Last JM: A Dictionary of Epidemiology. New York, Oxford University
Press, 1988.
  3. Prevoo MLL, Van’t Hof MA, et al: Modified disease activity scores
that include twenty-eight-joint counts: Development and ­validation
in a prospective longitudinal study of patients with rheumatoid
arthritis. Arthritis Rheum 38:44-48, 1995.
4. Fries JF, Spitz PW, Young DY: The dimensions of health outcomes:
The Health Assessment Questionnaire, Disability and Pain Scales.
J Rheumatol 9:19203, 1982.
5. Ware JE Jr, Sherbourne CD: The MOS 36-Item Short-Form Health
Survey (SF-36), I: Conceptual framework and item selection. Med
Care 30:473-483, 1992.
6. Fries JF: The hierarchy of outcome assessment. J Rheumatol 20:
546-547, 1993.
7. Wolfe F, Lassere M, van der Heijde D, et al: Preliminary core set
of domains and reporting requirements for longitudinal observa-
tional studies in rheumatology. J Rheumatol 26:484-489, 1999.
8. van der Heijde D, van der Linden S, Bellamy N, et al: Which
domains should be included in a core set for endpoints in ankylos-
ing spondylitis? Introduction to the ankylosing spondylitis module of
OMERACT IV. J Rheumatol 26:945-947, 1999.
9. Gladman DD, Mease PJ, Healy P, et al: Outcome measures in psori-
atic arthritis (PsA). J Rheumatol 34:1159-1166, 2007.
10. Gladman DD, Mease PJ, Strand V, et al: Consensus on a core set of
domains for psoriatic arthritis. OMERACT 8 PsA Module Report.
J Rheumatol 34:1167-1170, 2007.
11. Sambrook PN, Cummings SR, Eisman JA, et al: Guidelines of osteo-
porosis trials (workshop report). J Rheumatol 24:1234-1236, 1997.
12. Gladman DD, Strand V, Mease PJ, et-al: OMERACT 7 psoriatic
arthritis workshop: Synopsis. Ann Rheum Dis 64:ii-115-ii-116, 2005.
13. Bellamy N, Kirwan J, Boers M, et al: Recommendations for a core set
of outcome measures for future phase III clinical trials in knee, hip,
and hand osteoarthritis: Consensus development at OMERACT III.
J Rheumatol 24:799-802, 1997.
14. Smolen JS, Strand V, Cardiel M, et al: Randomized clinical trials and
longitudinal observational studies in systemic lupus erythematosus:
Consensus on a preliminary core set of outcome domains. J Rheuma-
tol 26:504-507, 1999.
15. Boers M, Tugwell P, Felson DT, et al: World Health Organization
and International League of Associations for Rheumatology core
endpoints for symptom modifying antirheumatic drugs in rheumatoid
arthritis clinical trials. J Rheumatol 21:86-89, 1994.
16. Felson DT, Anderson JJ, Boers M, et al: The American College of
Rheumatology preliminary core set of disease activity measures for
rheumatoid arthritis clinical trials. The Committee on Outcome
Measures in Rheumatoid Arthritis Clinical Trials. Arthritis Rheum
36:729-740, 1993.
17. van der Heijde D, Landewe R: Selection of a method for scoring radio-
graphs for ankylosing spondyolitis clinical trials, by the Assessment
in Ankylosing Spondylitis working groups (ASAS) and OMERACT.
J Rheumatol 32:2048-2049, 2005.
18. Guidelines of osteoporosis trials (workshop report). J Rheumatol
24:1234–1236, 1997.
19. Strand V, Gladman DD, Isenberg D, et al: Outcome measures to be
used in clinical trials in systemic lupus erythematosus. J Rheumatol
26:490-497, 1999.
20. Patrick DL, Deyo RA: Generic and disease-specific measures in
assessing health status and quality of life. Med Care 27(Suppl):
S217-S232, 1989.
21. Bergner M, Bobbitt RA, Pollard WE, et al: The sickness impact pro-
file: Validation of a health status measure. Med Care 14:57-67, 1976.
22. Ware JE Jr: SF-36 health survey update. Spine 25:3130-3139, 2000.
23. Ware JE Jr, Snow KK, Kosinski M, et al: SF-36 Health Survey Man-
ual and Interpretation Guide. Boston, The Health Institute, 1993.
24. Beaton DE, Bombardier C, Hogg-Johnson SA: Measuring health in
injured workers: A cross-sectional comparison of five generic health
status instruments in workers with musculoskeletal injuries. Am J Ind
Med 29:618-631, 1996.
25. Beaton DE, Hogg-Johnson S, Bombardier C: Evaluating changes in
health status: Reliability and responsiveness of five generic health
status measures in workers with musculoskeletal disorders. J Clin Epi-
demiol 50:79-93, 1997.
26. Visser MC, Fletcher AE, Parr G, et al: A comparison of three qual-
ity of life instruments in subjects with angina pectoris: The Sickness
Impact Profile, the Nottingham Health Profile, and the Quality of
Well Being Scale. J Clin Epidemiol 47:157-163, 1994.
27. Revicki DA, Kaplan RM: Relationship between psychometric and
utility-based approaches to the measurement of health-related qual-
ity of life. Qual Life Res 2:477-487, 1993.
28. Feeny D: Preference-based measures: utility and quality-adjusted life
years. In Fayers P, Hays R (eds): Assessing Quality of Life in Clinical
Trials, 2nd ed. New York, Oxford University Press, 2005, pp 405-429.
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
29. Farrar JT, Portenoy RK, Berlin JA, et al: Defining the clinically
important difference in pain outcome measures. Pain 88:287-294,
2000.
30. Garrett S, Jenkinson T, Kennedy LG, et al: A new approach to defin-
ing disease status in ankylosing spondylitis: The BATH Ankylosing
Spondylitis Disease Activity Index. J Rheumatol 21:2286-2291, 1994.
31. Fries JF: The hierarchy of quality-of-life assessment, the Health Assess-
ment Questionnaire (HAQ), and issues mandating development of a
toxicity index. Controlled Clinical Trials 12:106S-117S, 1991.
32. Meenan RF, Gertman PM, Mason JH: Measuring health status in
arthritis: The Arthritis Impact Measurement Scales. Arthritis Rheum
23:146-152, 1980.
33. Meenan RF, Mason JH, Anderson JJ, et al: Aims2: The content and
properties of a revised and expanded arthritis impact measurement
scales health status questionnaire. Arthritis Rheum 35:1-10, 1992.
34. Bellamy N, Buchanan WW, Goldsmith CH, et al: Validation study
of WOMAC: A health status instrument for measuring clinically-
important patient-relevant outcomes following total hip or knee
arthroplasty in osteoarthritis. J Orthop Rheum 1:95-108, 1988.
35. Bellamy N, Campbell J, Haraoui B, et al: Clinimetric properties of the
AUSCAN osteoarthritis hand index: An evaluation of reliability, valid-
ity and responsiveness. Osteoarthritis Cartilage 10:863-869, 2002.
36. Van Gestel AM, Prevoo MLL, Van’t Hof MA, et al: Development
and validation of the European League Against Rheumatism response
criteria for rheumatoid arthritis. Arthritis Rheum 39:34-40, 1996.
37. Vrijhoef HJM, Diederiks JPM, Spreeuwenberg C, et al: Applying low
disease activity criteria using the DAS28 to assess stability in patients
with rheumatoid arthritis. Ann Rheum Disease 62:419-422, 2003.
38. Aletaha D, Ward MM, Machold KP, et al: Remission and active
disease in rheumatoid arthritis: Defining criteria for disease activity
states. Arthritis Rheum 52:2625-2636, 2005.
39. Lassere M, van der Heijde D, Johnson K, et al: Robustness and gen-
eralizability of smallest detectable difference in radiological progres-
sion. J Rheumatol 28:911-913, 2001.
40. Ravaud P, Giraudeau B, Auleley GR, et al: Assessing smallest detect-
able change over time in continuous structural outcome measures:
Application to radiological change in knee osteoarthritis. J Clin Epi-
demiol 52:1225-1230, 1999.
41. Lassere M, Johnson K, Van Santen S, et al: Generic patient self-
report and investigator report instruments of therapeutic safety and
tolerability. J Rheumatol 32:2033-2036, 2005.
42. U.S. Department of Health and Human Services Food and Drug
Administration Center for Drug Evaluation and Research (CDER):
Guidance for industry: Patient-reported outcome measures: Use
in medical product development to support labeling claims: Draft
guidance. Available at: http://www.fda.gov/cder/gdlns/prolbl.htm.
Accessed November 3, 2006.
43. Woodworth T, Furst DE, Alten R, et al: Standardizing assessment and
reporting of adverse effects in rheumatology clinical trials, II: Rheu-
matology Common Toxicity Criteria v2.0. J Rheumatol 34:1401-
1414, 2007.
44. Kristjansson E, Tugwell PS, Wilson AJ, et al: Development of the
effective musculoskeletal consumer scale. J Rheumatol 34:1392-
1400, 2007.
45. Escorpizo R, Bombardier C, Boonen A, et al: Worker productivity
outcome measures in arthritis. J Rheumatol 34:1372-1380, 2007.
46. Lerner D, Amick BC III, Rogers WH, et al: The work limitations
questionnaire. Med Care 39:72-85, 2001.
47. Gignac MAM, Badley EM, Lacaille D, et al: Managing arthritis and
employment: Making arthritis-related work changes as a means of
adaptation. Arthritis Care Res 51:909-916, 2004.
48. Gilworth G, Chamberlain AM, Harvey A, et al: Development of
a work instability scale for rheumatoid arthritis. Arthritis Care Res
49:349-354, 2003.
49. Backman C, Kennedy SM, Chalmers A, et al: Participation in paid
and unpaid work by adults with rheumatoid arthritis. J Rheumatol
31:47-57, 2004.
50. Tugwell P, Bombardier C, Buchanan WW, et al: The MACTAR
Patient Preference Disability Questionnaire—an individualized
functional priority approach for assessing improvement in physi-
cal disability in clinical trials in rheumatoid arthritis. J Rheumatol
14:446-451, 1987.
51. Buchbinder R, Bombardier C, Yeung M, et al: Which outcome mea-
sures should be used in rheumatoid arthritis clinical trials? Clinical
and quality-of-life measures’ responsiveness to treatment in a ran-
domized controlled trial. Arthritis Rheum 38:1568-1580, 1995.
473
52. O’Boyle CA, Hofer S, Ring L: Individualized quality of life. In ­Fayers
P, Hays R (eds): Assessing Quality of Life in Clinical Trials: Methods
and Practice, 2nd ed. New York, Oxford University Press, 2005,
pp 225-242.
53. Buchbinder R, Bombardier C, Yeung M, et al: Which outcome
measures should be used in rheumatoid arthritis clinical trials?
Arthritis Rheum 38:1568-1580, 1995.
54. Solomon DH, Bates DW, Horsky J, et al: Development and valida-
tion of a patient satisfaction scale for musculoskeletal care. Arthritis
Care Res 12:96-100, 1999.
55. Hudak PL, McKeever PD, Wright JG: Understanding the meaning of
satisfaction with treatment outcome. Med Care 42:718-725, 2004.
56. Jette AM, Haley SM: Contemporary measurement technique for
rehabilitation outcome assessment. J Rehabil 37:339-345, 2005.
57. Jette AM, Keysor JJ: Disability models: Implications for arthritis
exercise and physical activity interventions. Arthritis Care Res 49:
114-120, 2003.
58. Wilson IB, Cleary PD: Linking clinical variables with health-related
quality of life: A conceptual model of patient outcomes. JAMA
273:59-65, 1995.
59. Nagi SZ: A study in the evaluation of disability and rehabilitation
potential. Am J Public Health 54:1568-1579, 1964.
60. Verbrugge LM, Jette AM: The disablement process. Soc Sci Med
38:1-14, 1994.
61. World Health Organization: International Classification of Function-
ing, Disability and Health. Geneva, World Health Organization, 2001.
62. Arts DGT, Keizer NF, Scheffer G: Defining and improving data qual-
ity in medical registries: A literature review, case study and generic
framework. J Am Med Inform Assoc 9:600-611, 2002.
63. Stucki G, Boonen A, Tugwell P, et al: The World Health Organi-
sation International Classification of Functioning, Disability and
Health (ICF): A conceptual model and interface for the OMERACT
process. J Rheumatol 34(3):600-606, 2007.
64. Jette AM: Toward a common language for function, disability and
health. Phys Ther 86:726-734, 2006.
65. Jette AM, Keysor JJ: Uses of evidence in disability outcomes and
effectiveness research. Milbank Q 80:325-345, 2002.
66. McHorneyCA, Tarlov AR: Individual patient monitoring in clini-
cal practice: Are available health status surveys adequate? Qual
Life Res 4:293, 1995.
67. Lohr KN, Aaronson NK, Alonso J, et al: Evaluating quality-of-life
and health status instruments: Development of scientific review cri-
teria. Clin Therap 18:979-992, 1996.
68. McDowell I, Jenkinson C: Development standards for health mea-
sures. J Health Serv Res Policy 1:238-246, 1996.
69. Boers M, Brooks P, Strand V, et al: The OMERACT Filter for out-
come measures in rheumatology. J Rheumatol 25:198-199, 1998.
70. Kane RA, Kane RL: Assessing the Elderly: A Practical Guide to mea-
surement. Toronto, Lexington Books, 1981, pp13-17.
71. Bergner M: Health status measures: An overview and guide for selec-
tion. Ann Rev Public Health 8:191-210, 1987.
72. Law M: Measurement in occupational therapy: Scientific criteria for
evaluation. Can J Occup Ther 54:133-138, 1987.
73. Scientific Advisory Committee of the Medical Outcomes Trust:
Assessing health status and quality of life instruments: Attributes
and review criteria. Qual Life Res 11:193-205, 2002.
74. Hays RD, Revicki D: Reliability and validity (including responsive-
ness). In Fayers P, Hays R (eds): Assessing Quality of Life in Clinical
Trials: Methods and Practice, 2nd ed. New York, Oxford University
Press, 2005, pp 25-39.
75. Stratford PW, Binkley JM: Applying the results of self-report mea-
sures to individual patients: An example using the Roland-­Morris
Questionnaire. J Orthop Sports Physical Ther 29:232-239,
1999.
76. Verhoeven A, Boers M, van der Linden S: Responsiveness of the core
set, response criteria, and utilities in early rheumatoid arthritis. Ann
Rheum Dis 59:966-974, 2000.
77. DeyoRA, Centor RM: Assessing the responsiveness of functional
scales to clinical change: An analogy to diagnostic test perfor-
mance. J Chronic Dis 39:897-906, 1986.
78. Kirwan J: Minimum clinically important difference: The crock of
gold at the end of the rainbow? J Rheumatol 28:439-444, 2001.
79. Deyo RA, Carter WB: Strategies for improving and expand-
ing the application of health status measures in clinical set-
tings: A researcher-developer viewpoint. Med Care 30(5 Suppl):
MS176-MS186, 1992.
 474 BEAToN  |  Assessment of Health Outcomes
80. Tubach F, Dougados M, Falissard B, et al: Feeling good rather than
feeling better matters more to patients. Arthritis Care Res 55:
526-530, 2006.
81. Beaton DE, Tarasuk V, Katz JN, et al: Are you better? A qualitative
study of the meaning of being better. Arthritis Care Res 7:313-320,
2001.
82. Boers M, Anderson JJ, Felson D: Deriving an operational definition
of low disease activity state in rheumatoid arthritis. J Rheumatol
30:1112-1114, 2003.
83. Tubach F, Wells GA, Ravaud P, et al: Minimal clinically important
difference, low disease activity state and patient acceptable symptom
state: Methodological issues. J Rheumatol 32:2025-2029, 2005.
84. Tubach F, Ravaud P, Baron G, et al: Evaluation of clinically relevant
states in patient reported outcomes in knee and hip osteoarthrits:
The patient acceptable symptom state. Ann Rheum Dis 64:34-37,
2005.
85. Felson DT, Furst DE, Boers M: Rationale and strategies for reeval-
uating the ACR20. J Rheumatol 34:1184-1187, 2007.
86. Beaton DE, Boers M, Wells GA: Many faces of the minimal clini-
cally important difference (MCID): A literature review and direc-
tions for future research. Curr Opin Rheumatol 14:109-114,
2002.
87. Hays RD, Woolley JM: The concept of clinically meaningful differ-
ence in health-related quality of life research. PharmacoEconomics
18:419-423, 2000.
88. Wells GA, Beaton DE, Shea B, et al: Minimal clinically important
differences: Review of methods. J Rheumatol 28:406-412, 2001.
89. Norman GR, Sloan JA, Wyrwich KW: Interpretation of changes in
health-related quality of life: The remarkable universality of half a
standard deviation. Med Care 41:582-592, 2003.
90. Salaffi F, Stancati A, Silvestri CA, et al: Minimal clinically impor-
tant changes in chronic musculoskeletal pain intensity measures on a
numerical rating scale. Eur J Pain 8:283-291, 2004.
91. Stucki G, Daltroy L, Katz JN, et al: Interpretation of change scores in
ordinal clinical scales and health status measures: The whole may not
be equal to the sum of the parts. J Clin Epidemiol 49:711-717, 1996.
92. Angst F, Aeschlimann A, Stucki G: Smallest detectable and minimal
clinically important differences of rehabilitation intervention with
their implications for required sample sizes using WOMAC and SF-
36 quality of life measurement instruments in patients with osteo-
arthritis of the lower extremities. Arthritis Care Res 45:384-391,
2001.
93. Tubach F, Ravaud P, Baron G, et al: Evaluation of clinically relevant
changes in patient reported outcomes in knee and hip osteoarthritis:
The minimal clinically important improvement. Ann Rheum Dis
64:29-33, 2005.
94. Jacobson NS, Roberts LJ, Berns SB, et al: Methods for defining and
determining the clinical significance of treatment effects: Descrip-
tion, application, alternatives. J Consult Clin Psychol 67:300-307,
1999.
95. Norman G: Hi! How are you? Response shift, implicit theories and
differing epistemologies. Qual Life Res 12:249, 2003.
96. National Institutes of Health: Patient Reported Outcome Measure-
ment Information System (PROMIS) network. 2006. Available at:
http://www.nihpromis.org/.
97. Athale N, Sturley A, Koczen Z, et al: A web-compatible instrument
for measuring self-reported disease activity in arthritis. J Rheumatol
31:223-228, 2004.
98. Fransen J, Stucki G, Twisk J, et al: Effectiveness of a measurement
feedback system on outcome in rheumatoid arthritis: A controlled
clinical trial. Ann Rheum Dis 62:624-629, 2003.
99. Bischoff-Ferrari HF, Vandechend M, Bellamy N, et al: Validation
and patient acceptance of a computer touch screen version of the
WOMAC 3.1 osteoarthritis index. Ann Rheum Dis 64:80-84, 2004.
100. Shaul MP: From early twinges to mastery: The process of adjustment in
living with rheumatoid arthritis. Arthritis Care Res 8:290-297, 1995.
101. Schwartz C, Sprangers M, Fayers P: Response shift: You know it’s
there but how do you capture it? Challenges for the next phase of
research. In Fayers P, Hays R (eds): Assessing Qualify of Life in Clini-
cal Trials: Methods and Practice, 2nd ed. New York, Oxford Univer-
sity Press, 2005, pp 275-290.
 32
KEY POINTS
Uniform definitions of disease are essential for biomarker
validation and comparison between studies.
The validation process of a biomarker depends on the specific
purpose of its use.
For tissue homeostasis, the balance between anabolic and
catabolic process is important; extracellular matrix remodeling
biomarkers should reflect these different processes.
Understanding tissue source, formation, and clearance of a
biomarker is important for correctly interpreting biomarker
data.
Analyses of serial synovial biopsy specimens can potentially
be used as a screening method to test new drug candidates
requiring relatively small numbers of patients.
Panels of biomarkers or biomarker profiles are potentially
more powerful than single biomarkers.
Biomarkers are anatomic, physiologic, biochemical, or
molecular parameters associated with the presence and
severity of specific diseases and are detectable by a vari-
ety of methods, including physical examination, labora-
tory assays, and imaging. Here the focus is on biomarkers
in senso stricto: markers that can be measured in patient
samples, such as blood, urine, synovial fluid, and synovial
tissue. Such molecules often first appear in studies on dis-
ease mechanisms, their application as biomarkers being
secondary. Realizing the importance and potential of bio-
logic markers, the identification, characterization, and
validation of novel biomarkers is often a primary objective
in many studies nowadays. Biomarkers are applied for vari-
ous purposes, including diagnosis, prognosis, monitoring
of disease progression, selection of patient populations in
clinical trials, assessment of the efficacy of treatment, or
unraveling of the pathobiology of a disease in the clini-
cal and in a preclinical setting. More recently, biomarker
validation level of evidence schemes have been proposed
to optimize efficient use of biomarkers in these diverse
research areas.1,2
For osteoarthritis and RA, the most important common
feature is progressive destruction of articular tissues, result-
ing in impaired joint function and pain. Diagnosis is based
on clinical symptoms and laboratory tests in combination
with radiography, to visualize often irreversible degenerative
and destructive changes in the joint. Radiologic evaluation
Biologic Markers
Jeroen Degroot  • 
Anne-Marie Zuurmond  • 
Paul P. Tak
of joints mainly images bone and is relatively insensitive:
A follow-up period of at least 1 year is needed to assess dis-
ease progression and effect of therapy. Magnetic resonance
imaging (MRI) has the ability to visualize all joint tissues
simultaneously; it is currently being optimized, but has yet
not reached its full potential.3 All imaging techniques pro-
vide a cumulative historical view of damage that has already
occurred, rather than assessing the current rate of disease
progression (Fig. 32-1).
Alternative methods that detect changes in the joints
in an early stage of the disease in a quantitative, reliable,
and sensitive manner are needed. The World Health Orga-
nization in its report Priority Medicine for Europe and the
World states that biomarkers are essential for arthritis in
general and osteoarthritis research in particular, and that
the lack of adequate markers constitutes a major hurdle for
osteoarthritis drug development.4 Molecular markers (i.e.,
markers that are used to monitor molecular events occur-
ring during disease) are well suited for this purpose. A good
marker is disease-specific, reflects actual disease activity, is
sensitive to change after therapy, and can predict disease
outcome. Most likely, all of these requirements are not met
by a single marker: Different (combinations of) markers
would have to be applied for different purposes. The fact
that imaging techniques often focus on one joint (e.g., knee
or hip) or joint group (e.g., hands, lumbar spine), whereas
urine-derived or blood-derived biomarkers are determined
by all of the joints in the body, further underscores the
importance of using imaging and biomarker approaches
as complementary tools. The molecular markers that are
described for joint diseases can be arbitrarily classified as
follows:
1. Immunologic and inflammatory markers (including acute-
phase proteins)
2. Markers that reflect extracellular matrix remodeling
(Table 32-1)
3. Markers present in synovial tissue biopsy specimens
4. “Omics”-based biomarkers (e.g., genomics, transcrip-
tomics, proteomics, metabolomics)
5. Genetic markers that have been shown to provide impor-
tant information on risk factors for development and
progression of osteoarthritis and RA; these are reviewed
elsewhere5-8 and are not included in this chapter.
IMMUNOLOGIC AND INFLAMMATORY
BIOMARKERS
Extensively studied markers in inflammatory arthritis that
are routinely used in clinical practice include acute-phase
proteins such as C-reactive protein (CRP) and measure-
ments such as the erythrocyte sedimentation rate (ESR),
475
 476 Degroot  |  Biologic Markers
Radiograph
Marker
A not show a clinical response (i.e., ACR20 nonresponders)
Radiograph/time
B tis biomarkers). In osteoarthritis patients, serum nitrate and
X-ray
AUC of marker
Time
Figure 32-1  Relationship between radiology and biomarkers. Radi-
ology and MRI provide a cumulative historical view of joint destruc-
tion, whereas biomarkers provide dynamic information on the current
rate of joint destruction or disease activity. A, At any given time, the
slope of the x-ray versus time plot should be compared with biomarker
levels. B, Consequently, the progression of x-ray damage (Δ x-ray)
must be related to the time-integrated marker levels (area under the
curve [AUC]).
both of which provide information about the systemic in­­
flammatory process. Although such markers of inflamma-
tion are neither disease-specific nor tissue-specific, they may
reflect disease activity,9 reflect the effects of immunosuppres-
sive and immunomodulatory therapies,10 and, to a certain
extent, predict disease progression in RA.11 A more disease-
specific marker for RA is the presence of anticitrullinated
protein/peptide antibodies.12 It predicts with high prob-
ability the development of RA in patients with no clinical
symptoms, distinguishes between RA and other rheumatic
diseases, and is a valuable tool for prognostic prediction of
joint destruction.13
RA patients often show elevated levels of a variety of
cytokines, chemokines, and their receptors (for details see
Chapter 23). Many of these molecules are subsequently
used as disease biomarkers or studied as targets for inter-
vention, analogous to the experience with tumor necrosis
factor (TNF)-α. This cytokine has been shown to play a
pivotal role in arthritis and has emerged as a major thera-
peutic target. TNF-α inhibitors (neutralizing antibodies or
soluble receptors) strongly reduce clinical symptoms, joint
inflammation, and biomarkers of inflammation and bone
destruction in RA patients.10,14,15 Neither the serum lev-
els of the TNF receptors nor those of interleukin-10 were
reduced, however, in response to treatment of RA patients
with methotrexate and anti-TNF treatment, despite a clear
improvement in clinical disease parameters and a reduction
of CRP and ESR.16 In contrast, in synovial tissue, treat-
ment with these drugs did reduce the local TNF level, indi-
cating that for biomarker analysis it is important to select
the right compartment.17,18 The observation that anti-TNF
treatment may block joint destruction in patients who do
suggests that, similar to osteoarthritis, in RA uncoupling
of inflammation and joint destruction may occur, which
may affect the selection of biomarkers to monitor treat-
ment effects.
Although synovial inflammation is often regarded as a
secondary process in osteoarthritis, cytokines and other sig-
nal molecules also have been proposed as markers in this
disease. In clinical studies, often highly sensitive analysis of
CRP is included because it is associated with osteoarthritis
of the knee19,20 and hip,21 but validation of this biomarker
to monitor treatment efficacy is hampered by the lack of
effective treatments (as is the difficulty with all osteoarthri-
nitrite (reflecting nitric oxide levels) are increased compared
with healthy controls, but not as much as in inflammatory
conditions such as RA.22 Also, similar to RA, high levels
of soluble receptors of TNF-α are associated with reduced
physical function and worse radiologic knee osteoarthritis.23
In addition, using a genomics approach, chondrocytes from
osteoarthritis cartilage have been shown to upregulate the
transcription of a variety of inflammatory genes.24 Syno-
vial tissue from osteoarthritis patients also shows signs of
hyperplasia and inflammation.25 Although osteoarthritis
traditionally is viewed as a noninflammatory arthropathy,
an osteoarthritic joint could be considered a mildly inflamed
organ.
The added value of inflammation biomarkers measured
in peripheral blood in joint diseases is still undecided.
Although the levels of many inflammatory molecules may
change in various (stages of) diseases, such change does not
mean that the molecule is directly involved in the disease
process or is sensitive to change after intervention. Serum
cytokine levels often do not predict clinical response. As
such, it is crucial to understand the complex biologic net-
works and the mode of action of treatments to be able to
select the right (relevant) molecules to use as biomarkers.
In view of this, the ultimate application of inflammation
biomarkers in osteoarthritis studies is likely to be different
from RA studies.
EXTRACELLULAR MATRIX REMODELING
BIOMARKERS
Because of the aforementioned issues, the focus of many
biomarker studies is on markers that mirror disease-related
changes in the joints, especially markers for remodel-
ing (i.e., degradation and synthesis) of the extracellular
matrix.26 Concomitant changes may occur in all joint tis-
sues (cartilage, bone, and synovium), and for a compre-
hensive assessment of these changes molecular markers
are needed for each of these tissues.27,28 For the markers
derived from these different tissues, there can be a substan-
tial difference in which biologic fluid biomarker levels are
determined.
The concentration of markers in body fluids not only
reflects the dynamics of the disease, but also the rate of
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES 477

Table 32-1  Molecular Markers That Reflect Extra Cellular Matrix Remodeling
Marker Joint Tissue References
Synthesis
Collagen type I
  N-propeptide (PINP) Bone, soft tissues 23, 131
  C-propeptide (PICP) Bone, soft tissues 23
Collagen type II
  N-propeptide (PIINP; PIIANP) Cartilage 27, 40
  C-propeptide (PIICP; chondrocalcin) Cartilage 27, 56, 61, 62, 72
Collagen type III
  N-propeptide (PIIINP) Soft tissues 39, 63-65, 131
Proteoglycans and GAGs
  Chondroitin sulfate (epitopes 846, 3-B-3, 7-D-4) Cartilage 51, 56, 57, 71-74
Miscellaneous
  Bone-specific alanine phosphatase Bone 23
  Osteocalcin Bone 24
  YKL-40 (CYLK-40, gp-39, chondrex) Cartilage 131
  Hyaluronan Cartilage, synovium 42, 80-84, 131
Degradation
Collagen type I
  Cross-linked N-telopeptide (NTx) Bone 33-35
  Cross-linked C-telopeptide (CTx) Bone 24, 32, 33, 36, 131
  Cross-linked C-telopeptide (ICTP) Soft tissues 29-31, 33
  Collagenase cleavage neoepitope (C1, 2C) Bone, soft tissues 27
Collagen type II
  Cross-linked C-telopeptide (CTx-II; 2B4 epitope) Cartilage 24, 38-46, 131
  Collagenase cleavage neoepitope (9A4, C2C, C1, 2C) Cartilage 27, 47-54
Pyridinolines
  Hydroxylysylpyridinoline (HP, PYR) Bone, cartilage 32, 33, 37, 55-57
  Lysylpyridinoline (LP, D-PYR, DPD) Bone 32-35, 55-57
  Glucosylgalactosyl-hydroxypyridinoline (GGHP) Synovium 40, 58
Proteoglycans and GAGs
  Aggrecan core protein (fragments) Cartilage 23
  Keratan sulfate (epitope 5-D-4, AN9P1) Cartilage 51, 56, 57, 66-70
  Chondroitin sulfate Cartilage 23
Miscellaneous
  Matrix metalloproteinases Cartilage, synovium 40, 72, 75-77, 94, 96-102, 131
  Aggrecanases Cartilage 75, 78, 79, 103-107
  Cartilage oligomeric matrix protein (COMP) Cartilage, possibly synovium 39, 56, 72, 85-95
  Bone sialoprotein (BSP) Bone 56
GAGs, glycosaminoglycans.

clearance and the amount of remaining tissue. Cartilage- COLLAGEN MARKERS

derived markers diffuse out of the tissue and enter the
synovial fluid, which may vary in volume depending on
the severity of ongoing inflammation. Synovial fluid urea
levels may be used to correct for this “dilution.”29 Clear-
ance from the synovial fluid predominantly occurs via lym-
phatic drainage, and partial degradation may occur in the
lymph nodes, depending on the marker studied. Synovial
clearance may depend on the severity of the ongoing syno-
vitis and be determined by the permeability of the synovial
membrane microvasculature.30 When the marker enters the
systemic circulation, dilution occurs (e.g., 5 mL of synovial
fluid ­versus ­approximately 5 L of blood), and marker levels
are confounded by molecules from other affected, or non-
affected, joints or even from nonarticular cartilage.
Bone-derived and synovium-derived markers also may
enter the circulation directly. When the marker enters the
systemic circulation, it is diluted, mixed with markers derived
from other joints or tissues, and potentially metabolized in
the liver and kidneys. Excretion in the urine depends on the
marker and the above-mentioned metabolic processes. In
the discussion that follows, examples are given from studies
of osteoarthritis and RA to illustrate their use in assessing
disease-related tissue remodeling.
The main constituent of the extracellular matrix of con-
nective tissues is collagen, which plays an essential role
in the maintenance of tissue shape, strength, and integ-
rity. Fibrillar collagen types I, II, and III are synthesized as
propeptide-containing α chains that are post-translation-
ally modified by hydroxylation of lysyl and prolyl resi-
dues and by glycosylation of hydroxylysyl residues. Triple
helical collagen molecules are secreted from the cell, the
propeptides are cleaved off, and fibrils are formed that
are stabilized by intermolecular cross-links. This unique
sequence of events provides a variety of tools that are
used to study collagen synthesis (especially propeptides)
and degradation (especially cross-links) in rheumatic dis-
eases (Fig. 32-2).31
In bone, type I collagen constitutes 90% of the organic
matrix, and markers of bone collagen turnover have proven
valuable in monitoring diseases such as osteoporosis.32 For
some bone markers, such as NTx (bone degradation [see
later]) and osteocalcin (bone formation), assays have been
approved by the U.S. Food and Drug Administration to
monitor efficacy of antiresorptive therapies or bone forma-
tion, sometimes even as point-of-care test. For these bone
 478 Degroot  |  Biologic Markers

Procollagen
synthesis

Glc Glc Gal

Gal Gal OH OH
Hyl Hyl Lys
Lys Post-translational hydroxylation,
Pro Pro glycosylation and helix formation

OH OH
Intracellular

Secretion
Extracelllular

OH O-Gal-Glc O-Gal-Glc OH O-Gal

N-terminal C-terminal
Figure 32-2  Collagen-based mark- propeptide propeptide
ers for joint destruction. Collagen is OH O-Gal O-Gal-Glc OH OH OH
synthesized as propeptide-containing
α chains that are post-translationally
modified by hydroxylation of lysyl and
prolyl residues and by glycosylation
of hydroxylysyl residues. These modi- ICTP
fications cease when three α chains CTx
­entwine to form a collagen triple HP/LP
N tx
­helix. Triple helical collagen molecules HP/LP
are secreted from the cell, and the
propeptides are cleaved off extracel-
lularly. Subsequently, collagen mol-
ecules spontaneously assemble into
fibrils with quarter-staggered overlap
of the individual triple helices. Finally,
the ­ fibrils are stabilized by formation
Collagenase cleavage
of ­ intermolecular pyridinoline cross-
neoepitopes
links.

metabolites, there is a significant menopausal effect that
requires properly matched control groups and careful inter-
pretation of the data.
Several assays have been used to assess collagen type I
degradation in RA and osteoarthritis. The cross-linked
carboxyterminal telopeptide (ICTP) is released by matrix
metalloproteinase (MMP) cleavage of type I collagen, and its
levels reflect MMP-mediated soft tissue degradation.33 The
NTx/CTx-I assay and ICTP assay detect type I collagen deg-
radation, but by different proteases. Cathepsin K–mediated
osteoclastic bone resorption destroys ICTP antigenicity.33
Slightly elevated serum ICTP levels are found in RA com-
pared with controls and are associated with disease activity
measured by ESR, CRP levels, and swollen joint counts.34
In osteoarthritis, fourfold increased ICTP levels have been
detected in patients with rapid progressive hip osteoarthritis
compared with patients with slowly progressive disease.35
When the carboxyterminal or the aminoterminal telo-
peptide of type I collagen (CTx or NTx) is released from
bone degraded by cathepsin K, an epitope is generated
that is different from the MMP-mediated ICTP epitope. In
osteoarthritis patients, serum and urinary CTx levels are
decreased compared with controls, which suggests decreased
bone remodeling in osteoarthritis.28,36 In RA, urinary NTx
and CTx levels are increased compared with healthy con-
trols and are sensitive to change after treatment.37-39
Although on initial examination CTx and ICTP seem to
provide identical information, closer inspection reveals that
these two markers, although based on the same principle
of detecting type I collagen telopeptides, may provide valu-
able complementary information. CTx and NTx levels are
very low in patients with pyknodysostosis, which is caused
by a deficient activity of cathepsin K, whereas ICTP levels
are elevated in this condition. It also has been shown that
in postmenopausal women, anti–bone resorption therapy by
hormone replacement reduced serum CTx levels, whereas
ICTP levels did not change.40
In cartilage, type II collagen constitutes 80% of the dry
weight of the tissue. Damage to the collagen network (colla-
gen degradation and subsequent denaturation) is one of the
first features of osteoarthritis and contributes significantly to
the decreased mechanical properties of osteoarthritic carti-
lage.41 Using monoclonal antibodies, release of cross-linked
type II collagen telopeptides (C-telopeptide, CTx-II) was
shown to reflect cartilage degradation with high tissue speci-
ficity.42 Urinary CTx-II levels were significantly increased
in RA and osteoarthritis patients compared with healthy
controls, although the ranges overlapped considerably.42-44
In a population-based study, subjects with a CTx-II level in
the highest quartile had a 4.2-fold increased risk of having
radiographic osteoarthritis of the knee and hip (compared
with subjects in the lowest quartile), and a 6-fold (knee) or
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
8.4-fold (hip) increased risk for progression of osteoarthri-
tis.45 In osteoarthritis patients, CTx-II levels correlated with
radiologic joint space narrowing and joint surface area, but
did not correlate with WOMAC indices of clinical status
in these osteoarthritis patients.28 In patients with hip osteo-
arthritis, urinary CTx-II levels greater than 346 ng/mmol
creatinine were associated with a twofold increase in radio-
graphic disease progression compared with patients whose
levels were less than 346 ng/mmol creatinine.46 Treatment
of these patients with a candidate antiosteoarthritis drug
(diacerein) seemed to modulate the CTx-II levels consis-
tent with the effects on disease progression.46,47 In a study of
patients with radiographic osteoarthritis in multiple joints,
there was a significant association between the total Radio-
graphic OA score and urinary CTx-II levels. Subsequent
multivariate analysis showed that the joint site–specific
ROA score at all joint sites except for spinal disk degenera-
tion contributed independently to this association.48
In RA patients, increased baseline CTx-II levels were
associated with progression of joint damage, which was inde-
pendent of baseline damage, treatment, and disease activity.44
In patients with active RA, the decrease in urinary CTx-II
levels that was observed 3 months after initiation of treat-
ment predicted the radiographic disease progression over 5
years, suggesting that urinary CTx-II levels may be used as
an early marker of treatment efficacy.49 CTx-II also can be
used to monitor therapy effects: In osteoporosis, patients’
CTx-II levels suggested that bisphosphonates attenuate
not only bone degradation, but also cartilage degradation.50
These initial results indicate that type II collagen–specific
markers may reflect ongoing cartilage destruction, and that
such markers eventually may be used in clinical practice.
Apart from telopeptide fragments, neoepitopes resulting
from the cleavage of type II collagen by collagenases (MMP-1,
MMP-8, and MMP-13) have been used to monitor cartilage
collagen damage.51-53 Urinary excretion of collagen frag-
ments containing the carboxyterminal neoepitope (TIINE
assay, using antibody 9A4) was increased in osteoarthritis
patients compared with age-matched healthy controls.54 In
synovial fluid, levels of a similar carboxyterminal neoepi­
tope (C2C epitope, using the Col2-3/4Clong antibody) were
significantly higher in osteoarthritis than in RA.55 In RA
patients, serum levels of these biomarkers (C2C and C1,2C,
a similar epitope present in types I and II collagen) were
associated with progression of radiographic joint damage.56
In a randomized, double-blind, placebo-controlled glucos-
amine discontinuation trial of 137 subjects with knee osteo-
arthritis, neither C2C level nor C1,2C level, or their ratio,
was affected by the treatment.57 Urinary levels of the 622-
632 peptide of type II collagen (also known as HELIX-II)
were increased in patients with primary knee osteoarthritis
(281 ng/mmol creatinine) and RA patients (409 ng/mmol
creatinine) compared with healthy controls (180 ng/mmol
creatinine).53 These data indicate that collagen-derived
markers also are promising candidates to serve as biomarkers
for cartilage degradation and disease progression for osteoar-
thritis and RA. HELIX-II and CTx-II levels independently
predicted radiographic progression in RA patients (Sharp
score)53 and patients with hip osteoarthritis,58 which suggests
that although both markers reflect collagen type II degrada-
tion, they reflect independent processes (spatially, tempo-
rally, or mechanistically) in cartilage destruction.
479
Collagen Cross-Links
Degradation of fibrillar collagen types I, II, and III results in
the quantitative excretion of the cross-links hydroxylysyl-
pyridinoline (HP), derived from bone and soft tissue, includ-
ing cartilage, and lysylpyridinoline (LP), derived from bone,
in the urine. In RA patients who received ­ combination
therapy (sulfasalazine, methotrexate, and ­ prednisolone
in the COBRA trial), time-integrated urinary HP ­ levels
correlated with the progression of radiographic joint
damage (Sharpe/van der Heide score) and bone mineral
density.59 In osteoarthritis patients, urinary HP and LP
­levels were increased compared with age-matched con-
trols.60 In the same patient population, followed for 1 year,
only the cluster of baseline bone metabolism markers
(comprising HP, LP, and serum bone sialoprotein)—out of
the 14 molecular markers measured—significantly corre-
lated with baseline clinical scores for pain, stiffness, and
disease activity.61 None of the baseline cartilage or bone
metabolism markers correlated with disease progression
after 1 year of follow-up.61 In these patients, HP that is
not derived from bone (extraosseous HP) showed a highly
significant correlation with the acute-phase response, sug­
gesting that in osteoarthritis, cartilage degradation is related
to the degree of inflammation.60
The glycosylated analogue of HP, glucosyl-galactosyl-
pyridinoline (GGHP), is present in human synovial tis-
sue and is released during its degradation in vitro. GGHP
is virtually absent in bone, whereas low levels have been
detected in muscle and liver, and intermediate levels have
been detected in cartilage. Methodologic problems, such
as the stability of HP during the alkaline hydrolysis that is
needed to liberate glycosylated molecules from tissues, have
so far prevented solid data on tissue distribution.62 Urinary
GGHP levels were increased in early RA patients versus
controls, and baseline levels correlated with disease progres-
sion, albeit weakly.44 Pending definite information on its tis-
sue distribution, urinary GGHP might prove to be a marker
for synovial tissue degradation.
Overall, collagen cross-links seem useful in measuring
bone and cartilage catabolism and contribute to under-
standing of the pathophysiology of osteoarthritis and RA. In
this respect, age-related changes in articular cartilage may
significantly affect its susceptibility to proteinase-mediated­
­degradation63,64; this underscores the use of proper ­ age-
matched control groups. Results obtained in studies with
young animals cannot be extrapolated to the adult human
situation.
Collagen Synthesis
In an attempt to repair tissue damage, collagen synthesis is
increased in osteoarthritis cartilage, leading to increased tis-
sue levels of the C-terminal propeptide of type II collagen
(PIICP, chondrocalcin).65 The rate of PIICP release is pro-
portional to the rate of collagen synthesis; the propeptide
has a half-life of approximately 18 hours.65 Synovial fluid
PIICP levels also are increased and correlate with osteo-
arthritis severity and body mass index.66 PIICP levels were
lower in serum of osteoarthritis patients than in that of
healthy controls.60 Similarly, the serum N-propeptide of col-
lagen type IIA (PIIANP, an alternative splice variant that
 480 Degroot  |  Biologic Markers
is expressed in embryonic and osteoarthritic cartilage) is
lowered in osteoarthritis serum versus controls. Preliminary
studies indicate that the balance between cartilage synthesis
(PIIANP) and cartilage degradation (urinary CTx-II) could
be used to discriminate between patients with rapid com-
pared with slow progression.44
In inflamed synovial tissue, the synthesis of type III col-
lagen is upregulated, resulting in the production of its ami-
noterminal propeptide. In osteoarthritis patients and RA
patients with knee involvement, serum PIIINP levels are
increased compared with age-matched controls.43,67,68 In RA
patients, prednisolone treatment that resulted in clinical
improvement also reduced serum PIIINP levels by 25%, and
levels remained suppressed until treatment was withdrawn.69
Thus far, collagen type III specific degradation markers have
not been described, and because type III collagen has a broad
distribution in soft tissues and blood vessels, its potential as
a specific biomarker seems limited.
PROTEOGLYCAN MARKERS
The main noncollagenous constituent of articular carti-
lage is aggrecan, a large proteoglycan composed of a core
protein to which glycosaminoglycan chains (e.g., kera-
tan sulfate and chondroitin sulfate) are attached. A vari-
ety of assays to measure aggrecan metabolism have been
described, but the available information is not always con-
sistent. Depending on the antibodies used, serum keratan
sulfate levels were reported to be either increased (antibody
5D4)70-72 or decreased (antibody AN9P1)73 in osteoarthri-
tis patients compared with controls. Additionally, previous
work has suggested that serum 5D4 reactive keratan sulfate
levels are either similar72 or higher in osteoarthritis than in
RA.55 In one study, the serum keratan sulfate levels (anti-
body AN9P1) were 30% lower in osteoarthritis patients
than in age-matched controls.60,61 For RA patients, a nega-
tive correlation between serum keratan sulfate levels and
inflammation has been found.74
The aggrecan epitope 846, which reflects the synthesis
of proteoglycans in an attempt to repair, was increased in
cartilage of osteoarthritis patients,75 and synovial fluid lev-
els correlated with other markers, such as ­cartilage oligo-
meric matrix protein (COMP), PIICP, tissue inhibitor of
metalloproteinases 1 (TIMP-1), MMP-1, and MMP-3,
and with the degree of radiologic damage.76 The epitope
846 levels in serum were lower, however, in osteoarthritis
patients than in healthy controls61,77 and RA patients.55,61
In the RA patients, elevated levels of epitope 846 could
predict a benign course of the disease.78 Taken together,
none of the aggrecan-derived markers has shown suffi-
cient power to discriminate between patients and con-
trols or to provide consistent information that can be
used in clinical studies. This effect may be partly caused
by diurnal variation of these biomarkers, which may
obscure relevant differences between study groups, espe-
cially when serum or urine sampling is not standardized.79
Similarly, increased motility of patients after initiation of
successful treatment may affect circulating proteoglycan
biomarker levels.
The identification of two members of the ADAM-TS fam-
ily of proteases (ADAM-TS4 and ADAM-TS5) as aggre­can­
ases80 supplied new tools to develop aggrecan-based markers
for cartilage destruction. Antibodies directed at aggrecanase
and MMP-generated neoepitopes in aggrecan core protein
have been produced and applied in a variety of in vitro ­studies
aimed at unraveling mechanisms of cartilage destruction in
osteoarthritis. In synovial fluid from patients with a variety
of joint diseases, MMP and aggrecanase-released aggrecan
fragments have been detected.81-83 These studies also have
shown that these two groups of proteases may have distinct
roles in articular cartilage catabolism.84
HYALURONAN
The glucuronic acid chain hyaluronan (hyaluronic acid) is
a constituent of cartilage and synovium and is synthesized
by many cell types. It functions as anchor for proteoglycans
such as aggrecan, allowing the formation of the large aggre-
gates that are responsible for the resilience of cartilage. In
the synovial membrane, hyaluronan is synthesized by syno-
vial fibroblasts and secreted into the synovial fluid to provide
lubrication of the joint and to facilitate joint movement.
Synovial fluid hyaluronan levels are decreased in osteo-
arthritis patients85 and may partly explain impaired joint
function and pain. This provides the rationale for visco-sup-
plementation therapy in osteoarthritis patients, consisting
of intra-articular injections with hyaluronan derivatives.
Elevated blood hyaluronan levels have been reported for
osteoarthritis patients and RA patients. In RA, some stud-
ies failed to show a relationship between plasma hyaluro-
nan levels and measures of disease activity,86 whereas others
showed significant correlations between serum ­hyaluronan
levels and a variety of measures of inflammation and destruc-
tion (e.g., CRP, ESR, Ritchie index, radiologic damage).87
In osteoarthritis patients, elevated serum hyaluronan levels
correlated weakly with the degree of cartilage degeneration.86
In addition, baseline hyaluronan levels could predict pro-
gression of osteoarthritis,46,88 and serum levels were shown
to increase with disease severity.89 These results suggest
that an increase in circulating systemic hyaluronan levels
could reflect synovial inflammation rather than cartilage
­destruction, prompting care in use of hyaluronan as a joint
destruction marker. Also, the observation that diet and
increased physical activity can influence its serum levels
should be considered when using hyaluronan as a biomarker
in joint diseases.79,90
CARTILAGE OLIGOMERIC MATRIX PROTEIN
Since its discovery in the early 1990s, COMP has received
much attention as a putative cartilage destruction marker.
Although its exact function is unclear, COMP has been
implicated in collagen fibrillogenesis. Increased COMP
levels have been detected in the synovial fluid of osteoar-
thritis patients.91 In addition, COMP levels are increased in
the serum of osteoarthritis patients compared with healthy
controls43,92 and are associated with progression of radio-
graphic signs of osteoarthritis.93 In osteoarthritis patients,
the serum COMP levels were even higher than in RA
patients.94 In early RA patients, increased serum COMP
levels were identified as a strong predictor of early large
joint destruction.95-97 These data generated interest in the
use of measurement of COMP levels as a selective cartilage
destruction marker.
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
The expression of COMP in joint tissues other than
cartilage, including synovium, tendons, ligaments, and
menisci, raised concerns about its tissue specificity. High
expression of COMP mRNA has been shown in murine
osteoarthritis, indicating that synovial fluid COMP levels
may reflect not only tissue degradation, but also the rate
of new synthesis.98 The concerns about the use of COMP
measurement as a specific cartilage ­degradation marker are
fueled further by a study showing that the extent of syno-
vial inflammation is one of the factors determining the
serum COMP levels.99 Other ­investigators did not observe
a relationship between markers of inflammation and serum
COMP levels (using a polyclonal antiserum ­ recognizing
all COMP forms) in RA patients.100 In this same study,
COMP levels did not have any prognostic value with
respect to progression of joint damage.100 ­ Similar to the
CTx and ICTP assays for collagen degradation, the use
of antibodies or antiserum recognizing different epitopes
within ­(fragments of) the COMP molecule might explain
the apparent inconsistent results in the literature.101
METALLOPROTEINASES
In addition to cartilage breakdown products, metallopro-
teinases (MMPs and aggrecanases) and their endogenous
inhibitors (TIMPs) that are involved in the pathologic
­degradation of joint tissues could serve as useful markers.
Data on MMP levels as a predictor for the progression of
joint erosion in early RA are rapidly accumulating. Serum
and synovial fluid MMP-3 (stromelysin) levels are increased
in RA patients compared with controls.44,102 MMP-3 levels
correlate with inflammation markers and disease ­ activity
in patients with untreated active RA103 and in early RA
patients who received nonsteroidal anti-inflammatory
drugs only.100,102 In these studies, serum MMP-3 levels were
not related to radiographic progression.100 Another study
revealed an association between serum proMMP-3 concen-
trations at disease onset and progression of joint destruc-
tion, which was independent of known risk factors, such
as the presence of the shared epitope and serum levels of
rheumatoid factor and CRP.104 In line with these observa-
tions, MMP-1 (collagenase) levels also have been shown
to indicate joint erosion independent of ­inflammation. In
early RA patients, there was a positive correlation between
the area under the curve measurements of MMP-1 serum
levels (but not the area under the curve of CRP levels)
and the number of new joint erosions after 18 months of
follow-up.102 Arthritic patients treated with anti-TNF-α
antibodies (infliximab) for 14 weeks did not show reduced
MMP-2 and MMP-9 levels (assessed by zymography, which
does not reliably detect the other MMPs) despite clear
clinical improvement.105 These data suggest that MMP
levels, although they may be correlated with parameters
of inflammation, do not reflect exactly the same pathways
as do acute-phase reactants and could provide valuable
­additional information on joint destruction in RA.
Extrapolated to osteoarthritis, in which secondary
inflammation is usually mild, these data suggest that MMP
levels may provide valuable predictive markers. In a cross-
sectional study in osteoarthritis patients, MMP-3 serum
­levels were similar, however, to levels in healthy controls.106
In a subset (120 patients) of 431 patients participating in a
481
randomized, placebo-controlled trial evaluating the effects
of ­ doxycycline treatment on unilateral knee osteoarthri-
tis, baseline plasma MMP-3 levels predicted joint space
­narrowing.107 In another study, the serum levels of TIMP-1
did not differ significantly between patients with ­unilateral
or bilateral hip osteoarthritis and healthy controls.108 Within
the osteoarthritis group, patients with rapid disease progres-
sion (joint space ­narrowing >0.6 mm/yr; 1-year follow-up)
had significantly lower serum TIMP-1 levels than patients
with slow progression (joint space narrowing <0.6 mm/yr).108
Apart from these, comprehensive studies for ­ predictive
MMP markers in osteoarthritis are not yet ­ available, and
information about the use of MMP levels in osteoarthritis
is still incomplete.
In recent years, more data on the role of aggrecanase in
degradation of the major proteoglycan of cartilage (aggre-
can) became available. Its pivotal role in tissue destruction
is now widely accepted.80,109,110 Because of the difficulties in
measuring aggrecanase activity in biologic samples, how-
ever, their value as a biomarker to monitor joint destruction
is not yet fully understood. It is hoped that the more recent
development of several aggrecanase assays111-113 will facili-
tate its use as a biomarker.
BIOLOGIC MARKERS IN SYNOVIAL TISSUE
Because many inflammatory arthropathies, including RA,
­primarily involve the synovial tissue, there has been
increased interest in investigations of the pathologic changes
in synovial biopsy specimens. This development has been
stimulated further by technical advances, such as the advent
of new methods to obtain synovial tissue ­ specimens from
actively inflamed joints and clinically quiescent joints under
local anesthesia, and because of the development of immu-
nohistologic methods, in situ hybridization, ­ quantitative
polymerase chain reaction, and microarray technology.
Previous work has shown the relationship between the
features of rheumatoid synovial tissue on the one hand
and arthritis activity114 and joint destruction115 on the
other (see also Chapter 48). The importance of evalua-
tion of synovial tissue samples has been underscored by the
­observation that clinical signs of arthritis activity are asso-
ciated with histologic signs of synovitis after treatment of
RA patients with the monoclonal antibody alemtuzumab
(Campath-1H), despite profound depletion of circulating
lymphocytes.116 Similarly, rituximab treatment leads to a
rapid and significant decrease in synovial B cell numbers in
only a subset of RA patients, whereas circulating B cells are
completely depleted in nearly all patients (Fig. 32-3).117
Several methodologic questions needed to be answered
before serial synovial biopsy could be used to screen for
potentially relevant effects after antirheumatic treatment.118
It has been shown in cross-sectional studies that biopsy
samples can be acquired by blind needle technique and
by miniarthroscopy.119 There are limitations and disadvan-
tages, however, of the use of serial blind needle biopsy in
the evaluation of treatment. It is usually restricted to larger
joints, such as the knee joint; the operator is not able to
select the tissue visually, causing potential sampling error;
and it is not always possible to obtain adequate tissue sam-
ples. This is especially true when clinically quiescent joints
are investigated (e.g., after successful therapy). Comparison
 482 Degroot  |  Biologic Markers
A C
Figure 32-3  Variable tissue response to treatment
with the anti-CD20 antibody rituximab in patients with
rheumatoid arthritis. A-D, Arthroscopic samples were
obtained before (A and B) and 4 weeks after (C and D)
initiation of treatment. Rheumatoid synovial tissue from
paired biopsy samples stained for CD22+ B cells. Single
staining, peroxidase technique, counterstaining with B D
Mayer’s Hämalaunlösung. Original magnification 200×.
of the features of synovial inflammation in biopsy samples
from inflamed knee joints and paired inflamed small joints
of RA patients revealed that inflammation in one inflamed
joint is generally representative of the inflammation in
other inflamed joints.120 It is possible to use serial samples
from the same joint, selecting either large or small joints, for
the evaluation of antirheumatic therapy. Sampling error can
be reduced by selecting at least six biopsy specimens from
multiple regions, resulting in variance of less than 10%.17,121
When the biopsy samples are taken from an actively
inflamed joint, there is on average no clear-cut difference
in the features of synovial inflammation or the expression of
mediators of inflammation and destruction at the pannus-
cartilage ­junction compared with other regions away from
the pannus-cartilage junction.122-124
An extensive quality control system is required to allow
reliable analysis by immunohistochemistry, tissue enzyme-
linked immunosorbent assay, quantitative polymerase chain
reaction, or microarray analysis. Finally, sophisticated com-
puter-assisted image analysis systems allow reliable and
efficient evaluation of the synovial cell infiltrate and the
expression of adhesion molecules, cytokines, and MMPs in
innovative clinical trials.125
Using this approach, successful treatment with disease-
modifying antirheumatic drugs, such as gold,126 methotrex-
ate17,127,128 and leflunomide,128 was shown to be associated
with decreased mononuclear cell infiltration. Similarly, suc-
cessful treatment of RA patients with infliximab,18,129,130
etanercept,131 and anakinra132 results in reduced synovial
inflammation. The number of macrophages in the synovium
was significantly decreased 48 hours after initiation of inflix-
imab treatment.133 Similarly, high-dose intravenous meth-
ylprednisolone reduced expression of TNF-α in synovial
biopsy samples 24 hours after treatment, a result that cor-
related with a clinical response to, and subsequent relapse
after, methylprednisolone therapy.134
More recently, a randomized trial was designed to
address formally the question of which feature in RA
synovial tissue samples could be used as a biomarker for
clinical ­ efficacy in relatively small studies of short dura-
tion.135 Patients received either prednisolone according to
the COBRA ­ regimen or placebo for 2 weeks. This study
identified ­ sublining ­ macrophages as the best biomarker
associated with the clinical response to corticosteroids.
Next, the utility of macrophages in the synovial sublining
as a candidate biomarker was tested across discrete inter-
ventions and kinetics.125 A strong correlation between the
mean change in disease activity score (Δ DAS28) and the
mean change in the number of sublining macrophages was
observed. When patients from all actively treated studies
were grouped (n = 70), the standardized response mean, a
measure of sensitivity to change, was 1.16 for the change in
DAS28 and 0.83 for the change in sublining macrophages,
indicating good sensitivity to change for both variables.
For the patients from the placebo groups, the standard-
ized response mean was −0.23 (for DAS28) and 0.30 (for
macrophages), consistent with the notion that the bio-
marker is less susceptible to placebo effects or expectation
bias than clinical evaluation, which includes subjective
measures of disease activity.125 In addition to its role as a
marker of response to effective treatment, the change in
numbers of sublining macrophages could potentially help
to distinguish effective from ineffective treatment. Taken
together, these studies suggest that analyses of serial biopsy
samples can be used as a screening method to test new drug
candidates requiring relatively small numbers of subjects.
The absence of changes after treatment would suggest that
the therapy is probably not effective. The demonstration of
biologic changes at the site of inflammation could provide
the rationale for larger, placebo-controlled trials, how-
ever. Most of the biopsy studies have been performed in
RA patients, but more recent work suggests that the same
approach can be used for the evaluation of novel therapies
in patients with other rheumatologic disease, such as spon-
dyloarthritis.136-138
As an alternative to immunohistologic and in situ hybrid-
ization methods, quantitative polymerase chain ­reaction on
small synovial biopsy specimens can be employed to ­evaluate
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
drug effects in clinical trials.118,121 Using this approach,
­prednisolone was shown to reduce expression of interleukin-8
and MMP-1 in synovial biopsy specimens of RA patients
after 2 weeks of treatment.138a Because the biopsy specimens
contain various cell types, it is important to realize that a
change in gene expression level also may reflect a change in
cellular composition of the biopsy, and not a change in the
expression level within a certain cell type.
BIOMARKER PANELS
Almost none of the markers for osteoarthritis and RA that
are currently used can distinguish successfully between
patients and controls on an individual basis, although aver-
age marker levels differ among groups. Principal component
analysis of 14 biochemical markers revealed that the mark-
ers segregate into five clusters: inflammation (interleukin-6,
CRP, TNF receptor I, TNF receptor II, and eosinophil cat-
ionic protein), bone (HP, LP, and BSP), cartilage synthesis
(CPII, epitope 846, and hyaluronan), cartilage degradation
(COMP and keratan sulfate), and transforming growth fac-
tor-β1 (which is independent of all other markers).60 The
combination of three of the markers (TNF receptor II,
COMP, and epitope 846) from the independent clusters
inflammation, cartilage degradation, and cartilage synthesis
could discriminate correctly between osteoarthritis patients
and controls in approximately 90% of the cases.60 In a study
of 376 patients with hip osteoarthritis, a similar approach
resulted in five different clusters with similar makeup: a
cartilage and bone cluster (PINP, CTx-I, and CTx-II), a
putative synovitis cluster (COMP, PIIINP, and HA), a puta-
tive systemic inflammation cluster (CRP and YKL-40), and
MMP-1 and MMP-3 as two independent factors.67 Similarly,
in RA, a combination of seven clinical scores and molecu-
lar markers provided a clinical prediction model that could
discriminate, at the first visit, between three forms of arthri-
tis—self-limiting arthritis, persistent nonerosive arthritis,
and persistent erosive arthritis.139 Different clusters of bio-
markers may relate to osteoarthritis at different joint sites,
suggesting that pathophysiologic processes may be different
between those sites.48 These studies support the notion that
panels of biomarkers may provide a valuable additional tool
in the monitoring of osteoarthritis patients and RA patients
and in helping to understand disease processes.
The progressive destruction of the articular cartilage is
considered a major determinant of disability in patients with
joint disease. A report showed that the balance between
cartilage synthesis and degradation discriminates between
osteoarthritis patients with rapid versus slow progression, as
assessed by the change in joint space width and arthroscopi-
cally scored chondropathy.44 These studies support the
hypo­thesis that the “-omics” approaches, combining even
more markers than the few used previously, not only may
be ­successful in the identification of disease-specific finger-
prints, but also may provide tools to monitor tissue-specific
degradation.
“-OMICS”-BASED BIOMARKERS
The current technical progress in genomics, ­transcriptomics,
proteomics, and metabolomics, in combination with advan­
ced bioinformatics,140 makes it possible to analyze numerous
483
markers in one sample, which could be urine, serum, syno-
vial fluid, or (synovial) tissue. The resulting profile com-
bines the levels of a variety of markers to create a “disease
fingerprint” that could serve as a powerful marker by itself.
In addition to specific markers for early diagnosis, markers
are needed that specifically reflect cartilage degradation.
These technologies can arbitrarily be separated into three
primary levels: genomics, which deals with variations in
DNA composition (e.g., single nucleotide polymorphisms)
and expression levels (differences in mRNA levels, also
known as transcriptomics); proteomics, which analyzes the
proteome, or total of proteins in a sample; and metabolo-
mics, which focuses on metabolites. Variations on these
themes include technologies such as lipidomics (profiling
the lipids and free fatty acids in a cell or biologic fluid),
degradomics (the study of protein degradation products),
and toponomics (the study of the localization of molecules
within a cell).
GENOMICS
Of all the “-omics” technologies, genomics was the first to
evolve in the footsteps of the human genome project, and
various aspects of genomics approaches to study joint dis-
eases have been extensively reviewed.141-143 Comparisons
in gene expression levels between controls, osteoarthri-
tis, and RA have been made for a variety of tissues, such
as ­ chondrocytes, blood-derived cells, and synovial tissue.
Within a group of RA patients, cDNA microarray analy-
sis with a focus on immune-related genes could separate
classes of patients with potentially different pathogenicity
based on the expression of genes involved in the adaptive
immune response versus genes involved in tissue remodel-
ing.144 In osteoarthritis, chondrocytes from cartilage have
been shown to upregulate the transcription of a variety of
inflammatory genes.24 In another study, 3543 genes were
differentially expressed by blood cells of patients with mild
knee osteoarthritis compared with healthy controls.145,146
Logistic ­regression indicated that nine of these genes were
discriminatory between subjects with mild osteoarthritis and
controls, with a sensitivity of 86% and specificity of 83% in
a training set of 78 samples. The optimal biomarker combi-
nations were evaluated using a blind test set (67 subjects),
which showed 72% sensitivity and 66% specificity for the
diagnosis of osteoarthritis.145 These data underscore that the
combination of biomarkers (in this case the expression of
nine genes) may be useful in differential diagnosis.
Also in other rheumatic diseases, expression profiling has
contributed to the understanding of disease pathways. In
patients with systemic lupus erythematosus, several ­studies
have shown that interferon-regulated genes are highly upreg-
ulated in peripheral blood cells and in kidney ­glomeruli.147
One of the ultimate uses of the identification of differ-
entially expressed genes in a disease is illustrated by the
antitumor drug trastuzumab (a recombinant monoclonal
antibody against the human epidermal growth factor recep-
tor 2 [HER2] protein), which would not have reached the
marker if not for the accompanying prognostic test.148 Nor-
mal cells express low levels of HER2 protein on their plasma
membrane. In approximately 25% of breast cancer patients,
HER2 is overexpressed, changing the growth control of
these cells. The prognostic test measures the expression
 484 Degroot  |  Biologic Markers
l­evels of HER2, assisting in the selection of patients who
would benefit from trastuzumab treatment.
PROTEOMICS
Similar to the genomics revolution, which was partly
driven by technology that allowed the production of gene-
chip and high-throughput DNA sequencing methods, the
proteomics field was boosted by the development of bet-
ter two-dimensional electrophoresis technologies and the
rapid improvements in the area of mass spectroscopy, both
of which facilitated the reproducible analysis of a panel of
proteins within a sample. Two-dimensional gel electropho-
resis has been used to identify proteins secreted into the
culture medium of normal and osteoarthritic cartilage sam-
ples,149 but also to analyze the protein composition of the
mitochondria of healthy human chondrocytes.150,151 Using
surface-enhanced laser desorption/ionization time-of-flight
mass spectroscopy, 103 serum samples of RA patients,
osteoarthritis patients, patients with non-RA inflamma-
tory conditions (psoriatic arthritis, asthma, Crohn’s dis-
ease), and controls were analyzed. This approach yielded
several signals in the mass spectrum that contributed to
the separation between RA patients and controls.152 A
different approach was taken by Xiang and coworkers,153
who first separated human chondrocyte proteins by two-
dimensional electrophoresis, then blotted the proteins to
a membrane, and finally incubated these membranes with
serum of osteoarthritis patients, RA patients, and ­controls
to identify which chondrocyte-derived autoantigens are
present in these patients. This approach yielded triose­
phosphate isomerase as a potential osteoarthritis-specific
biomarker. Yet another approach focuses on the panels
of autoantibodies present in patients with various auto-
immune diseases to act as biomarkers.154 Using an array
of 30 antigens known to be expressed in the glomeruli,
the clusters of autoantibodies that occur in the serum of
lupus patients were studied and shown to be related to the
patients’ disease activity.155,156 All biomarkers that are iden-
tified by the above-described examples studies naturally
need further validation to establish their true usefulness
for diagnostic, prognostic, or disease-monitoring applica-
tion in patients with joint disease.
METABOLOMICS
Biologic fluids, such as urine, blood, and synovial fluid,
­contain numerous metabolites that may provide valuable
­information on the metabolism of an organism and about its
health ­status. Metabolic profiling, also referred to as metabolo-
mics, ­metabonomics, or related terms, is defined as the quan-
titative and qualitative analysis of the whole complement of
small molecules in a sample (e.g., cell, tissue, body fluids).157
The technology has emerged from approaches used to pro-
file body fluids that were developed many decades ago for the
study of inborn errors of metabolism and the effects of nutri-
tion. A wide array of analytic methods is used to analyze the
various metabolites. Gas chromatography–mass spectroscopy
and nuclear magnetic resonance (NMR) can be employed
for a global insight into a broad range of metabolites, such
as (phosphorylated) sugars, amino acids, fatty acids, nucleo-
bases and nucleosides, amines, higher alcohols, and bile acids.
­Liquid chromatography–mass spectroscopy methods can be
used not only to zoom in on free fatty acids and lipids, but
also to ­analyze amino acids, peptides, sugars, and aminosug-
ars and hormones and steroids. All of these analytic measures
need to be combined with data preprocessing to obtain clean
data. This is necessary to analyze these very large metabolite
profiles reliably and to relate relevant changes in metabolites
to biologic processes, using multivariate statistics.
The application of metabolomics in the area of joint
diseases is relatively recent. 1H-NMR (500 MHz) has
been used to compare the effects of unilateral knee joint
denervation on the biochemical profiles of synovial fluid
in a ­bilateral canine anterior cruciate ligament transection
model of osteoarthritis. Increases in glycerol, hydroxybuty­
rate, glutamine/glutamate, creatinine/creatine, acetate, and
N-acetyl-glycoprotein concentrations were observed in sy-
novial fluids from denervated osteoarthritis knees compared
with normally innervated osteoarthritis knees.158 These
metabolite profiles of denervated osteoarthritis knees sup-
port the idea of neurogenic acceleration of osteoarthritis
in that the observed differences in metabolite concentra-
tions found in the denervated knee fluids seem to corre-
late with metabolic changes resulting from aggravation of
the osteoarthritis process caused by joint denervation.159
Using 1H-NMR (300 MHz) and multivariate data analy-
sis, a metabolite profile was detected, which was strongly
­associated with osteoarthritis in 10- and 12-month-old
Hartley guinea pigs that ­spontaneously develop osteoarthri-
tis.160 1H-NMR also revealed a urinary metabolite profile
that could distinguish osteoarthritis patients from healthy
individuals.161 The human urine profile largely resembles the
Dunkin Hartley guinea pig profile; the presence of hydroxy-
butyrate, pyruvate, creatine/creatinine, and glycerol in the
metabolite profile could point to an enhanced use of fat and
altered energy use, consistent with the canine synovial fluid
­composition.158,159,161
SYSTEMS BIOLOGY
Although each is already tremendously powerful on its own,
the combination of genomics (transcriptomics), proteomics,
and metabolomics theoretically could deliver a full picture
of a living system (cell, organ, or organism). Such a sys-
tems biology approach162 would provide insight into which
disturbances of a healthy system cause it to shift toward a
pathologic phenotype, which mechanisms are employed by
the organism to maintain its equilibrium, and which fac-
tors indicate a point of no return, followed by failure of the
intrinsic balancing mechanisms and disease initiation. As
such, a systems biology approach would help to identify the
most promising molecules that describe this shift and can
act as biomarkers, while concomitantly key molecules can
be detected that, when normalized, could rebalance the sys-
tem, acting as therapeutic targets. The first steps in this area
are being made for RA,163 but steps for osteoarthritis projects
also have been initiated.
BIOMARKER VALIDATION
Following or parallel to the crucial investigations to identify
relevant biomarkers for disease, biologic validation studies
need to be performed: Does the biomarker reflect the disease
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
process, and how does it change with endogenous or drug-
induced changes in pathophysiologic processes? In addition,
the actual application of such biomarkers in preclinical or
clinical studies requires an in-depth analytic validation.
The obvious reason for this is that to draw conclusions
based on biomarker data, it is essential to be able to trust
that a measured value is reliable and reproducible. Some
of the essential steps in biomarker validation are described
next. Details on validation procedures and requirements
can be found elsewhere (http://www.fda.gov/cder/guidance/
4252fnl.htm).164 The fundamental parameters to show that
a given biomarker can be quantitatively measured in a given
biologic matrix (e.g., serum, urine, synovial fluid, saliva)
include the following:
1. Accuracy—how close is the mean measured concen-
tration of at least five replicates to the true value of
the analyte.
2. Precision—what is the variation between individual
measurements of one sample. Typically, at each test
concentration, the precision should not exceed the
15% of the coefficient of variation.
3. Selectivity or specificity—how well does the analytic
method distinguish between the analyte of interest
and other components of the samples.
4. Sensitivity—what is the smallest amount (or the larg-
est amount) of analyte that can be reliably detected.
5. Reproducibility—how well can the measurements be
repeated on a different day, by different operators, or
by using different equipment and still result in the
same measured values.
6. Stability—how stable is the analyte of interest in a
certain matrix, tube, or storage condition. This also
includes studies of the stability of the analyte on
repeated freeze-thaw cycles and short-term and long-
term storage at various temperatures.
Other factors that are important in validating biomarker
assays are the availability of references or calibrators that are
used as external standards to quantify the results. Also the
development of standard operating procedures and docu-
mentation is essential for optimal assay performance.
CONCLUSION
Over the years, many reports have been published that
employ molecular markers in body fluids to assess inflamma-
tion and tissue destruction in joint diseases. Starting from
single markers with limited tissue or disease specificity, pan-
els of biomarkers are increasingly used, and these become
more and more tissue specific. The novel markers include
collagen-based markers (telopeptides, neoepitopes, and cross-
links), COMP, and MMPs, which are often included in clin-
ical trials. Apart from well-known markers, such as CRP,
ESR, rheumatoid factor, anticitrullinated protein/peptide
antibodies, and a few other autoantibodies, none of the
markers has made it to the clinic for routine evaluation of
patient disease status. The requirements for such a clini-
cally useful marker are high because it should be superior to
existing markers in being able to predict disease progression
or monitor therapy efficacy in individual patients. In this
respect, biomarkers for osteoarthritis provide the greatest
challenge: Joint destruction often proceeds without signs
of inflammation. Validation of osteoarthritis biomarkers is
485
hampered by the absence of a generally accepted, effective
treatment for the disease. In early RA patients, suppression
of inflammation often, although not always, coincides with
decreased joint destruction, and monitoring inflammation
may fulfill its role as surrogate destruction marker. In later
stages of the disease, when inflammation and destruction
seem more uncoupled,165 specific destruction markers also
are needed for RA patients. In general, the combination of
multiple markers holds most promise to meet these needs to
increase disease specificity or tissue specificity, or both, and
to reduce the extensive overlap in marker levels that exists
between patients and controls.
Analysis of molecular markers in synovial tissue is increas-
ingly used, especially in clinical trials on targeted therapies.
Tissue specificity is not a problem, and examination of serial
biopsy samples may be used to monitor the response in indi-
vidual patients and screen for interesting biologic effects at
the site of inflammation. This approach is generally well tol-
erated by patients, but it requires a more demanding setup.
It can be anticipated that future development will include
the use of more extensive markers of joint degradation—in
addition to the available markers of inflammation—and the
use of panels of biomarkers in synovial tissue samples.
As illustrated by studies described in this chapter, many
investigators measure different sets of biomarkers and may
use different definitions of disease (or progression or both).
In addition, common terminology to describe a biomarker
is lacking, and investigators may have a historical bias in
favor of (or against) certain biomarkers. In combination,
these issues may slow down the urgently needed progress
in the development of clinically applicable biomarkers for
joint diseases. To solve this, further collaboration between
researchers of various disciplines, and the execution of
large, unbiased studies incorporating a wide panel of avail-
able (or newly developed) biomarkers and complementary
methods, including imaging and patient assessments, are
needed.166
REFERENCES
  1. Lassere MN, Johnson KR, Boers M, et al: Definitions and validation
criteria for biomarkers and surrogate endpoints: Development and
testing of a quantitative hierarchical levels of evidence schema. J
Rheumatol 34:607-615, 2007.
   2. Bauer DC, Hunter DJ, Abramson SB, et al: Classification of osteo-
arthritis biomarkers: A proposed approach. Osteoarthritis Carti-
lage 14:723-727, 2006.
  3. Biswal S, Hastie T, Andriacchi TP, et al: Risk factors for progressive
cartilage loss in the knee. Arthritis Rheum 46:2884-2892, 2002.
  4. Kaplan W, Laing R: Priority Medicines for Europe and the World.
WHO/EDM/PAR/2004.7. Geneva, World Health Organization, 2004.
  5. Peach CA, Carr AJ, Loughlin J: Recent advances in the genetic
investigation of osteoarthritis. Trends Mol Med 11:186-191, 2005.
  6. Brandi ML, Gennari L, Cerinic MM, et al: Genetic markers of osteo-
articular disorders: Facts and hopes. Arthritis Res 3:270-280, 2001.
  7. Williamson AA, McColl GJ: Early rheumatoid arthritis: Can we pre-
dict its outcome? Intern Med J 31:168-180, 2001.
  8. Loughlin J: Polymorphism in signal transduction is a major route
through which osteoarthritis susceptibility is acting. Curr Opin
Rheumatol 17:629-633, 2005.
  9. Otterness IG: The value of C-reactive protein measurement in rheu-
matoid arthritis. Semin Arthritis Rheum 24:91-104, 1994.
10. Maini R, St Clair EW, Breedveld F, et al: Infliximab (chimeric anti-
tumour necrosis factor alpha monoclonal antibody) versus placebo
in rheumatoid arthritis patients receiving concomitant methotrex-
ate: A randomised phase III trial. ATTRACT Study Group. Lancet
354:1932-1939, 1999.
 486 Degroot  |  Biologic Markers
11. Scott DL: Prognostic factors in early rheumatoid arthritis. Rheuma-
tology (Oxf) 39(Suppl 1):24-29, 2000.
12. Avouac J, Gossec L, Dougados M: Diagnostic and predictive value of
anti-cyclic citrullinated protein antibodies in rheumatoid arthritis: A
systematic literature review. Ann Rheum Dis 65:845-851, 2006.
13. van Venrooij WJ, Zendman AJ, Pruijn GJ: Autoantibodies to citrul-
linated antigens in (early) rheumatoid arthritis. Autoimmun Rev 6:
37-41, 2006.
14. Lipsky PE, van der Heijde DM, St Clair EW, et al: Infliximab and
methotrexate in the treatment of rheumatoid arthritis. Anti-Tumor
Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant
Therapy Study Group. N Engl J Med 343:1594-1602, 2000.
15. Torikai E, Kageyama Y, Takahashi M, et al: The effect of infliximab
on bone metabolism markers in patients with rheumatoid arthritis.
Rheumatology (Oxf) 45:761-764, 2006.
16. Macias I, Garcia-Perez S, Ruiz-Tudela M, et al: Modification of pro-
and antiinflammatory cytokines and vascular-related molecules by
tumor necrosis factor-α blockade in patients with rheumatoid arthri-
tis. J Rheumatol 32:2102-2108, 2005.
17. Dolhain RJ, Tak PP, Dijkmans BA, et al: Methotrexate reduces
inflammatory cell numbers, expression of monokines and of adhesion
molecules in synovial tissue of patients with rheumatoid arthritis. Br
J Rheumatol 37:502-508, 1998.
18. Ulfgren AK, Andersson U, Engstrom M, et al: Systemic anti-tumor
necrosis factor alpha therapy in rheumatoid arthritis down-regulates
synovial tumor necrosis factor alpha synthesis. Arthritis Rheum
43:2391-2396, 2000.
19. Sowers M, Jannausch M, Stein E, et al: C-reactive protein as a
­biomarker of emergent osteoarthritis. Osteoarthritis Cartilage
10:595-601, 2002.
20. Spector TD, Hart DJ, Nandra D, et al: Low-level increases in serum
C-reactive protein are present in early osteoarthritis of the knee and
predict progressive disease. Arthritis Rheum 40:723-727, 1997.
21. Cohen MP, Wu VY, Cohen JA: Glycated albumin stimulates fibro-
nectin and collagen IV production by glomerular endothelial cells
under normoglycemic conditions. Biochem Biophys Res Commun
239:91-94, 1997.
22. Ersoy Y, Ozerol E, Baysal O, et al: Serum nitrate and nitrite levels in
patients with rheumatoid arthritis, ankylosing spondylitis, and osteo-
arthritis. Ann Rheum Dis 61:76-78, 2002.
23. Penninx BW, Abbas H, Ambrosius W, et al: Inflammatory markers
and physical function among older adults with knee osteoarthritis.
J Rheumatol 31:2027-2031, 2004.
24. Attur MG, Dave M, Akamatsu M, et al: Osteoarthritis or osteoar-
throsis: The definition of inflammation becomes a semantic issue
in the genomic era of molecular medicine. Osteoarthritis Cartilage
10:1-4, 2002.
25. Smith MD, Triantafillou S, Parker A, et al: Synovial membrane
inflammation and cytokine production in patients with early osteoar-
thritis. J Rheumatol 24:365-371, 1997.
26. Saxne T, Heinegard D: Matrix proteins: Potentials as body fluid
markers of changes in the metabolism of cartilage and bone in arthri-
tis. J Rheumatol Suppl 43:71-74, 1995.
 27. Garnero P, Rousseau JC, Delmas PD: Molecular basis and clinical
use of biochemical markers of bone, cartilage, and synovium in
joint diseases. Arthritis Rheum 43:953-968, 2000.
28. Garnero P, Piperno M, Gineyts E, et al: Cross sectional evaluation of
biochemical markers of bone, cartilage, and synovial tissue metabo-
lism in patients with knee osteoarthritis: Relations with disease
­activity and joint damage. Ann Rheum Dis 60:619-626, 2001.
29. Kraus VB, Huebner JL, Fink C, et al: Urea as a passive transport
marker for arthritis biomarker studies. Arthritis Rheum 46:420-427,
2002.
30. Myers SL, Brandt KD, Eilam O: Even low-grade synovitis significantly
accelerates the clearance of protein from the canine knee: Implica-
tions for measurement of synovial fluid “markers” of osteoarthritis.
Arthritis Rheum 38:1085-1091, 1995.
31. Elsaid KA, Chichester CO: Review: Collagen markers in early
arthritic diseases. Clin Chim Acta 365(1-2):68-77, 2006.
 32. Szulc P, Delmas PD: Biochemical markers of bone turnover in
men. Calcif Tissue Int 69:229-234, 2001.
33. Sassi ML, Eriksen H, Risteli L, et al: Immunochemical characteriza-
tion of assay for carboxyterminal telopeptide of human type I ­collagen:
Loss of antigenicity by treatment with cathepsin K. Bone 26:367-373,
2000.
34. Jensen T, Hansen M, Madsen JC, et al: Serum levels of parathyroid
hormone and markers of bone metabolism in patients with rheu-
matoid arthritis: Relationship to disease activity and glucocorticoid
treatment. Scand J Clin Lab Invest 61:491-501, 2001.
35. Berger CE, Kroner A, Stiegler H, et al: Elevated levels of serum type
I collagen C-telopeptide in patients with rapidly destructive osteoar-
thritis of the hip. Int Orthop 29:1-5, 2005.
36. Takahashi M, Suzuki M, Naitou K, et al: Comparison of free and
­peptide-bound pyridinoline cross-links excretion in rheumatoid
arthritis and osteoarthritis. Rheumatology (Oxf) 38:133-138,
1999.
37. St Clair EW, Moak SA, Wilkinson WE, et al: A cross sectional
analysis of 5 different markers of collagen degradation in rheumatoid
arthritis. J Rheumatol 25:1472-1479, 1998.
38. al-Awadhi AM, Olusi SO, al-Zaid NS, et al: Urine levels of type 1
collagen cross-linked N-telopeptides and deoxypyridinoline correlate
with disease activity in rheumatoid arthritis. Clin Exp Rheumatol
16:569-572, 1998.
39. Seriolo B, Ferretti V, Sulli A, et al: Serum osteocalcin levels in
premenopausal rheumatoid arthritis patients. Ann N Y Acad Sci
966:502-507, 2002.
40. Rosenquist C, Fledelius C, Christgau S, et al: Serum CrossLaps One
Step ELISA: First application of monoclonal antibodies for measure-
ment in serum of bone-related degradation products from C-terminal
telopeptides of type I collagen. Clin Chem 44:2281-2289, 1998.
41. Bank RA, Soudry M, Maroudas A, et al: The increased swelling and
instantaneous deformation of osteoarthritic cartilage is highly cor-
related with collagen degradation. Arthritis Rheum 43:2202-2210,
2000.
42. Christgau S, Garnero P, Fledelius C, et al: Collagen type II
C-telopeptide fragments as an index of cartilage degradation. Bone
29:209-215, 2001.
43. Garnero P, Christgau S, Delmas PD: The bisphosphonate zoledronate
decreases type II collagen breakdown in patients with Paget’s disease
of bone. Bone 28:461-464, 2001.
44. Garnero P, Gineyts E, Christgau S, et al: Association of baseline
levels of urinary glucosyl-galactosyl-pyridinoline and type II collagen
C-telopeptide with progression of joint destruction in patients with
early rheumatoid arthritis. Arthritis Rheum 46:21-30, 2002.
45. Reijman M, Hazes JM, Bierma-Zeinstra SM, et al: A new marker for
osteoarthritis: Cross-sectional and longitudinal approach. Arthritis
Rheum 50:2471-2478, 2004.
46. Mazieres B, Garnero P, Gueguen A, et al: Molecular markers of car-
tilage breakdown and synovitis at baseline as predictors of structural
progression of hip osteoarthritis. The ECHODIAH Cohort. Ann
Rheum Dis 65:354-359, 2006.
47. Dougados M: Disease controlling antirheumatic therapy in spondylo-
arthropathy. J Rheumatol Suppl 62:16-20, 2001.
48. Meulenbelt I, Kloppenburg M, Kroon HM, et al: Urinary ­ CTX-
II levels are associated with radiographic subtypes of osteoarthri-
tis in hip, knee, hand, and facet joints in subject with familial
­osteoarthritis at multiple sites: The GARP study. Ann Rheum Dis
65:360-365, 2006.
49. Landewe R, Geusens P, Boers M, et al: Markers for type II collagen
breakdown predict the effect of disease-modifying treatment on long-
term radiographic progression in patients with rheumatoid arthritis.
Arthritis Rheum 50:1390-1399, 2004.
50. Lehmann HJ, Mouritzen U, Christgau S, et al: Effect of bisphosphonates
on cartilage turnover assessed with a newly developed assay for collagen
type II degradation products. Ann Rheum Dis 61:530-533, 2002.
51. Otterness IG, Downs JT, Lane C, et al: Detection of collagenase-
induced damage of collagen by 9A4, a monoclonal C-terminal neo-
epitope antibody. Matrix Biol 18:331-341, 1999.
52. Billinghurst RC, Wu W, Ionescu M, et al: Comparison of the
­degradation of type II collagen and proteoglycan in nasal and ­articular
cartilages induced by interleukin-1 and the selective ­ inhibition
of type II collagen cleavage by collagenase. Arthritis Rheum 43:
664-672, 2000.
53. Charni N, Juillet F, Garnero P: Urinary type II collagen helical pep-
tide (HELIX-II) as a new biochemical marker of cartilage degrada-
tion in patients with osteoarthritis and rheumatoid arthritis. Arthritis
Rheum 52:1081-1090, 2005.
54. Downs JT, Lane CL, Nestor NB, et al: Analysis of collagenase-
­cleavage of type II collagen using a neoepitope ELISA. J Immunol
Methods 247(1-2):25-34, 2001.
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
55. Ishiguro N, Ito T, Oguchi T, et al: Relationships of matrix metal-
loproteinases and their inhibitors to cartilage proteoglycan and col-
lagen turnover and inflammation as revealed by analyses of synovial
fluids from patients with rheumatoid arthritis. Arthritis Rheum
44:2503-2511, 2001.
56. Verstappen SM, Poole AR, Ionescu M, et al: Radiographic joint dam-
age in rheumatoid arthritis is associated with differences in cartilage
turnover and can be predicted by serum biomarkers: An evaluation
from 1 to 4 years after diagnosis. Arthritis Res Ther 8:R31, 2006.
57. Cibere J, Thorne A, Kopec JA, et al: Glucosamine sulfate and ­cartilage
type II collagen degradation in patients with knee ­ osteoarthritis:
Randomized discontinuation trial results employing biomarkers.
J Rheumatol 32:896-902, 2005.
58. Garnero P: Use of biochemical markers to study and follow patients
with osteoarthritis. Curr Rheumatol Rep 8:37-44, 2006.
59. Verhoeven AC, Boers M, Tekoppele JM, et al: Bone turnover, joint
damage and bone mineral density in early rheumatoid arthritis treated
with combination therapy including high-dose prednisolone. Rheu-
matology (Oxf) 40:1231–1237, 2001.
  60. Otterness IG, Swindell AC, Zimmerer RO, et al: An analysis of
14 molecular markers for monitoring osteoarthritis: Segregation of
the markers into clusters and distinguishing osteoarthritis at base-
line. Osteoarthritis Cartilage 8:180-185, 2000.
61. Otterness IG, Weiner E, Swindell AC, et al: An analysis of 14 molec-
ular markers for monitoring osteoarthritis: Relationship of the mark-
ers to clinical end-points. Osteoarthritis Cartilage 9:224-231, 2001.
62. Gineyts E, Garnero P, Delmas PD: Urinary excretion of glucosyl-
galactosyl pyridinoline: A specific biochemical marker of synovium
degradation. Rheumatology (Oxf) 40:315-323, 2001.
63. DeGroot J, Verzijl N, Jacobs KM, et al: Accumulation of advanced
glycation endproducts reduces chondrocyte-mediated extracellular
matrix turnover in human articular cartilage. Osteoarthritis Cartilage
9:720-726, 2001.
64. DeGroot J, Verzijl N, Wenting-van Wijk MJG, et al: Age-related
decrease in susceptibility of human articular cartilage to matrix
metalloproteinase-mediated degradation: The role of advanced gly-
cation end products. Arthritis Rheum 44:2562-2571, 2001.
65. Nelson F, Dahlberg L, Laverty S, et al: Evidence for altered synthe-
sis of type II collagen in patients with osteoarthritis. J Clin Invest
102:2115-2125, 1998.
66. Kobayashi T, Yoshihara Y, Samura A, et al: Synovial fluid concentrations
of the C-propeptide of type II collagen correlate with body mass index in
primary knee osteoarthritis. Ann Rheum Dis 56:500-503, 1997.
67. Garnero P, Mazieres B, Gueguen A, et al: Cross-sectional association
of 10 molecular markers of bone, cartilage, and synovium with disease
activity and radiological joint damage in patients with hip osteoar-
thritis: The ECHODIAH cohort. J Rheumatol 32:697-703, 2005.
68. Hakala M, Aman S, Luukkainen R, et al: Application of markers of
collagen metabolism in serum and synovial fluid for assessment of dis-
ease process in patients with rheumatoid arthritis. Ann Rheum Dis
54:886-890, 1995.
69. Sharif M, Salisbury C, Taylor DJ, et al: Changes in biochemical
markers of joint tissue metabolism in a randomized controlled trial
of glucocorticoid in early rheumatoid arthritis. Arthritis Rheum
41:1203-1209, 1998.
70. Sweet MB, Coelho A, Schnitzler CM, et al: Serum keratan sulfate
levels in osteoarthritis patients. Arthritis Rheum 31:648-652, 1988.
71. Campion GV, McCrae F, Schnitzer TJ, et al: Levels of keratan sulfate
in the serum and synovial fluid of patients with osteoarthritis of the
knee. Arthritis Rheum 34:1254-1259, 1991.
72. Mehraban F, Finegan CK, Moskowitz RW: Serum keratan sulfate:
Quantitative and qualitative comparisons in inflammatory versus
noninflammatory arthritides. Arthritis Rheum 34:383-392, 1991.
73. Poole AR, Ionescu M, Swan A, et al: Changes in cartilage metabo-
lism in arthritis are reflected by altered serum and synovial fluid levels
of the cartilage proteoglycan aggrecan: Implications for pathogenesis.
J Clin Invest 94:25-33, 1994.
74. Poole AR, Witter J, Roberts N, et al: Inflammation and cartilage
metabolism in rheumatoid arthritis: Studies of the blood markers
hyaluronic acid, orosomucoid, and keratan sulfate. Arthritis Rheum
33:790-799, 1990.
75. Rizkalla G, Reiner A, Bogoch E, et al: Studies of the articular carti-
lage proteoglycan aggrecan in health and osteoarthritis: Evidence for
molecular heterogeneity and extensive molecular changes in disease.
J Clin Invest 90:2268-2277, 1992.
487
76. Lohmander LS, Ionescu M, Jugessur H, et al: Changes in joint carti-
lage aggrecan after knee injury and in osteoarthritis. Arthritis Rheum
42:534-544, 1999.
77. Saltarelli MJ, Vasques-Abad M, Pickering E, et al: Type II collagen
neo-epitope (TIINE) levels in urine of patients with rheumatoid
arthritis decrease 90 days after the start of methotrexate treatment
following clinical response. Arthritis Rheum 43:5184(706), 2000.
78. Mansson B, Carey D, Alini M, et al: Cartilage and bone metabolism
in rheumatoid arthritis: Differences between rapid and slow progres-
sion of disease identified by serum markers of cartilage metabolism. J
Clin Invest 95:1071-1077, 1995.
79. Kong SY, Stabler TV, Criscione LG, et al: Diurnal variation of serum
and urine biomarkers in patients with radiographic knee osteoarthri-
tis. Arthritis Rheum 54:2496-2504, 2006.
80. Tortorella MD, Burn TC, Pratta MA, et al: Purification and cloning
of aggrecanase-1: A member of the ADAMTS family of proteins. Sci-
ence 284:1664-1666, 1999.
81. Fosang AJ, Last K, Maciewicz RA: Aggrecan is degraded by matrix
metalloproteinases in human arthritis: Evidence that matrix metal-
loproteinase and aggrecanase activities can be independent. J Clin
Invest 98:2292-2299, 1996.
82. Little CB, Hughes CE, Curtis CL, et al: Matrix metalloproteinases
are involved in C-terminal and interglobular domain processing of
cartilage aggrecan in late stage cartilage degradation. Matrix Biol
21:271-288, 2002.
83. Sandy JD, Flannery CR, Neame PJ, et al: The structure of aggrecan
fragments in human synovial fluid: Evidence for the involvement
in osteoarthritis of a novel proteinase which cleaves the Glu 373-
Ala 374 bond of the interglobular domain. J Clin Invest 89:1512-
1516, 1992.
84. Sandy JD, Verscharen C: Analysis of aggrecan in human knee carti-
lage and synovial fluid indicates that aggrecanase (ADAMTS) activ-
ity is responsible for the catabolic turnover and loss of whole aggrecan
whereas other protease activity is required for C-terminal processing
in vivo. Biochem J 358(Pt 3):615-626, 2001.
85. Brandt KD, Smith GNJ, Simon LS: Intraarticular injection of hyal-
uronan as treatment for knee osteoarthritis: What is the evidence?
Arthritis Rheum 43:1192-1203, 2000.
86. Goldberg RL, Huff JP, Lenz ME, et al: Elevated plasma levels of
hyaluronate in patients with osteoarthritis and rheumatoid arthritis.
Arthritis Rheum 34:799-807, 1991.
87. Paimela L, Heiskanen A, Kurki P, et al: Serum hyaluronate level as
a predictor of radiologic progression in early rheumatoid arthritis.
Arthritis Rheum 34:815-821, 1991.
88. Georges C, Vigneron H, Ayral X, et al: Serum biologic markers as pre-
dictors of disease progression in osteoarthritis of the knee. Arthritis
Rheum 40:590-591, 1997.
89. Sharif M, Osborne DJ, Meadows K, et al: The relevance of chondroi-
tin and keratan sulphate markers in normal and arthritic synovial
fluid. Br J Rheumatol 35:951-957, 1996.
90. Rossler A, Laszlo Z, Kvas E, et al: Plasma hyaluronan concentration:
No circadian rhythm but large effect of food intake in humans. Eur J
Appl Physiol Occup Physiol 78:573-577, 1998.
91. Neidhart M, Hauser N, Paulsson M, et al: Small fragments of cartilage
oligomeric matrix protein in synovial fluid and serum as markers for
cartilage degradation. Br J Rheumatol 36:1151-1160, 1997.
92. Clark AG, Jordan JM, Vilim V, et al: Serum cartilage oligomeric
matrix protein reflects osteoarthritis presence and severity: The John-
ston County Osteoarthritis Project. Arthritis Rheum 42:2356-2364,
1999.
93. Vilim V, Olejarova M, Machacek S, et al: Serum levels of cartilage
oligomeric matrix protein (COMP) correlate with radiographic pro-
gression of knee osteoarthritis. Osteoarthritis Cartilage 10:707-713,
2002.
94. Volck B, Johansen JS, Stoltenberg M, et al: Studies on YKL-40 in
knee joints of patients with rheumatoid arthritis and osteoarthritis:
Involvement of YKL-40 in the joint pathology. Osteoarthritis Carti-
lage 9:203-214, 2001.
95. Kuhne SA, Neidhart M, Everson MP, et al: Persistent high serum
levels of cartilage oligomeric matrix protein in a subgroup of patients
with traumatic knee injury. Rheumatol Int 18:21-25, 1998.
96. Mansson B, Geborek P, Saxne T: Cartilage and bone macromolecules
in knee joint synovial fluid in rheumatoid arthritis: Relation to devel-
opment of knee or hip joint destruction. Ann Rheum Dis 56:91-96,
1997.
 488 Degroot  |  Biologic Markers
97. Wollheim FA, Eberhardt KB, Johnson U, et al: HLA DRB1* typ-
ing and cartilage oligomeric matrix protein (COMP) as predictors of
joint destruction in recent-onset rheumatoid arthritis. Br J Rheuma-
tol 36:847-849, 1997.
98. Salminen H, Perala M, Lorenzo P, et al: Up-regulation of cartilage
oligomeric matrix protein at the onset of articular cartilage degenera-
tion in a transgenic mouse model of osteoarthritis. Arthritis Rheum
43:1742-1748, 2000.
99. Vilim V, Vytasek R, Olejarova M, et al: Serum cartilage oligomeric
matrix protein reflects the presence of clinically diagnosed ­synovitis
in patients with knee osteoarthritis. Osteoarthritis Cartilage 9:
612-618, 2001.
100. Roux-Lombard P, Eberhardt K, Saxne T, et al: Cytokines, metal-
loproteinases, their inhibitors and cartilage oligomeric matrix pro-
tein: Relationship to radiological progression and inflammation in
early rheumatoid arthritis: A prospective 5-year study. Rheumatol-
ogy (Oxf) 40:544-551, 2001.
101. Vilim V, Lenz ME, Vytasek R, et al: Characterization of monoclo-
nal antibodies recognizing different fragments of cartilage oligomeric
matrix protein in human body fluids. Arch Biochem Biophys 341:
8-16, 1997.
102. Cunnane G, Fitzgerald O, Beeton C, et al: Early joint erosions and
serum levels of matrix metalloproteinase 1, matrix metalloproteinase 3,
and tissue inhibitor of metalloproteinases 1 in rheumatoid arthritis.
Arthritis Rheum 44:2263-2274, 2001.
103. Ribbens C, Andre B, Jaspar JM, et al: Matrix metalloproteinase-3
serum levels are correlated with disease activity and predict clinical
response in rheumatoid arthritis. J Rheumatol 27:888-893, 2000.
104. Tchetverikov I, Lard LR, Verzijl N, et al: ProMMP-3 levels predict
joint destruction rate in early rheumatoid arthritis. Trans Orthop Res
Soc 28, page 15, 2003.
105. Giannelli G, Iannone F, Marinosci F, et al: Infliximab therapy does
not modify MMP-2 and MMP-9 serum concentrations in chronic
arthritis. Clin Exp Rheumatol 23:867-872, 2005.
106. Ribbens C, Porras M, Franchimont N, et al: Increased matrix metal-
loproteinase-3 serum levels in rheumatic diseases: Relationship with
synovitis and steroid treatment. Ann Rheum Dis 61:161-166, 2002.
107. Lohmander LS, Brandt KD, Mazzuca SA, et al: Use of the plasma strome-
lysin (matrix metalloproteinase 3) concentration to ­predict joint space
narrowing in knee osteoarthritis. Arthritis Rheum 52:3160-3167, 2005.
108. Chevalier X, Conrozier T, Gehrmann M, et al: Tissue inhibitor of
metalloprotease-1 (TIMP-1) serum level may predict progression of
hip osteoarthritis. Osteoarthritis Cartilage 9:300-307, 2001.
109. Stanton H, Rogerson FM, East CJ, et al: ADAMTS5 is the major
aggrecanase in mouse cartilage in vivo and in vitro. Nature 434:
648-652, 2005.
110. Glasson SS, Askew R, Sheppard B, et al: Deletion of active
ADAMTS5 prevents cartilage degradation in a murine model of
osteoarthritis. Nature 434:644-648, 2005.
111. Will H, Dettloff M, Bendzko P, et al: A quantitative assay for aggre-
canase activity. J Biomolec Tech 16:459-472, 2005.
112. Pratta MA, Su JL, Leesnitzer MA, et al: Development and character-
ization of a highly specific and sensitive sandwich ELISA for detec-
tion of aggrecanase-generated aggrecan fragments. Osteoarthritis
Cartilage 14:702-713, 2006.
113. Thomas M, Sabatini M, Bensaude F, et al: A microplate assay for the
screening of ADAMTS-4 inhibitors. Matrix Biol 25:261-267, 2006.
114. Tak PP, Smeets TJ, Daha MR, et al: Analysis of the synovial cell infil-
trate in early rheumatoid synovial tissue in relation to local disease
activity. Arthritis Rheum 40:217-225, 1997.
115. Cunnane G, Fitzgerald O, Hummel KM, et al: Synovial tissue prote-
ase gene expression and joint erosions in early rheumatoid arthritis.
Arthritis Rheum 44:1744-1753, 2001.
116. Ruderman EM, Weinblatt ME, Thurmond LM, et al: Synovial ­tissue
response to treatment with Campath-1H. Arthritis Rheum 38:
254-258, 1995.
117. Vos K, Thurlings RM, Wijbrandts CA, et al: Early effects of rituximab
on the synovial cell infiltrate in patients with rheumatoid arthritis.
Arthritis Rheum 56:772-778, 2007.
118. Gerlag D, Tak PP: Synovial biopsy. Best Pract Res Clin Rheumatol
19:387-400, 2005.
119. Youssef PP, Kraan M, Breedveld F, et al: Quantitative microscopic
analysis of inflammation in rheumatoid arthritis synovial membrane
samples selected at arthroscopy compared with samples obtained
blindly by needle biopsy. Arthritis Rheum 41:663-669, 1998.
120. Kraan MC, Reece RJ, Smeets TJ, et al: Comparison of synovial tis-
sues from the knee joints and the small joints of rheumatoid arthritis
patients: Implications for pathogenesis and evaluation of treatment.
Arthritis Rheum 46:2034-2038, 2002.
121. Boyle DL, Rosengren S, Bugbee W, et al: Quantitative biomarker
analysis of synovial gene expression by real-time PCR. Arthritis Res
Ther 5:R352-R360, 2003.
122. Smeets TJ, Kraan MC, Galjaard S, et al: Analysis of the cell infil-
trate and expression of matrix metalloproteinases and granzyme B in
paired synovial biopsy specimens from the cartilage-pannus junction
in patients with RA. Ann Rheum Dis 60:561-565, 2001.
123. Kirkham B, Portek I, Lee CS, et al: Intraarticular variability of
­synovial membrane histology, immunohistology, and cytokine mRNA
­expression in patients with rheumatoid arthritis. J Rheumatol 26:
777-784, 1999.
124. Kane D, Jensen LE, Grehan S, et al: Quantitation of metalloprotein-
ase gene expression in rheumatoid and psoriatic arthritis synovial
tissue distal and proximal to the cartilage-pannus junction. J Rheu-
matol 31:1274-1280, 2004.
125. Haringman JJ, Gerlag DM, Zwinderman AH, et al. Synovial tissue
macrophages: A sensitive biomarker for response to treatment in
patients with rheumatoid arthritis. Ann Rheum Dis 64:834-838,
2005.
126. Rooney M, Whelan A, Feighery C, et al: Changes in lymphocyte
infiltration of the synovial membrane and the clinical course of
rheumatoid arthritis. Arthritis Rheum 32:361-369, 1989.
127. Firestein GS, Paine MM, Boyle DL: Mechanisms of methotrexate
action in rheumatoid arthritis: Selective decrease in synovial col-
lagenase gene expression. Arthritis Rheum 37:193-200, 1994.
128. Kraan MC, Reece RJ, Barg EC, et al: Modulation of inflammation and
metalloproteinase expression in synovial tissue by leflunomide and
methotrexate in patients with active rheumatoid arthritis: ­Findings
in a prospective, randomized, double-blind, parallel-design ­clinical
trial in thirty-nine patients at two centers. Arthritis Rheum 43:
1820-1830, 2000.
129. Tak PP, Taylor PC, Breedveld FC, et al: Decrease in cellularity and
expression of adhesion molecules by anti-tumor necrosis factor
alpha monoclonal antibody treatment in patients with rheumatoid
arthritis. Arthritis Rheum 39:1077-1081, 1996.
130. Taylor PC, Peters AM, Paleolog E, et al: Reduction of chemokine
levels and leukocyte traffic to joints by tumor necrosis factor alpha
blockade in patients with rheumatoid arthritis. Arthritis Rheum
43:38-47, 2000.
131. Verschueren PC, Markusse HM, Smeets TJ, et al: Reduced cel-
lularity and expression of adhesion molecules and cytokines after
treatment with soluble human recombinant TNF receptor (P75) in
rheumatoid arthritis patients. Arthritis Rheum 42:S197, 1999.
132. Cunnane G, Madigan A, Murphy E, et al: The effects of treatment
with interleukin-1 receptor antagonist on the inflamed synovial mem-
brane in rheumatoid arthritis. Rheumatology (Oxf) 40:62-69, 2001.
133. Smeets TJ, Kraan MC, van Loon ME, et al: Tumor necrosis factor
alpha blockade reduces the synovial cell infiltrate early after initia-
tion of treatment, but apparently not by induction of apoptosis in
synovial tissue. Arthritis Rheum 48:2155-2162, 2003.
134. Youssef PP, Triantafillou S, Parker A, et al: Variability in cytokine
and cell adhesion molecule staining in arthroscopic synovial biop-
sies: Quantification using color video image analysis. J Rheumatol
24:2291-2298, 1997.
135. Gerlag DM, Haringman JJ, Smeets TJ, et al: Effects of oral pred-
nisolone on biomarkers in synovial tissue and clinical improvement
in rheumatoid arthritis. Arthritis Rheum 50:3783-3791, 2004.
136. Kruithof E, De Rycke L, Vandooren B, et al: Identification of synovial
biomarkers of response to experimental treatment in early-phase clin-
ical trials in spondylarthritis. Arthritis Rheum 54:1795–1804, 2006.
137. Goedkoop AY, Kraan MC, Teunissen MB, et al: Early effects of
tumour necrosis factor alpha blockade on skin and synovial tissue in
patients with active psoriasis and psoriatic arthritis. Ann Rheum Dis
63:769-773, 2004.
138. Kraan MC, Van Kuijk AW, Dinant HJ, et al: Alefacept treatment
in psoriatic arthritis: Reduction of the effector T cell population in
peripheral blood and synovial tissue is associated with improvement
of clinical signs of arthritis. Arthritis Rheum 46:2776-2784, 2002.
138a. Gerlag DM, Boyle DL, Rosengren S, et al: Real-time quantitative PCR
to detect changes in synovial gene expression in rheumatoid arthritis
after corticosteroid treatment. Ann Rheum Dis 66:545-547, 2007.
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
139. Visser H, le Cessie S, Vos K, et al: How to diagnose rheumatoid
arthritis early: A prediction model for persistent (erosive) arthritis.
Arthritis Rheum 46:357-365, 2002.
140. McDonald WH, Yates 3rd JR: Shotgun proteomics and biomarker
discovery. Dis Markers 18:99-105, 2002.
141. Aigner T, Haag J, Zimmer R: Functional genomics, evo-devo and
systems biology: A chance to overcome complexity? Curr Opin Rheu­
matol 19(5):463-470, 2007.
142. Aigner T, Stoss H, Weseloh G, et al: Activation of collagen type II
expression in osteoarthritic and rheumatoid cartilage. Virchows Arch
B Cell Pathol Mol Pathol 62:337-345, 1992.
143. Criswell LA, Gregersen PK: Current understanding of the genetic
aetiology of rheumatoid arthritis and likely future developments.
Rheumatology (Oxf) 44(Suppl 4):iv-9-iv-13, 2005.
144. van der Pouw Kraan TC, van Gaalen FA, Kasperkovitz PV, et al:
Rheumatoid arthritis is a heterogeneous disease: Evidence for differ-
ences in the activation of the STAT-1 pathway between rheumatoid
tissues. Arthritis Rheum 48:2132-2145, 2003.
145. Marshall KW, Zhang H, Yager TD, et al: Blood-based biomarkers for
detecting mild osteoarthritis in the human knee. Osteoarthritis Car-
tilage 13:861-871, 2005.
146. Marshall KW, Zhang H, Nossova N: Chondrocyte genomics: Impli-
cations for disease modification in osteoarthritis. Drug Discov Today
11(17-18):825-832, 2006.
147. Feng X, Wu H, Grossman JM, et al: Association of increased inter-
feron-inducible gene expression with disease activity and lupus
nephritis in patients with systemic lupus erythematosus. Arthritis
Rheum 54:2951-2962, 2006.
148. Fisler R: Biomarkers in clinical development: Implications for per-
sonalized medicine and streamlining R&D. Cambridge Healthtech
Advisors, report 47, March 2005. (http:www.insightpharmareports.
com/reports/2005/47_Biomarkers/overview.asp)
149. Hermansson M, Sawaji Y, Bolton M, et al: Proteomic analysis of
articular cartilage shows increased type II collagen synthesis in osteo-
arthritis and expression of inhibin betaA (activin A), a regulatory
molecule for chondrocytes. J Biol Chem 279:43514-43521, 2004.
150. Ruiz-Romero C, Lopez-Armada MJ, Blanco FJ: Proteomic charac-
terization of human normal articular chondrocytes: A novel tool for
the study of osteoarthritis and other rheumatic diseases. Proteomics
5:3048-3059, 2005.
151. Ruiz-Romero C, Lopez-Armada MJ, Blanco FJ: Mitochondrial pro-
teomic characterization of human normal articular chondrocytes.
Osteoarthritis Cartilage 14:507-518, 2006.
152. deSeny D, Fillet M, Meuwis MA, et al: Discovery of new rheuma-
toid arthritis biomarkers using the surface-enhanced laser desorption/
ionization time-of-flight mass spectrometry ProteinChip approach.
Arthritis Rheum 52:3801-3812, 2005.
489
153. Xiang Y, Sekine T, Nakamura H, et al: Proteomic surveillance of
autoimmunity in osteoarthritis: Identification of triosephosphate
isomerase as an autoantigen in patients with osteoarthritis. Arthritis
Rheum 50:1511-1521, 2004.
154. Graham KL, Robinson WH, Steinman L, et al: High-throughput
methods for measuring autoantibodies in systemic lupus erythematosus
and other autoimmune diseases. Autoimmunity 37:269-272, 2004.
155. Li QZ, Xie C, Wu T, et al: Identification of autoantibody clusters that
best predict lupus disease activity using glomerular proteome arrays. J
Clin Invest 115:3428-3439, 2005.
156. Li QZ, Zhou J, Wandstrat AE, et al: Protein array autoantibody pro-
files for insights into systemic lupus erythematosus and incomplete
lupus syndromes. Clin Exp Immunol 147:60-70, 2007.
157. Nicholson JK, Lindon JC, Holmes E: ‘Metabonomics’: Understand-
ing the metabolic responses of living systems to pathophysiological
stimuli via multivariate statistical analysis of biological NMR spec-
troscopic data. Xenobiotica 29:1181-1189, 1999.
158. Damyanovich AZ, Staples JR, Marshall KW: 1H NMR investigation
of changes in the metabolic profile of synovial fluid in bilateral canine
osteoarthritis with unilateral joint denervation. Osteoarthritis Carti-
lage 7:165-172, 1999.
159. Damyanovich AZ, Staples JR, Chan AD, et al: Comparative study of
normal and osteoarthritic canine synovial fluid using 500 MHz 1H
magnetic resonance spectroscopy. J Orthop Res 17:223-231, 1999.
160. Lamers RJ, DeGroot J, Spies-Faber EJ, et al: Identification of disease-
and nutrient-related metabolic fingerprints in osteoarthritic Guinea
pigs. J Nutr 133:1776-1780, 2003.
161. Lamers RJ, van Nesselrooij JH, Kraus VB, et al: Identification of
an urinary metabolite profile associated with osteoarthritis. Osteo-
arthritis Cartilage 13:762-768, 2005.
162. van der Greef J, McBurney RN: Innovation: Rescuing drug dis-
covery: In vivo systems pathology and systems pharmacology. Nat
Rev Drug Discov 4:961-967, 2005.
163. Glocker MO, Guthke R, Kekow J, et al: Rheumatoid arthritis, a com-
plex multifactorial disease: On the way toward individualized medi-
cine. Med Res Rev 26:63-87, 2006.
164. Lee JW, Smith WC, Nordblom GD, et al: Validation of assays for
the bioanalysis of novel biomarkers: Practical recommendations for
clinical investigation of new drug entities. In Bloom JC, Dean RA
(eds): Biomarkers in Clinical Drug Development. New York, Marcel
Dekker, 2003, pp 119-148.
165. van den Berg WB, van Riel PL: Uncoupling of inflammation and
destruction in rheumatoid arthritis: Myth or reality? Arthritis Rheum
52:995-999, 2005.
166. Kraus VB: Do biochemical markers have a role in osteoarthritis
­diagnosis and treatment? Best Pract Res Clin Rheumatol 20:69-80,
2006.
 33
KEY POINTS
Some occupational and recreational activities have been
linked with musculoskeletal syndromes or disorders. These
include certain syndromes manifested by neck pain; ­shoulder,
elbow, hand, or wrist pain or tendinitis; carpal tunnel
­syndrome; and hand-arm vibration syndrome.
The concepts of so-called cumulative trauma disorders
and ­repetitive strain disorders, though perhaps intuitive,
are ­poorly supported by the literature. Causal relation-
ships ­between most occupations or activities and these
­“syndromes” should not be considered well established.
Some activities and mechanical stresses have been associated
with osteoarthritis at certain sites—for example, the hips of
­farmers, the knees of workers whose jobs involve frequent
knee bending, and the hands of workers doing repetitive
tasks with their hands (e.g., seamstresses, diamond workers,
textile ­workers).
Certain rheumatic disorders have been related to environ-
mental or occupational risks, such as Raynaud’s phenomenon
with vibration and polyvinyl chloride; autoimmune disease
with teaching school; systemic sclerosis with chlorinated
­hydrocarbons, organic solvents, and silica; scleroderma-like
syndromes with rapeseed oil and l-tryptophan; lupus syn-
dromes with canavanine, hydrazine, mercury, pesticides, and
solvents; lupus, scleroderma, and Paget’s disease with
pet ownership; rheumatoid arthritis with silica (Caplan’s
syndrome); and saturnine gout with lead exposure.
Putting a normal joint through its normal range of motion
is not necessarily harmful for a normal individual; however,
if the joint, motion, stress, or biomechanics are not normal,
there may be a risk of joint harm.
Most normal individuals comfortably engaging in reasonable
recreational activities can do so without evidence of lasting
soft tissue or articular damage; runners have been best stud-
ied. Conversely, individuals who exercise with pain, effusions,
underlying joint abnormalities (e.g., ligamentous or meniscal
damage), or abnormal or unusual biomechanics or as profes-
sional or elite athletes (e.g., boxers, American football or
soccer players) seem to be at increased risk of joint injury.
Performing artists, vocalists, dancers, and musicians seem to
have a risk of soft tissue and joint injury analogous to that of
athletes, but little information is available.
“The diseases of persons incident to this craft arise from three
causes: first constant sitting, second the perpetual motion of
the hand in the same manner, and thirdly the attention and
application of the mind. . . . Constant writing also considerably
fatigues the hand and whole arm on account of the continual
and almost tense tension of the muscles and tendons. I knew
a man who, by perpetual writing, began first to complain of
an ­excessive weariness of his whole right arm, which could be
Occupational
and Recreational
Musculoskeletal
Disorders
Richard S. Panush
removed by no medicines, and which was at last succeeded by a
perfect palsy of the whole arm.”
—Ramazzini 17131
“When job demands . . . repeatedly exceed the biomechanical
capacity of the worker, the activities become trauma-inducing.
Hence, traumatogens are workplace sources of biomechanical
strain that contribute to the onset of injuries affecting the mus-
culoskeletal system.”
—National Institute for Occupational Safety and Health (1986)2
This chapter discusses the possible association of certain
occupational and recreational activities with musculoskel-
etal disorders. It has been conventional wisdom that “wear
and tear” from at least some activities lead to reversible
or irreversible damage to the musculoskeletal system.2-5
Despite the apparent logic that work or recreational activi-
ties might cause rheumatic and musculoskeletal disorders
or soft tissue syndromes, this putative association is contro-
versial and perhaps seriously flawed. There are confounding
aspects to much of the available data, including imprecise
diagnostic labels, the subjectivity of complaints, anecdotal
and survey data, inadequate controls, differing definitions of
disease and disability, limited duration of follow-up obser-
vations, inadequate epidemiology, inferential observations,
difficulty quantifying activities and defining health effects,
assumptions of the validity of claims data, variable quality
of reported observations, psychological factors influencing
symptoms, and conflicting data.
OCCUPATION-RELATED
MUSCULOSKELETAL DISORDERS
Many presumptive work-related musculoskeletal ­ disorders
have been described and are summarized in Table
33-1.1-11 These have been reported as sprains, strains,
inflammations, dislocations, and irritations. The cost of
work-related disability from musculoskeletal disorders
has been equivalent to approximately 1% of the United
States’ gross national product, making these entities of
considerable societal interest.12 Industries with the high-
est rates of musculoskeletal disorders include meatpacking,
knit-­underwear manufacture, motor vehicle manufacture,
poultry processing, mail and message distribution, health
assessment and treatment, construction, butchery, food
processing, machine operation, dental hygiene, data entry,
hand grinding and polishing, carpentry, industrial truck
and tractor operation, nursing assistance, and houseclean-
ing. There have been imprecise associations between work-
related musculoskeletal syndromes and age, gender, fitness,
and weight.6,10,11
491
 492 PANUSH  |  Occupational and Recreational Musculoskeletal Disorders
Table 33-1  Occupation-Related Musculoskeletal   Table 33-2  Selected Literature Describing Regional
Syndromes
Cherry pitter’s thumb Gamekeeper’s thumb
Staple gun carpal tunnel syndrome Espresso maker’s wrist
Bricklayer’s shoulder Espresso elbow
Carpenter’s elbow Pizza maker’s palsy
Janitor’s elbow Poster presenter’s thumb
Stitcher’s wrist Rope maker’s claw hand
Cotton twister’s hand Telegraphist’s cramp
Writer’s cramp Waiter’s shoulder
Bowler’s thumb Ladder shins
Jeweler’s thumb Tobacco primer’s wrist
Carpet layer’s knee
A number of work-related regional musculoskeletal
­syndromes have been described. These include disorders of
the neck, shoulder, elbow, hand and wrist, lower back, and
lower extremities10 (Table 33-2); some of these are discussed
in greater detail in other chapters. Neck musculoskeletal
disorders are associated with repetition, forceful exertion,
and constrained or static postures. Shoulder musculoskeletal­
disorders occur with work at or above shoulder height, lift-
ing of heavy loads, static postures, hand-arm vibration, and
repetitive motion. For elbow epicondylitis, risk factors are
overexertion of finger and wrist extensors with the elbow
in extension, as well as posture. Hand-wrist tendinitis and
work-related carpal tunnel syndrome are noted with repeti-
tive work, forceful activities, flexed wrists, and duration­
of continual effort.1,10 Hand-arm vibration syndrome
­(Raynaud-like phenomenon)13 has been linked to the inten-
sity and duration of vibrating exposure. Work-related lower
back disorders are associated with repetition, the weight of
objects lifted, twisting, and poor biomechanics of lifting.13,14
Other risk factors for work-related musculoskeletal disorders
involving the back include awkward posture, high static
muscle load, high-force exertion at the hands and wrists,
sudden applications of force, work with short cycle times,
little task variety, frequent tight deadlines, inadequate rest
or recovery periods, high cognitive demands, little control
over work, cold work environment, localized mechanical
stresses to tissues, and poor spinal support.1
The development of recommended treatment methods
(rehabilitation) for these so-called occupational musculoskel-
etal disorders has included collaboration by workers, employ-
ers, insurers, and health professionals. The process has been
divided into three phases: protection from and resolution of
symptoms, restoration of strength and dynamic stability, and
return to work. This process includes symptomatic therapies,
physical therapy, and ergonomic evaluation.7 Prognosis for
these maladies has not been well studied or defined.8
Until recently, the prevailing view was that musculo-
skeletal disorders were consistently and predictably work
related. That understanding has now come under consid-
erable scrutiny and criticism.2,15-24 Despite the quantity of
published information (see Table 33-2), the previously cited
literature about occupational musculoskeletal disorders is
now considered flawed; its quality was uneven and perhaps
poor in some cases. Definitions of musculoskeletal disorders
were ­imprecise; diagnoses, by rheumatologic standards, were
 
Occupation-Related Musculoskeletal Syndromes
No. of Epidemiologic Odds Ratio/
Syndrome Studies Relative Risk
Neck pain 26 0.7-6.9
Shoulder tendinitis 22 0.9-13
Elbow tendinitis 14 0.7-5.5
Hand-wrist tendinitis 16 0.6-31.7
Carpal tunnel   22 1-34
syndrome
Hand-arm vibration 8 0.5-41
syndrome
Data from references 5 and 6.
infrequent; studies were usually not prospective, and there
were selection biases; psychological influences and second-
ary gain were often ignored; questionnaires were often used
without validation of subjective complaints; and quantifi-
cation of putative causative factors was difficult. Indeed, a
review of this literature concluded that none of the pub-
lished studies satisfactorily established a causal relationship
between work and distinct medical entities.20 In fact, certain
experiences argued powerfully against the notion of work-
related musculoskeletal disorders. In Lithuania, for example,
where insurance was limited and disability was not a societal
expectation or entitlement, “whiplash” from auto accidents
did not exist.19 In Australia, when legislation for compen-
sability was made more stringent, an epidemic of whiplash
and repetitive-strain injuries abated.21,23 In the United States,
too, expressed symptoms correlated closely with the likeli-
hood of obtaining compensation.25 In other cases, ergo-
nomic interventions had no effect on alleged work-related
symptoms, and close analysis of epidemics of work-related
musculoskeletal disorders revealed serious inconsistencies.17
These concerns led the American Society for Surgery of
the Hand to editorialize that “the current medical litera-
ture does not provide the information necessary to establish
a causal relationship between specific work activities and
the development of well-recognized disease entities. Until
scientifically valid studies are conducted, the society urges
the government to exercise restraint in considering regula-
tions designed to reduce the incidence of these conditions,
as premature regulations could have far-reaching legal and
economic effects, and could have an adverse impact on the
care of workers.”18
One review summarized that “most believe scientific data
are insufficient to establish a definite causal relationship of
these so-called cumulative trauma disorders to the worker’s
occupation, and many believe the issue has become a socio-
political problem.”9 Hadler2,15-17 has written particularly
forcefully that popular notions about work-related muscu-
loskeletal disorders have been based on inadequate science.
Others, too, have expressed serious reservations about the
cumulative trauma disorder hypothesis, including the Indus-
trial Injuries Committee of the American Society for Surgery
of the Hand, the Working Group of the British Orthopaedic
Association, and the World Health Organization.2,15-17,20,22 An
appreciation of the importance of psychosocial factors influ-
encing work disability has emerged. These factors include
lack of job control, fear of layoff, monotony, job ­dissatisfaction,
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
unsatisfactory performance appraisals, distress and unhap-
piness with coworkers or supervisors, poor coping abilities,
divorce, low income, and less education.2,15-25 This is remi-
niscent of the story of silicone breast implants and their
putative association with rheumatic disease. In this
instance—as seems to be the case with work-related muscu-
loskeletal disorders—there was a coalescence of naively sim-
plistic assumptions, untested hypotheses, confusion between
the repetition of hypotheses and their scientific valida-
tion, media exaggeration, and public advocacy intertwined
with politics and governmental regulatory agencies, dollar
jackpots, litigation, and inadequate science. All these ele-
ments confounded and perverted the silicone breast implant
story26,27 and may have confused the interpretation of evi-
dence-based work-related musculoskeletal disorders as well.
More research is needed to learn about work-related muscu-
loskeletal disorders and to clearly identify the ­circumstances
in which they occur. Work-related musculoskeletal disorders
probably do exist, but they are likely to be less pervasive and
less noxious than originally thought.
OCCUPATION-RELATED RHEUMATIC
DISEASES
Work-related rheumatic diseases have not been consistently
well studied, but associations between occupations and well-
defined rheumatic disorders are clearer than those involving
musculosketetal disorders. This topic also recapitulates the
simplistic notion that joints deteriorate with use. However,
this perception is neither necessarily logical nor correct.
The discussion focuses largely on osteoarthritis (OA); see
also Chapters 90 and 91.
OSTEOARTHRITIS
Is OA caused, at least in part, by mechanical stress? One
analytic approach to determining a possible relation-
ship between activity and joint disease is to consider the
­epidemiologic evidence that degenerative arthritis may
Table 33-3  Occupational Physical Activity and Possible Associations with Osteoarthritis
Occupation Involved Joints
Miner Elbow, knee, spine
Pneumatic driller Shoulder, elbow, wrist, MCP
Dockworker Intervertebral disk, DIP,   Increased
elbow, knee
Cotton mill worker Hand
Diamond worker Hand
Shipyard laborer Knee
Foundry worker Lumbar spine
Seamstress Hand
Textile worker Hand
Manual laborer MCP
Occupations requiring   Knee
knee bending
Farmer Hip, knee
DIP, distal interphalangeal joint; MCP, metacarpophalangeal joint; OA, osteoarthritis.
493
f­ollow ­ repetitive trauma. Most discussions of the patho-
genesis of OA include a role for “stress.”28-31 Several studies
have suggested an increased prevalence of OA of the elbows,
knees, and spine in miners32-34; of the shoulders, elbows,
wrists, and metacarpophalangeal joints in pneumatic drill
operators35; of the intervertebral disks, distal interphalangeal
joints, elbows, and knees in dockworkers33; of the hands in
cotton workers,36 diamond cutters,32,37 seamstresses,37 and tex-
tile workers38,39; of the knees and hips in farmers; of the knees
in shipyard workers and a variety of occupations involv-
ing knee bending; and of the spine in foundry workers40-43
(Table 33-3). Population studies have noted increased hip
OA in farmers, firefighters, mill workers, dockworkers,
female mail carriers, unskilled manual laborers, fishermen,
and miners and have reported increased knee OA in farm-
ers, firefighters, construction workers, house and hotel clean-
ers, craftspeople, laborers, and service workers.40-43 Activities
leading to an increased risk for premature OA involve power
gripping, carrying, lifting, increased physical loading,
increased static loading, kneeling, walking, and bending.40-43
Studies of skeletons of several populations have suggested
that age at onset, ­ frequency, and location of osteoarthritic
changes were directly related to the nature and degree of
physical activities.44 ­However, not all these studies adhered to
­contemporary standards, nor have they been confirmed. One
report, for example, failed to find an increased incidence of
OA in pneumatic drill users and criticized inadequate sample
sizes, lack of statistical analyses, and omission of appropri-
ate control populations in previous reports.34 The investi-
gators further commented that earlier work was “frequently
­misinterpreted” and that their studies suggested that “impact,
without injury or preceding abnormality of either joint con-
tour or ligaments, is unlikely to produce osteoarthritis.”35
Do epidemiologic studies of OA implicate physical
or mechanical factors related to disease predisposition
or development? The first national Health and Nutrition
Examination Survey of 1971 to 1975 (HANES I) and the
Framingham studies explored cross-sectional associations
between ­ radiographic OA of the knee and possible risk
Risk of OA References
Increased Lawrence33 (1955), Kellgren & Lawrence34 (1958),
Felson43,44 (1997, 1998)
Increased/none Jurmain (1977) (cited in ref 40), Burke et al35 (1977)
Lawrence33 (1955)
Increased Lawrence36 (1961)
Increased Kellgren & Lawrence32 (1957), Tempelaar  
& van Breeman37 (1932)
Increased Goldberg & Montgomery (1987) (cited in refs 43, 44)
Increased Lawrence et al (1966) (cited in refs 43, 44)
Increased Tempelaar & Van Breeman37 (1932)
Increased Hadler et al39 (1978)
Increased Williams et al (1987) (cited in refs 43, 44)
Increased Felson et al40-43 (1988, 1991, 1997, 1998)
Increased Felson40-43 (1988, 1991, 1997, 1998)
 494 PANUSH  |  Occupational and Recreational Musculoskeletal Disorders
Table 33-4  Other Occupation-Related Rheumatic   forces, either congenital or secondary to joint injury, are
Diseases
Disease or Syndrome Occupation or Risk Factor
Reflex sympathetic dystrophy Trauma
Raynaud’s phenomenon Vibration
Chemicals (polyvinyl chloride)
Autoimmune disease Teaching school
Systemic sclerosis Chlorinated hydrocarbons
Organic solvents
Silica
Scleroderma-like syndromes Rapeseed oil
l-Tryptophan
Systemic lupus   Canavanine, hydrazine,   with degenerative arthritis? Several animal studies have
erythematosus mercury, pesticides, solvents
Lupus, scleroderma,   Pet ownership
and Paget’s disease
Rheumatoid arthritis   Silica
(Caplan’s syndrome)
Gout (saturnine) Lead
f­actors.40-46 Strong associations were noted between knee
OA and obesity and those occupations involving the stress
of knee bending, but not all habitual physical activities and
leisure-time physical activities (running, walking, team
sports, racquet sports, and others) were linked with knee
OA.28-30
OTHER OCCUPATIONAL RHEUMATOLOGIC
DISORDERS
Certain rheumatic diseases other than repetitive strain
or cumulative trauma disorders have been associated with
occupational risks. These include reports of reflex sympa-
thetic dystrophy after trauma; Raynaud’s phenomenon with
vibration or exposure to chemicals (polyvinyl chloride);
autoimmune disease from teaching school43,47; systemic
sclerosis from chemicals and silica; scleroderma-like syn-
dromes from rapeseed oil and l-tryptophan; systemic lupus
erythematosus from canavanine, hydrazine, mercury, pes-
ticides, and solvents48; lupus, scleroderma, and Paget’s dis-
ease from pets49; rheumatoid arthritis (Caplan’s syndrome)
with silica; and gout (saturnine) with lead intoxication50
(Table 33-4).
RECREATION- AND SPORTS-RELATED
MUSCULOSKELETAL DISORDERS
Do recreational or sports-related activities lead to mus-
culoskeletal disorders? Patients with sports injuries (such
as from downhill skiing and football) to the anterior cru-
ciate and medial collateral ligaments frequently develop
the chondromalacia patellae and radiographic abnor-
malities of OA (20% to 52%).28-30 Retrospective studies
suggest that the development of OA may be associated
with varus deformity, previous meniscectomy, and relative
body weight.51,52 Both partial and total meniscectomies
have been associated with degenerative changes. Early
joint stabilization and direct meniscus repair surgery may
decrease the incidence of premature OA. These observa-
tions support the concept that abnormal ­ biomechanical
important factors in the development of exercise-related
OA.28-30 Other factors considered important in the devel-
opment of sports-related OA include certain physical
characteristics of the participant, biomechanical­ and bio-
chemical factors, age, gender, hormonal influences, nutri-
tion, characteristics of the playing surface, unique features
of particular sports, and duration and intensity of exer-
cise participation, as has been reviewed extensively else-
where.28-30 It is increasingly recognized that biomechanical
factors have an important role in the ­ pathogenesis
of OA.
Is regular participation in physical activity associated
suggested, but not proved, a possible relationship between
exercise and OA. It has been stated that the husky breed
of dog has increased hip and shoulder arthritis associated
with ­ pulling sleds, that tigers and lions develop foreleg
OA related to sprinting and running, and that racehorses
and workhorses develop OA in the forelegs and hind legs,
respectively, consistent with their physical stress patterns.28-30
Rabbits with experimentally induced arthritis in one hind
limb did not develop progressive OA when exercised on
treadmills,54-59 but sheep in normal health walking on con-
crete did develop OA.53 Later studies found that beagle dogs
running 4 to 20 km a day did not develop OA.60 Although
these observations were not entirely consistent, they sug-
gested, but did not prove, that physical activities in some
circumstances might predispose to degenerative joint
­disease.
There have been some pertinent, largely anecdotal, obser-
vations in human studies28-30 (Table 33-5). Wrestlers were
reported to have an increased incidence of OA of the lumbar
spine, cervical spine, knees, and elbows; boxers, of the car-
pometacarpal joints; baseball pitchers, of the shoulders and
elbows; parachutists, of knees, ankles, and spine; cyclists, of
the patella; cricketers, of the fingers; and gymnasts, of the
shoulders, elbows, and wrists.28-30 Most of these reports were
observational, and not all of them reflected confirmed asso-
ciations. Soccer players have been reported to have talar
joint, ankle, cervical spine, knee, and hip OA.28-30,61 Few
studies of American football players have been reported,
but it has been suggested that they are susceptible to OA
of the knees, particularly those who sustained knee injuries
while playing football. Among football players (average age
23 years) competing for a place on a professional team, 90%
had radiographic abnormalities of the foot or ankle, com-
pared with 4% of an age-matched control population; line-
men had more changes than did ball carriers or linebackers,
who in turn had more changes than did flankers or defen-
sive backs. All those who had played football for 9 years
or longer had abnormal findings on radiography.28-31 Most of
these studies suffered in several respects: criteria for OA (or
“osteoarthrosis,” “degenerative joint disease,” or “abnormal-
ity”) were not always clear, specified, or consistent; duration
of follow-up was often not indicated or was inadequate to
determine the risk of musculoskeletal problems at a later
age; intensity and duration of physical activity were variable
and difficult to quantify; selection bias toward individuals
exercising or participating versus those not exercising or
participating was not weighted; other possible risk factors
and predispositions to musculoskeletal disorders were rarely
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES 495

Table 33-5  Sports Participation and Alleged Associations with Osteoarthritis

Sport Site (Joint) References* Risk
Ballet Talus Ottani & Betti (1953), Coste et al  
(1960), Brodelius (1961),  
Miller et al (1975)
Ankle
Probably increased
Cervical spine Washington (1978), Ende & Wickstrom (1982)
Hip
Knee
Metatarsophalangeal Washington (1978)
Baseball Elbow Adams (1965), Hansen (1982)
Shoulder Bennett (1941)
Boxing Hand   Iselin (1960)
(carpometacarpal joints)
Cricket Finger Vere Hodge (1971)
Cycling Finger Bagneres (1967)
American football Ankle
Vincelette et al (1972)
Foot
Rall et al (1964)
Knee
Spine Ferguson et al (1975), Albright et al  
(1976), Moretz et al (1984)
Probably increased
Gymnastics Elbow
Shoulder Bozdech (1971)
Wrist
Hip Murray & Duncan (1971)
Lacrosse Ankle
Thomas (1971)
Knee
Martial arts Spine Rubens-Duval et al (1960)
Parachuting Ankle
Murray & Duncan (1971)
Knee
Spine Murray-Leslie et al (1977a)
Rugby Knee Slocum (1960)
Running Knee McDermott & Freyne (1983),  
Lane et al (1986, 1987, 1998),  
Panush et al (1986)
Hip Puranen et al (1975), de Carvalho  
& Langfeldt (1977), McDermott   Small
& Freyne (1983), Lane et al (1986,  
1987, 1998), Panush et al (1986),  
Konradsen et al (1990)
Ankle Konradsen et al (1990), Marti et al (1990)
Soccer Ankle-foot Pellissier et al (1952), Pellegrini et al (1964)
Sortland et al (1982)
Hip Klunder et al (1980)
Knee Pellissier et al (1952), Solonen (1966),   Possibly increased
Klunder et al (1980)
Talus Brodelius (1961), Solonen (1966)
Talofibular Burel et al (1960)
Weightlifting Spine Aggrawal et al (1965), Muenchow  
& Albert (1969), Fitzgerald  
& McLatchie (1980)
Possibly increased
Wrestling Cervical spine
Elbow Layani et al (1960)
Knee
*Cited in Panush RS, Lane NE: Exercise and the musculoskeletal system. Baillieres Clin Rheumatol 8:79, 1994; Panush RS: Physical activity, fitness, and osteoar-
thritis. In Bouchard C, Shephard RJ, Stephens T (eds): Physical Activity, Fitness, and Health. International Proceedings and Consensus Statement. Champaign, Ill,
Human Kinetics Publishers, 1994, pp 712-723; Panush RS: Does exercise cause arthritis? Long-term consequences of exercise on the musculoskeletal system.
Rheum Dis Clin North Am 16:827, 1990.

considered; studies were not always properly controlled, and
examinations were not always “blind”; little information
regarding nonprofessional, recreational athletes was avail-
able; and little clinical information about functional status
was provided.
Several studies have now examined a possible relation-
ship between running and OA. Uncontrolled observa-
tions generally suggested that runners without underlying
­biomechanical problems of the lower extremity joints did
not develop arthritis at a different rate from a normal
 496 PANUSH  |  Occupational and Recreational Musculoskeletal Disorders

p­ opulation of nonrunners. However, those individuals who
had underlying articular biomechanical abnormalities from
a previously injured joint (and perhaps elite athletes, partic-
ularly women) did appear to be at greater risk for the subse-
quent development of OA. Early studies showed that groups
of long-duration, high-mileage runners and nonrunning
control subjects had a comparable (and low) prevalence of
OA and suggested that recreational running need not lead
inevitably to OA.54,62 These observations have, in general,
now been confirmed by others55-64 (Table 33-6). Eight- and
9-year follow-up observations were encouraging; most of
the original runners were still running, with a prevalence of
degenerative joint disease that was comparable with that of
the control subjects.54,56 Perhaps even more significant is the
growing evidence that running and other aerobic exercise
protect against the development of disability and early mor-
tality.64 Former college varsity long-distance runners were
compared with former college swimmers in another study65;
there was no association between moderate levels of run-
ning or number of years running and the development of
symptomatic OA. Other authors have concluded that run-
ning alone does not cause OA; rather, prior injuries and
anatomic variances are directly responsible for some of the
changes.62 Several additional reports have found that run-
ners are not at risk for the development of premature OA of
the knees or ankles.59,65-67
Studies examining degenerative hip diseases in former
athletes58,68-71 noted that former champion distance run-
ners had no more clinical or radiographic evidence of OA
than did nonrunners.66 However, another study found more
radiographic changes due to degenerative hip disease in for-
mer national team long-distance runners than in bobsled
competitors and control subjects.68 In all the subjects stud-
ied, age and mileage run in 1973 were strong predictors of
radiographic hip OA; for runners, running pace in 1973 was
the strongest predictor of subsequent radiographic hip OA

Table 33-6  Studies of Running and Risk of Developing Osteoarthritis

Mean No. of
References No. of Runners Mean Age (yr) Years Running Miles/Wk Comments
Minor et al (1989)   319 NA NA NA OA noted more frequently in former runners (with
(cited in refs 28-30) underlying anatomic “tilt” abnormality— 
epiphysiolysis) than in nonathletes
Puranen et al58 (1975) 74 56 21 NA Champion distance runners had no more hip OA
than did nonrunners in their sixth decade
De Carvalho   32 NA NA NA X-ray findings of runners’ hips and knees were
& Langfeldt59 (1977) similar to those of control subjects
Marti et al69 (1990) 20 35 13 48 OA occurred in runners with underlying anatomic
(biomechanical) abnormality
Sohn & Micheli65 (1985) 504 57 9-15 18-19 No association between moderate long-distance
running and future development of OA (of hip
and knees)
Panush et al62 (1986) 17 53 12 28 Comparable low prevalence of lower extremity
OA in runners and nonrunners
Lane et al55 (1986) 41 58 9 (5 hr/wk) No differences between runners and control
subjects in cartilage loss, crepitus, joint stability,
or symptoms
Lane et al (1987)   498 59 12 27 No differences between groups in ­conditions
(cited in refs 28-30) thought to predispose to OA and
­musculoskeletal disability
Marti et al68,69   27 42 NA 61 (in refer- More radiographic changes of hip OA in former
(1989, 1990) ence years) Swiss national team long-distance runners
than in bobsledders and control subjects;
few ­runners had clinical symptoms of OA; no
­difference in ankle joints
Konradsen et al66   30 58 40 12-24 No clinical or radiographic differences in hips,
(1990) knees, and ankles between runners and  
nonrunners
Vingard et al70 (1995) 114 50-80 NA NA Unvalidated questionnaire reported threefold
increase of hip arthrosis in former athletes
Kujala et al67 (1994) 342 NA NA NA More former athletes hospitalized with hip OA
than expected
Kujala et al61 (1995) 28 60 32 NA Women soccer players and weightlifters,  
nonrunners were at risk of premature OA
Panush et al54 (1995) 16 13 22 22 8-yr follow-up of original observations made in
1986 still found no differences between runners
and nonrunners
Lane et al56 (1998) 35 60 10-13 23-28 Running did not appear to influence the
­development of radiographic OA (with possible
exception of spur formation in women)
NA, not available; OA, osteoarthritis.
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
in 1988. These authors concluded that high-intensity, high-
mileage running should not be dismissed as a risk factor for
premature OA of the hip. Other reports found that former
top-level soccer players and weightlifters, but not runners,
were at risk for the development of knee OA,61,71 but it was
suggested elsewhere that former athletes seemed to be dis-
proportionately represented in hospital admissions for OA
of hip, knee, or ankle.71 A questionnaire of former elite and
track-and-field athletes noted more hip OA.70 Similarly,
radiographic OA of the hip and knee was reported in women
who were formerly runners and tennis players.71
Cross-sectional studies on the effect of weight-bearing
exercise on the development of OA of the hip, knee, or ankle
and foot must be interpreted with caution, however. The
radiographic scoring methods used by each group of inves-
tigators differ, and their reliability has not been adequately
tested. This information is important when the major end
points in the studies are radiographic features of OA.
PERFORMING ARTS–RELATED
MUSCULOSKELETAL DISORDERS
Musculoskeletal problems are common among perform-
ing artists. Performing artists—particularly musicians and
­dancers—have unique medical and musculoskeletal problems
that deserve special consideration. Injuries that might be triv-
ial to others may be catastrophic to such artists. These inju-
ries are usually associated with overuse—the consequences of
tissues stressed beyond anatomic or normal ­physical limits.
INSTRUMENTALISTS
The frequency of musculoskeletal problems in musicians
rivals the frequency of disability in athletes. For example,
one report found that 82% of orchestral musicians experi-
enced medical problems related to their occupation. Mus-
culoskeletal problems represented the bulk of the difficulties
encountered by musicians.72
The causes of, mechanisms of, and therapies for these
musculoskeletal problems are unclear (Table 33-7). An edi-
torial suggested that overuse, tendinitis, cumulative trauma
disorder, repetitive motion disorder, occupational cervico-
brachial disorder, and regional pain syndrome may be critical
risk factors in the development of joint laxity in musicians.72
Joint laxity declined with age and was associated with gender,
starting earlier in men but persisting in women through their
mid-40s. The presence or absence of hypermobility at certain
sites was associated with musicians’ complaints of associated
symptoms. Hypermobility in musicians might produce advan-
tages or disadvantages, depending on the site of the laxity and
the instrument played.73 Paganini, with his long fingers and
reported hyperextensibility, had a wider ­finger reach on the
violin than his contemporaries, but he may have had a predis-
position to OA because of this. A review of studies found that
66% of professional symphony orchestra members reported
severe musculoskeletal problems. Of interest and seemingly
unexplained was the high frequency of symptoms among
women (68% to 84%); perhaps this is related to their higher
incidence of hypermobility.72
One group of disorders has not been clearly character-
ized clinically, probably because of the overlap of com-
plaints. These include neurologic problems such as carpal
497
tunnel syndrome and ulnar nerve compression syndromes
at the wrist. On occasion, hand and wrist problems are due
to cervical or cervicobrachial radiculopathies. Stress is a
factor in all performance fields and contributes to motor
function problems, such as occupational cramps; dealing
with this problem often requires the best efforts of a team of
­physicians and therapists.72-75
VOCAL ARTISTS
Musculoskeletal problems among singers have not been
addressed extensively. In a report from the Royal Theater in
Copenhagen, the frequency of musculoskeletal problems was
the same in both instrumentalists and opera singers. How-
ever, singers had more hip, knee, and foot joint complaints,
perhaps reflecting the effects of prolonged standing.74
DANCERS
Dance has always been viewed as a demanding art form,
but only recently have the athletic rigors of this discipline
become widely appreciated. Classic ballet ranked first in
activities generating physical and mental stress, followed by
professional football and professional hockey. The dancer
and athlete have much in common, but there are impor-
tant differences in training and performance technique
that influence the nature of their injuries. Other important
sociocultural differences affect their care. Professional danc-
ers (as well as musicians and vocalists) have traditionally
been wary of physicians, and their conviction that physi-
cians know little about dance (and music) is still common.
Injured dancers seeking care have often been told that the
treatment is to stop dancing. Others, seeking assistance with
weight control, have been told to gain weight. Dancers fre-
quently underreport their injuries and seek care from non-
medical therapists.
It is difficult to generalize about dance injuries because
dance is not a monolithic effort. It is a broad-based hierar-
chic endeavor in which thousands of local school-based and
private amateur dance classes supply a much smaller num-
ber of university-based dance programs, which lead finally
to relatively few professional dance companies. This system
of training encompasses many forms of dance that are highly
divergent, ranging from classic ballet to break dancing. For-
tunately, the majority of injuries are from overuse and are
rarely catastrophic, regardless of the dance style or setting.
As with other overuse injuries in sports, they are influenced
by a variety of factors that may be classified as intrinsic, such
as biomechanical and anatomic variations, or extrinsic, such
as those related to occupation or equipment.72
There are few reliable data on the epidemiology of ama-
teur dance injuries, but a study of ballet dancers revealed
a high lifetime incidence of a variety of injuries, including
overuse injuries (reported by 63%), stress fractures (26%),
and major (51%) and minor (48%) problems, throughout
their careers. In a cumulative study of nine major surveys of
dance-related injuries in ballet, modern, jazz, and theatrical
dancers, more than 7000 injuries were classified. Not surpris-
ingly, the majority (60% to 80%) involved the ankle, foot,
or knee. The most common types of overuse injuries include
strains of muscles and tendons and tendinitis (particularly of
the Achilles and flexor hallucis longus tendons).
 498 PANUSH  |  Occupational and Recreational Musculoskeletal Disorders

Table 33-7  Musculoskeletal and Rheumatic Disorders Associated with Overuse in Performing Artists
Instrument Affliction (Common Name) References*
Piano, keyboard Myalgias Hochberg et al (1983), Knishkowy & Lederman (1986)
Tendinitis Hochberg et al (1983), Caldron et al (1986), Knishkowy & Lederman
(1986), Newmark & Hochberg (1987)
Synovitis Hochberg et al (1983), Knishkowy & Lederman (1986)
Contractures Hochberg et al (1983), Knishkowy & Lederman (1986)
Nerve entrapment
Median nerve   Hochberg et al (1983), Knishkowy & Lederman (1986)
(carpal tunnel– pronator syndrome)
Ulnar nerve Hochberg et al (1983), Knishkowy & Lederman (1986)
Brachial plexus Hochberg et al (1983), Knishkowy & Lederman (1986)
Posterior interosseous branch of radial nerve Hochberg et al (1983), Charness et al (1985)
Thoracic outlet syndrome Hochberg et al (1983), Knishkowy & Lederman (1986), Lederman (1987)
Motor palsies Hochberg et al (1983), Schott (1983), Caldron et al (1986), Knishkowy  
& Lederman (1986), Merriman et al (1986), Cohen et al (1987),
Jankovic & Shale (1989)
Osteoarthritis Bard et al (1984)
Strings
Violin, viola Myalgias Fry (1986b), Hiner et al (1987), Bryant (1989)
Tendinitis Fry (1986b), Hiner et al (1987)
Epicondylitis Fry (1986b), Hiner et al (1987)
Cervical spondylosis Fry (1986b), Hiner et al (1987)
Rotator cuff tears Fry (1986b), Newmark & Hochberg (1987)
Thoracic outlet syndrome Roos (1986), Lederman (1986)
Temporomandibular joint syndrome Hirsch et al (1982), Ward (1990), Kovera (1989)
Motor palsies Schott (1983), Knishkowy & Lederman (1986), Hiner et al (1987),
Jankovic & Shale (1989)
Garrod’s pads Bird (1987)
Nerve entrapment
Ulnar Knishkowy & Lederman (1986)
Interosseous Maffulli & Maffulli (1991)
Cello Myalgias Fry (1986b)
Tendinitis Caldron et al (1986), Fry (1986b)
Epicondylitis Fry (1986b)
Low back pain Fry (1986b)
Nerve entrapment Caldron et al (1986), Knishkowy & Lederman (1986)
Motor palsies Schott (1983)
Thoracic outlet syndrome Lederman (1987), Palmer et al (1991)
Bass Low back pain Fry (1986b)
Myalgias Fry (1986b)
Tendinitis Caldron et al (1986), Fry (1986b), Mandell et al (1986)
Motor palsies Caldron et al (1986)
Viola da gamba Saphenous nerve compression (gamba leg) Schwartz & Hodson (1980), Howard (1982)
Harp Tendinitis Caldron et al (1986)
Nerve entrapment Caldron et al (1986)
Woodwinds
Clarinet and   First web space muscle strain Fry (1986b), Newmark & Hochberg (1987)
oboe Tendinitis Dawson (1986), Fry (1986b)
Motor palsies Jankovic & Shale (1989)
Flute Myalgias Fry (1986b)
Spine pain Fry (1986b)
Temporomandibular joint syndrome La France (1985)
Tendinitis Patrone et al (1988)
Nerve entrapment
Digital Cynamon (1981)
Posterior interosseous Charness et al (1985)
Thoracic outlet syndrome Lederman (1987)
Brass
Trumpet,   Motor palsies Turner (1893), Dibbell (1977), Dibbell et al (1979)
cornet Orbicularis oris rupture (Satchmo’s syndrome) Planas (1982, 1988), Planas & Kaye (1982)
English horn de Quervain’s tenosynovitis Studman and Milberg (1982)
French horn Motor palsies James & Cook (1983), Jankovic & Shale (1989)
Saxophone Thoracic outlet syndrome Lederman (1987)
Percussion Osteoarthritis Caldron et al (1986)
Drums Tendinitis Fry (1986b), Caldron et al (1986)
Myalgias Fry (1986b)
Nerve entrapment Makin & Brown (1985)
Cymbals Bicipital tenosynovitis (cymbal player’s shoulder) Huddleston & Pratt (1983)
*As cited in Greer JM, Panush RS: Musculoskeletal problems of performing artists. Baillieres Clin Rheumatol 8:103, 1994.
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
Table 33-7  Musculoskeletal and Rheumatic Disorders Associated with Overuse in Performing Artists—cont’d
Instrument Affliction (Common Name)
Miscellaneous
Guitar Tendinitis
Synovitis
Motor palsies
Congas Pigmenturia
Spoons Tibial stress fracture (spoon player’s tibia)
*As cited in Greer JM, Panush RS: Musculoskeletal problems of performing artists. Baillieres Clin Rheumatol 8:103, 1994.
The distribution of injuries is strongly influenced by the
type and style of dance and the age and sex of the popu-
lation.73,76 For example, ballet dancers in companies whose
choreography emphasizes bravura technique with big jumps
and balances are more likely to develop Achilles tendinitis
than are those in companies that do not. Men are more likely
to have back injuries because of the requisite jumping and
lifting, whereas women who dance on pointe are more prone
to toe, foot, and ankle problems. The surface on which danc-
ers perform is also critical. Touring companies may encoun-
ter nonflexible surfaces, including concrete; this predisposes
dancers to injuries such as shin splints and stress fractures.
Many dance injuries, particularly among nonprofession-
als, stem from bodies that are poorly suited to perform the
many unphysiologic positions and maneuvers demanded by
a variety of dance techniques. For example, in ballet, the
most important physical feature is proper turnout ­(external
rotation) of the hip. The extent of a dancer’s turnout
is determined primarily by anatomic factors as well as by
the influence of training before the age of 10 or 11 years.
In would-be dancers who are older, attempts to “force” a
proper turnout generally result in severe strains to the lower
extremity, particularly the knee and foot. Other anatomic
factors are also important, including somatotype, lack of
flexibility in general, and particularly lack of ankle-instep
flexibility. Occupational factors, including long hours of
practice and rehearsal, pressures to return to work quickly
after an injury, and the “show must go on” mentality, must
also be ­considered in the care of dancers.72
Physicians caring for dancers, particularly ballet danc-
ers at any level, must be aware of the aesthetic pressures for
extreme leanness and the potential consequences. Numerous
studies have documented this leanness as well as the poor
dietary balance and high incidence of disordered eating pat-
terns among dancers.72,77,78 Dancers and other excessively lean
athletes may suffer serious reproductive and skeletal sequelae
as a result of such aberrant dietary practices. The combina-
tion of disordered eating, amenorrhea, and osteoporosis has
been referred to as the female athletic triad and should be
recognized by those caring for athletes, especially dancers.
Unfortunately, the dance world is not lacking in other seri-
ous medical problems, including ­mental illness, drug abuse,
and human immunodeficiency virus (HIV) infection.72
REFERENCES
  1. Buckle PW: Work factors and upper limb disorders. BMJ 315:1360,
1997.
  2. Hadler NM: Repetitive upper-extremity motions in the workplace
are not hazardous. J Hand Surg Am 22:19, 1997.
  3. Yassi A: Work-related musculoskeletal disorders. Curr Opin Rheuma-
tol 12:124-130, 2000.
499
References*
Newmark & Hochberg (1987)
Mortanroth (1978), Bird & Wright (1981)
Mladinich & De Witt (1974), Cohen et al (1987), Jankovic & Shale (1989)
Fenichel (1974), Furie & Penn (1974)
O’Donoghue (1984)
  4. Schouten SAG, de Bie RA, Swaen G: An update on the relationship
between occupational factors and osteoarthritis of the hip and knee.
Curr Opin Rheumatol 14:89-92, 2002.
  5. Mani L, Gerr F: Work-related upper extremity musculoskeletal disor-
ders. Primary Care 27:845-864, 2000.
  6. Colombini D, Occhipinti E, Delleman N, et al: Exposure assessment of
upper limb repetitive movements: A consensus document developed
by the technical committee on musculoskeletal disorders of Interna-
tional Ergonomics Association endorsed by International Commission
on Occupation Health. G Ital Med Lav Ergon 23:129-142, 2000.
  7. Straaton KV, Fine PR, White MB, Maisiak RS: Disability caused by
work-related musculoskeletal disorders. Curr Opin Rheumatol 10:141,
1998.
  8. Cole DC, Hudak PL: Prognosis of nonspecific work-related muscu-
loskeletal disorders of the neck and upper extremity. Am J Ind Med
29:657, 1996.
  9. Millender L, Tromanhauser SG, Gaynot S: A team approach to reduce
disability in work-related disorders. Orthop Clin 27:669, 1996.
10. Hales TR, Bernard BP: Epidemiology of work-related musculoskeletal
disorders. Orthop Clin North Am 27:679, 1996.
11. Malchaire N, Cook N, Vergracht S: Review of the factors associated
with musculoskeletal problems in epidemiologic studies. Arch Occup
Environ Health 74:79-90, 2001.
12. Harrington JM: Occupational medicine and rheumatic diseases. Br J
Rheumatol 36:153, 1997.
13. Hadler NM: Vibration white finger revisited. J Occup Environ Med
41:772, 1998.
14. Viikari-Juntura ERA: The scientific basis for making guidelines
and standards to prevent work-related musculoskeletal disorders.
Ergonomics 40:1097, 1997.
15. Hadler NM: Coping with arm pain in the workplace. Clin Orthop
351:57, 1998.
16. Hadler NM: A keyboard for “Daubert”. J Occup Environ Med 38:469,
1996.
17. Hadler NM: Occupational Musculosketal Disorders, 2nd ed., Philadel-
phia, Lippincott Williams & Wilkins, 1999.
18. Lister GD: Ergonomic disorders [editorial]. J Hand Surg Am 20:353,
1995.
19. Schrader H, Obelieniene D, Bovim G, et al: Natural evolution of late
whiplash syndrome outside the medicolegal context. Lancet 347:1207,
1996.
20. Vender MI, Kasdan ML, Truppa KL: Upper extremity disorders:
A literature review to determine work-relatedness. J Hand Surg Am
20:534, 1995.
21. Reilly PA, Travers R, Littlejohn GO: Epidemiology of soft tissue rheu-
matism: The influence of the law [editorial]. J Rheumatol 18:1448,
1991.
22. Panush RS: Osteoarthritis, regional rheumatic syndromes, fibromyal-
gia. In Sergent JS, LeRoy EC, Meenan RF, et al (eds): Yearbook of
Rheumatology. St. Louis, Mosby, 1994, pp 247-249.
23. Bell DS: “Repetition strain injury”: An iatrogenic epidemic of simu-
lated injury. Med J Austral 151:280, 1989.
24. Davis TR: Do repetitive tasks give rise to musculoskeletal disorders?
Occup Med 49:257-258, 1999.
25. Higgs PE, Edwards D, Martin DS, Weeks PM: Carpal tunnel surgery
outcomes in workers: Effect of workers’ compensation status. J Hand
Surg Am 20:354, 1995.
26. Panush RS: Introduction to Chapter 1: Health sciences, epidemiology,
and economics. In Panush RS, Hadler NM, Hellman D, et al (eds):
Yearbook of Rheumatology. St. Louis, Mosby, 1999.
27. Angell M: Science on Trial: The Clash of Medical Evidence and the
Law in the Breast Implant Case. New York, Norton, 1997.
 500 PANUSH  |  Occupational and Recreational Musculoskeletal Disorders
28. Panush RS, Lane NE: Exercise and the musculoskeletal system.
Baillieres Clin Rheumatol 8:79, 1994.
29. Panush RS: Physical activity, fitness, and osteoarthritis. In
Bouchard C, Shephard RJ, Stephens T (eds): Physical Activity, Fit-
ness, and Health: International Proceedings and Consensus State-
ment. Champaign, Ill, Human Kinetics, 1994, pp 712-723.
30. Panush RS: Does exercise cause arthritis? Long-term consequences of
exercise on the musculoskeletal system. Rheum Dis Clin North Am
16:827, 1990.
31. Callahan LF, Currey SS, Jonas Bl, et al: Osteoarthritis in retired
National Football League (NFL) players: The role of injuries and play-
ing position [abstract]. Arthritis Rheum 46:S415, 2002.
32. Kellgren JH, Lawrence JS: Radiological assessment of osteoarthrosis.
Ann Rheum Dis 16:494, 1957.
33. Lawrence JS: Rheumatism in coal miners. III. Occupational factors. Br
J Ind Med 1955; 12:249.
34. Kellgren JH, Lawrence JS: Osteoarthritis and disc degeneration in an
urban population. Ann Rheum Dis 12:5, 1958.
35. Burke MJ, Fear EC, Wright V: Bone and joint changes in pneumatic
drillers. Ann Rheum Dis 36:276, 1977.
36. Lawrence JS: Rheumatism in cotton operatives. Br J Ind Med 18:270,
1961.
37. Tempelaar HHG, Van Breeman J: Rheumatism and occupation. Acta
Rheumatol 4:36, 1932.
38. Hadler NM: Industrial rheumatology: Clinical investigations into the
influence of the pattern of usage on the pattern of regional musculo­
skeletal disease. Arthritis Rheum 21:1019, 1977.
39. Hadler NM, Gillings DB, Imbus HR: Hand structure and function in
an industrial setting: The influence of the three patterns of stereo-
typed, repetitive usage. Arthritis Rheum 21:210, 1978.
40. Anderson J, Felson DT: Factors associated with knee osteoarthritis
(OA) in the HANES I survey, evidence for an association with over-
weight, race and physical demands of work. Am J Epidemiol 128:179,
1988.
41. Felson DT, Hannan MTP, Naimark A, et al: Occupational physical
demands, knee bending and knee osteoarthritis. J Rheumatol 18:1587,
1991.
42. Felson DT, Zhang Y, Hannan MT, et al: Risk factors for incident
radiographic knee osteoarthritis in the elderly: The Framingham
Study. Arthritis Rheum 40:728, 1997.
43. Felson DT, Zhang Y: An update on the epidemiology of knee and
hip osteoarthritis with a view to prevention. Arthritis Rheum
41:1343, 1998.
44. Molleson T: The eloquent bones of Abu Hureyra. Sci Am 271:70,
1994.
45. Sutton AJ, Muir KR, Mocket S, et al: A care-control study to investi-
gate the relation between low and moderate levels of physical activity
and osteoarthritis of the knee using data collected as part of the allied
Dunbar national fitness survey. Ann Rheum Dis 60:756-764, 2001.
46. Manninen P, Riihimaki H, Heliovaara M, et al: Physical exercise and
risk of severe knee osteoarthritis requiring arthroplasty. Rheumatology
40:432-437, 2001.
47. Walsh SJ, DeChello LM: Excess autoimmune disease mortality among
school teachers. J Rheumatol 28:1537-1545, 2001.
48. Cooper GS, Parks CG, Dooley MA, et al: Occupational exposures
and risk of systemic lupus erythematosus [abstract]. Arthritis Rheum
46:S616, 2002.
49. Panush RS, Levine ML, Reichlin M: Do I need an ANA? Some
thoughts about man’s best friend and the transmissibility of lupus.
J Rheumatol 27:287-291, 2000.
50. Morse LH: Unusual occupational rheumatologic and musculoskeletal
disorders. Occup Med 7:423, 1992.
51. McDermott M, Freyne P: Osteoarthrosis in runners with knee pain.
Br J Sports Med 17:84, 1983.
52. Videman T: The effect of running on the osteoarthritic joint: An
experimental matched-pair study with rabbits. Rheumatol Rehabil
21:1, 1982.
53. Radin EL, Evre D, Schiller AL: Effect of prolonged walking on con-
crete on the joints of sheep [abstract]. Arthritis Rheum 22:649, 1979.
54. Panush RS, Hanson CS, Caldwell JR, et al: Is running associated with
osteoarthritis? An eight-year follow-up study. J Clin Rheum 1:35,
1995.
55. Lane NE, Bloch DA, Jones HH, et al: Long-distance running, bone
density and osteoarthritis. JAMA 255:1147, 1986.
56. Lane NE, Oehlert JW, Bloch DA, Fries JF: The relationship of run-
ning to osteoarthritis of the knee and hip and bone mineral density of
the spine: 9 year longitudinal study. J Rheumatol 25:334, 1998.
57. Murry RO, Duncan C: Athletic activity in adolescence as an etiological
factor in degenerative hip disease. J Bone Joint Surg Br 53:405, 1971.
58. Puranen J, Ala-Ketola L, Peltokalleo P, Saarela J: Running and pri-
mary osteoarthritis of the hip. BMJ 1:424, 1975.
59. De Carvalho A, Langfeldt B: [Running practice and arthrosis defor-
mans: A radiological assessment]. Ugeskr Laeger 139:2421, 1977.
60. Arokoski J, Kivirantal I, Jirvelin J, et al: Long-distance running causes
site-dependent decrease of cartilage glycosaminoglycan content in the
knee joints of beagle dogs. Arthritis Rheum 36:1451, 1993.
61. Kujala UM, Kettunen J, Paananen H, et al: Knee osteoarthritis in
former runners, soccer players, weight lifters, and shooters. Arthritis
Rheum 38:539, 1995.
62. Panush RS, Schmidt C, Caldwell J, et al: Is running associated with
degenerative joint disease? JAMA 255:1152, 1986.
63. Wang WE, Ramey DR, Schettler JD, et al: Postponed development of
disability in elderly runners: A 13-year longitudinal study. Arch Intern
Med 162:2285-2294, 2002.
64. Lane NE, Hochberg MC, Pressman A, et al: Recreational physical
activity and the risk of osteoarthritis of the hip in elderly women.
J Rheumatol 26:849-854, 1999.
65. Sohn RS, Micheli LJ: The effect of running on the pathogenesis of
osteoarthritis of the hips and knees. Clin Orthop Rel Res 198:106,
1985.
66. Konradesen L, Hansen EM, Sondegaard L: Long distance running and
osteoarthritis. Am J Sports Med 18:379, 1990.
67. Kujala UM, Kapriio J, Samo S: Osteoarthritis of weight-bearing joints
in former elite male athletes. BMJ 308:231, 1994.
68. Marti B, Knobloch M, Tschopp A, et al: Is excessive running predic-
tive of degenerative hip disease? Controlled study of former elite ath-
letes. BMJ 229:91, 1989.
69. Marti B, Biedert R, Howald H: Risk of arthrosis of the upper ankle
joint in long distance runners: Controlled follow-up of former elite
athletes. Sportverletz Sportschaden 4:175, 1990.
70. Vingard E, Sandmark H, Alfredsson L: Musculoskeletal disorders in
former athletes. Orthop Scand 66:289, 1995.
71. Specter TD, Harris PA, Hart DJ, et al: Risk of osteoarthritis associated
with long-term weight-bearing sports. Arthritis Rheum 39:988, 1996.
72. Baum J, Calabrese LH, Greer JM, Panush RS: Performing arts
rheumatology. Bull Rheum Dis 44:5, 1995.
73. Larsson L-G, Baum J, Mudholkar GS, Kollia GD: Benefits and dis-
advantages of joint hypermobility among musicians. N Engl J Med
329:1079, 1993.
74. Greer JM, Panush RS: Musculoskeletal problems of performing artists.
Baillieres Clin Rheumatol 8:103, 1994.
75. Hoppman RA: Instrumental musicians’ hazards. Occup Med 16:
619-631, 2001.
76. Zaza C: Playing-related musculoskeletal disorders in musicians: A sys-
tematic review of incidence and prevalence. CMAJ 158:1019, 1998.
77. Hoppmann RA, Reid RR: Musculoskeletal problems of performing
artists. Curr Opin Rheumatol 7:147, 1995.
78. Lockwood AH: Medical problems of musicians. N Engl J Med 320:221,
1989.
 34
KEY POINTS
Complementary and alternative medicine (CAM) is used in
some form by most rheumatology patients.
Most patients do not discuss CAM use or preferences unless
asked specifically about this by a clinician.
CAM use is strongly linked to health beliefs and is most com-
monly used in conjunction with conventional care.
Asking about patients’ current or potential use of CAM is
important for the following reasons:
Avoiding harmful or unproven therapies
Coordinating therapies that have some evidence for benefit
(e.g., acupuncture for knee osteoarthritis)
Reviewing therapies that have equivocal evidence, but
whose safety profile makes them worthy of a symptom-
atic trial
Defined in various ways, complementary and alternative
medicine (CAM) has become an increasingly popular
avenue of treatment and out-of-pocket expenditure for
patients with rheumatologic conditions. Because many of
these treatments traditionally have not been prescribed
or recommended by a health care provider, there is often
a lack of discussion and evidence-based coordination of
care. This chapter provides an evidence-based overview
of common treatments and insight into the prevalence,
rationale, and approach to a patient who is considering
CAM.
DEFINITION AND BACKGROUND
The definition of CAM has evolved more recently. Pre-
viously, CAM was defined as therapies not taught as part
of medical school training. This definition is no longer
relevant because more than 60% of allopathic medical
schools in the United States were providing some level of
instruction in CAM as of 1998.1 More recently, CAM has
been defined by the National Institutes of Health Center
for Complementary and Alternative Medicine (NCCAM)
as therapies or practices that are not typically part of the
conventional treatment paradigm. This definition is likely
to continue to evolve as clinicians incorporate selected
evidence-based CAM options into mainstream care in a
manner known as integrative medicine.2 The NCCAM
definition attempts to classify CAM into categories that
are helpful for discussion. These categories and examples
are listed in Table 34-1.
Integrative Medicine
in Rheumatology:
An Evidence-Based
Approach
ROBERT ALAN BONAKDAR  • 
DAVID C. ­LEOPOLD
PREVALENCE AND PREDICTORS OF USE
CAM generally has become extremely popular over the last
several decades. Total CAM usage was reported in one third
of the population in 1990 and increased to 42% by 1997.
This represented 628 million office visits and $27 million
spent, which far exceeded out-of-pocket expenditure for all
conventional care and the 328 million visits to primary care
providers in the same year. A follow-up analysis showed that
approximately one third of the total visits (628 million) to
CAM providers were for the treatment of musculoskeletal
pain.3 Most of the top 10 reasons for CAM usage in one
survey were related to musculoskeletal issues.4
Numerous surveys have examined CAM usage specifically
in a rheumatologic population.3-14 Prevalence of use varies
widely from approximately one third of those surveyed to
greater than 90% in some populations. The prevalence rate
also is highly influenced by the definition of CAM in vari-
ous surveys, which can include common practices such as
prayer that are not included in other surveys. Overall, most
surveys find a greater than 50% prevalence of CAM usage
by individuals with musculoskeletal problems.
Several condition-specific surveys have been completed
in the areas of osteoarthritis, rheumatoid arthritis (RA), sys-
temic lupus erythematosus, and fibromyalgia.15-18 Fibromy-
algia patients show the highest level of use (93%), and more
than two thirds of fibromyalgia patients have used multiple
CAM modalities concomitantly.
EPIDEMIOLOGY
The most common CAM modalities used by rheumato-
logic patients vary, although common choices found in
nearly all surveys include dietary supplements, mind/body
and spiritual practices, manual/manipulative therapies,
biostimulation (acupuncture, magnets), and topical oint-
ments. Several factors other than rheumatologic diagnoses
predict CAM use. Typical CAM users are more likely to be
women with a higher level of education and income, greater
severity and duration of symptoms, and greater comorbidi-
ties such as insomnia and functional impairment. African-
­Americans tend to have higher levels of spiritual practice
than other groups, and ethnic groups with traditional medi-
cine use, such as Hispanic and Asian populations, tend to
have higher levels of dietary supplement use.
The health values of individuals considering CAM
also are quite important. Individuals who are involved in
“active coping behaviors,” such as greater physical activ-
ity, tend to view CAM use in a similar manner.5 Several
surveys also show that individuals with a more “holistic
501
 502 bonakdar  |  Integrative Medicine in Rheumatology: An Evidence-Based Approach

Table 34-1  Categories of Complementary   Table 34-2  Regulation Resources for Complementary
and ­Alternative Medicine and Alternative Medicine
Category Overview Examples Organization/Agency Website
Alternative medical systems: Traditional Chinese medicine, Federation of State Medical http://www.fsmb.org/m_pub.
­Systems of care based on ayurveda, naturopathy, Boards html
­unifying health paradigms that ­homeopathy American Academy of Medical http://www.medicalacupuncture.
may incorporate ­individual Acupuncture org
treatments, including those
noted in the following National Certification   http://www.nccaom.org
­categories Commission for Acupuncture
and Oriental Medicine
Mind-body interventions: Diverse Biofeedback, meditation, yoga,
techniques that use ­cognitive, tai chi/qi gong, creative American Chiropractic   http://www.acatoday.org
behavioral, and movement therapies (art, music, or ­Association
therapies to modify and dance), ­relaxation, ­hypnosis, National Certification Board for http://www.ncbtmb.com
increase awareness between ­visualization/imagery, Therapeutic Massage and
mental and physiologic ­cognitive-behavioral therapies, Bodywork
­functioning group support, autogenic
­training, spirituality
Biologically based therapies: Dietary modification (dietary
Therapies that modify nutrient elimination, fasting, or medical board, department of consumer ­ affairs/­protection,
intake either through dietary specific dietary regimens), or therapy-specific national certification body (see resources
intervention or through dietary supplements (herbal listed in Table 34-2).
­supplementation supplements—white willow Dietary supplements are governed according to the
bark; nonherbal supplements—
­glucosamine, vitamins [vitamin
Dietary Supplement Health and Education Act of 1994.
D], minerals [selenium]) In contrast to prescription medications that must proceed
Manipulative and body-based Chiropractic manipulation,
through multiphase trials to gain premarketing approval
methods: Techniques that use ­osteopathic manipulation, from the Food and Drug Administration (FDA), supple-
manipulation, movement, or manual and massage therapy ments (with established ingredients) are not required to
stretching of one or more parts have safety, efficacy, or bioavailability data before market-
of the body ing. Ensuring these important qualities, along with having
Energy therapies (biofield thera- Acupuncture, qi gong, healing a clear and truthful label, is mainly the responsibility of the
pies): Techniques that involve touch, therapeutic touch manufacturer. Because of many factors, the role of the FDA
the application of human or
nonhuman energy fields has often begun after a supplement is marketed as it moni-
tors safety and label claims. The FDA must use adverse drug
reports and product analysis to prove that a supplement
outlook” wish to use complementary methods, which may poses significant health risks.8,9 Two more recent regulatory
take this viewpoint into consideration.6 Although there measures are expected to be helpful in this area. The Dietary
has been speculation that CAM use is a sign of dissatis- Supplement and Non-Prescription Drug Consumer Protec-
faction with conventional care, the more prevalent view tion Act (S. 3546) mandates the reporting of serious adverse
is that CAM is used as an adjunct to optimize care. Dis- events to the FDA beginning in December 2007 and should
satisfaction with conventional care did not predict use of allow for improved monitoring and enforcement. In addi-
CAM in a previous national survey, and less than 5% of tion, the FDA published long awaited guidelines for good
CAM users used CAM in isolation from conventional care. manufacturing practices for dietary supplements, which
Most CAM users state that their motivations for CAM use began in August 2007 and help to standardize the manufac-
include that it gives them more control over their health turing process.10
care, and 80% report substantial benefit from its use.5 As In addition to governmental oversight, several indepen-
a corollary, CAM users have been noted to have more fre- dent agencies offer testing and monitoring services that
quent relationships with a primary care physician, have allow manufacturers to demonstrate their adherence to
regular physician follow-up, and have good compliance regulatory standards. Manufacturers that pass inspection
with recommended preventive health behaviors such as may carry an independent “seal of approval” on their label
regular mammography.7 and advertising. Table 34-3 lists several government and
independent agencies currently involved in testing. Clini-
REGULATION cians should become familiar with well-regulated and well-
researched brands for the supplements that are likely to be
The regulation of CAM varies with the type of modality, discussed with patients.
the training of practitioners, and state laws. Acupuncture
done by a Licensed Acupuncturist or physician acupunc-
turist in the same state is regulated differently by the state
APPROACH TO THE PATIENT
board of oriental medicine, medical board, or department of Regardless of their personal beliefs about CAM, clinicians
consumer affairs. The level of training and oversight for acu- have an ethical obligation to discuss treatment alternatives
puncture varies widely by state, and the referring clinician with their patients. The interaction between patients and
should guide patients in confirming the CAM practitioner’s clinicians regarding CAM use typically creates a less than
credentials and certification whenever possible. Verification optimal environment for coordination of care. This situation
of licensure can be obtained by initially contacting the state’s is linked to several factors, which are reviewed subsequently,
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
Table 34-3  Governmental and Independent   Table 34-4  Selected Complementary and Alternative
Regulatory Agencies
Agency Website
Governmental
Food and Drug Administration www.fda.gov/medwatch
(FDA) Medwatch Program for
collecting adverse reactions to
prescription and over-  alternative and complementary medicine
the-counter medications and
dietary supplements
Federal Trade Commission (FTC) www.ftc.gov/ftc/complaint.htm
site for submitting ­complaints
on false or misleading
­advertising
American Association of Poison www.poison.org or 800-222-1222
Control Centers for reporting
and management of adverse
effects
Independent Laboratories Providing Supplement Testing
Consumerlab Product Review www.Consumerlab.com
Dietary Supplement Verification www.uspverified.org
Program (DSVP) through the
United States Pharmacopeia
(USP)
National Sanitation Foundation www.NSF.ORG/consumer/­
(NSF) dietary_supplements
including patient and clinician CAM knowledge base, level
of CAM discussion, and management strategies such as
charting and follow-up.
KNOWLEDGE BASE
The knowledge base of the average clinician and consumer
regarding CAM is suboptimal to poor. The clearest example
is found in the dietary supplement literature. Physician sur-
veys have found that physicians may have an insufficient
general understanding of commonly used supplements and
their safety and interaction profiles.11,12 Similarly, consum-
ers and patients tend to have misconceptions regarding
product claims and efficacy. As pointed out by a previous
Harris Poll, most consumers believed that the government
ensured a higher level of safety and regulation than actu-
ally exists.13 Inaccurate or biased information may create a
scenario of decreased perceived need for clinician guidance
regarding CAM.
One of the other key deficiencies in the CAM scenario
is the level of clinician/patient discussion. An initial survey
found that in approximately 70% of encounters, there was
no discussion of CAM use.2 More recently, the percentage
seems to be improving, but still leaves many encounters in
which neither the patient nor the clinician introduces the
topic.14 More concerning is the fact that if a patient is hos-
pitalized by a specialist, CAM use is not identified 88% of
the time.15
It is important to understand why patients do not discuss
CAM use. Surveys indicate that factors including antici-
pation of a negative or disinterested clinician response
and belief that the clinician would not provide useful
information motivated nondisclosure.16 Most important
may be clinician inquiry, however, because patients show
a willingness to disclose use of supplements, but only if
503
Medicine (CAM) Resources
Internet
National Center for Complementary and Alternative Medicine (NCCAM)
(http://nccam.nih.gov): Overview of CAM fields and information on
clinical trials and patient education materials
CAM on PubMed (www.nlm.nih.gov/medlineplus/alternativemedicine.
html): Focused search on PubMed for articles with a focus on
Cochrane Library (www.cochrane.org): Collection of systematic
reviews with a Complementary Medicine Field.
NIH Office of Dietary Supplements and International Bibliographic
Information on Dietary Supplements (IBIDS) (http://ods.od.nih.gov):
Collaborative database of available abstracts on dietary supple-
ments
FDA Division of Dietary Supplement (http://vm:cfsan.fda.gov/~dms/
supplmnt/html): Discussion of dietary supplement regulation,
including list of recalls and warnings
Natural Medicines Comprehensive Database (www.naturaldatabase.
com): Extensive collection of supplement information, including
uses, indication, efficacy, and adverse effects with a drug interac-
tions checker
Longwood Herbal Taskforce (www.mcp.edu/herbal): Collection of
clinically oriented monographs and reviewed Internet link
Peer-Reviewed Information for Patients
MedlinePlus (www.nlm.nih.gov/medlineplus/alternativemedicine.
html): Patient-oriented summary of Medline published research
and international health news
National Center for Complementary and Alternative Medicine (NCCAM)
(www.nccam.nih.gov): National Institutes of Health clearinghouse
of government-funded initiatives in CAM, including research, fel-
lowships, grants, and patient education materials
Natural Medicine Comprehensive Database patient handouts (www.
naturaldatabase.com)
asked by a clinician directly.17 A survey of physicians
found that few of those surveyed felt comfortable discuss-
ing CAM with their patients. One of the major reasons for
this lack of comfort was related to a need to improve one’s
knowledge base regarding CAM (84% of responders). It is
hypothesized that with improved education about CAM,
physicians may be more willing to discuss and counsel
patients.18
PATIENT AND CLINICIAN EDUCATION
Numerous resources are available to clinicians interested
in understanding CAM better as a means of increasing and
improving patient communication. These resources are
listed in Table 34-4 and include print and online informa-
tion on evidence-based use of CAM and continuing medi-
cal education courses available to clinicians. The HERBAL
mnemonic is an additional practice tool for alerting clini-
cians to the most important steps involved in managing
CAM use (Table 34-5).
NUTRITION AND DIETARY SUPPLEMENTS
DIET
Alterations in the “standard American diet” can result in
a significant decrease of inflammatory burden and may be
­especially significant for individuals with inflammation-
driven diseases. Dietary intake of high-quality food has
 504 bonakdar  |  Integrative Medicine in Rheumatology: An Evidence-Based Approach
Table 34-5  HERBAL Mnemonic*
H Hear the patient out with respect
E Educate the patient
R Record
B Be aware of potential interactions and side effects
A Agree to discuss and follow up
L Learn
*Copyright Robert Alan Bonakdar. Used with permission.
degenerated for most Western cultures over the past 100
years. Americans are eating an increased amount of pro-
cessed animal products and foods, often high in trans fatty
acids, omega-6 free fatty acids (O6FFAs), food additives,
preservatives, and high-glycemic-index foods. These factors
directly or indirectly stimulate inflammation and generally
result in increased levels of arachidonic acid, a key compo-
nent of many inflammatory mediators.19 Changes in omega-3
free fatty acid (O3FFA) and O6FFA ratios have resulted in
a shift to a “proinflammatory” physiologic state (estimates
place the ratio in the 1900s at 4:1, and now at 25:1 to 30:1).
In many native cultures, a severalfold increase in the ratio of
O6FFAs to O3FFAs has been observed as the dietary pattern
shifts away from traditional diets to a more Western diet.20
In our current medical paradigm, we have multiple ways to
deal with inflammation; most involve blockade of mediators
when they are formed. An anti-inflammatory diet decreases
stimulation of inflammatory pathways, provides block-
ade of mediators owing to high natural anti-­inflammatory
­components, provides novel ways of downregulating inflam-
matory mediator–producing cells, and provides “upstream”
blockade of inflammatory pathways.
An anti-inflammatory diet is a predominantly plant-
based diet, with proper O6FFA-to-O3FFA ratios, and high
in naturally occurring anti-inflammatories and antioxidants.
In addition, there is reduced or minimal intake of “proin-
flammatory” components of diet, such as trans and saturated
fatty acids, high glycemic foods, and foods causing known
allergenic or sensitivity reactions.
The essential fatty acids comprise a group of lipids that
must be acquired from exogenous sources, most often plants
or animals that have ingested plants rich in O3FFA (e.g.,
grasses, algae). Saturated fats include most animal products
and coconut and palm oils. Monounsaturated fats include
olive, avocado, and canola oils. Polyunsaturated fats include
the O6FFAs (linoleic acid, gamma linolenic acid, and ara-
chidonic acid), and the O3FFAs (alpha-linolenic acid,
eicosapentaenoic acid, docosahexaenoic acid).
Polyunsaturated fats have important effects on the pros-
taglandin pathways. The main essential fatty acids are lin-
oleic acid (O6FFA) and linolenic acid (O3FFA). Linoleic
acid can be elongated and desaturated to arachidonic acid,
whereas alpha-linolenic acid is elongated and desaturated
into eicosapentaenoic acid and docosahexaenoic acid. Eico-
sanoids derived from arachidonic acid are generally proin-
flammatory and prothrombotic, whereas O3FFAs tend to
be less inflammatory and inhibit platelet aggregation. The
Standard American diet provides a ratio of approximately
25:1 O6FFA to O3FFA, whereas an anti-inflammatory ratio
is considered to be approximately 4:1. There is evidence
that the monounsaturated fats also affect these pathways,
although not nearly as extensively. The anti-inflammatory
effects from olive oil are believed to be more directly related
to the oleanic acid components.21,22
The proposed mechanism of an anti-inflammatory diet is
multifactorial and is typically described as involving inhibi-
tion of cyclooxygenase (COX) and leukotriene enzymatic
pathways and inhibition of phospholipase A2. Bioactive
substances also may be found in an anti-inflammatory diet
that directly mediate promotion and inhibition of genetic
transcription and the encoding of proteins and enzymes.
These include peroxisome proliferator activated receptor
gamma and other mediators and are exemplified by the fol-
lowing substances:23,24
• Isoflavones found in soy inhibit tumor necrosis factor
(TNF)-α activation of nuclear factor κB in lympho-
cytes of healthy males.25
•  Epigallocatechin-3-gallate, found in colorful berries
and teas, inhibits interleukin (IL)-1 in chondrocytes
and inhibits COX-2 activity.26,27
•  Curcumin, found in turmeric, inhibits monocyte and
alveolar macrophage production of IL-8, monocyte
inflammatory protein-1, α-monocyte chemotactic
protein-1, and TNF-α.28
•  Ursolic acid, a pentacyclic triterpene found in apples,
Greek sage (Salvia triloba), oleander, rosemary, laven-
der, and thyme, is a moderate inhibitor of COX-2 and
lipoxygenase.
•  Oleanic acid, another pentacyclic triterpene found in
olives and olive leaves, seems to inhibit phospholipase
A2.
Evidence for the Anti-Inflammatory Diet
Several lines of evidence support the incorporation of an
anti-inflammatory diet to improve serologic and symptom-
atic markers of inflammation. The anti-inflammatory diet
can be characterized by the avoidance of proinflammatory
constituents, incorporation of whole dietary patterns (Med-
iterranean and vegetarian diets), and incorporation of spe-
cific anti-inflammatory constituents (e.g., fiber and essential
fatty acids).
Avoidance. A hallmark of the anti-inflammatory diet in-
cludes avoidance of excess and high-glycemic-load calories.
A study of 244 women identified a significant associa-
tion between high glycemic loads and elevated plasma
C-­reactive protein. These findings were found to be in-
dependent of heart disease risks.29 Inversely, RA patients
completing a week-long modified fast showed anti-inflam-
matory-related serologic changes, including decreases in
neutrophil release of lysozyme, aggregation of polymorpho-
nuclear neutrophils, and production of leukotriene B4.30
Mediterranean Diet. The Mediterranean diet is typically
characterized as a diet emphasizing high consumption of
fruits, vegetables, and cereals with incorporation of virgin
olive oil, fish, nuts, and small-to-moderate quantities of
wine. Several studies have shown that subjects maintain-
ing this diet, while adjusting for body mass index, age, dia-
betes, smoking, and ­ nonsteroidal anti-inflammatory drugs
(NSAIDs) are able to reduce markers of inflammation,
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
including C-reactive protein and IL-6.31,32 Studies of RA
patients randomly assigned to a Mediterranean diet versus
a standard Western diet for 3 months showed that patients
on a Mediterranean diet had improved symptoms, including
decreased joint swelling and functional ability.33,34
Vegetarian Diet. A vegetarian/vegan diet has been used in
several controlled fibromyalgia and RA trials. A 3-month,
randomized Finnish study of fibromyalgia patients found
that patients placed on a vegan diet showed improve-
ments in pain scores, joint stiffness, and sleep compared
with controls.35 RA patients followed on a vegan diet for
2 years improved on all assessments for pain and function-
ality, ­ including morning stiffness, number of swollen and
tender joints, and grip strength.36 Similarly, these dietary
interventions have been correlated with changes noted in
IgM, rheumatoid factor, and the complement components
C3 and C4.37 Finally, a systematic review of studies of RA
patients undergoing fasting followed by vegan diet of at least
3 months’ duration showed statistical benefit in long-term
management of symptoms.38
Fiber. An inverse relationship between fiber intake and
levels of C-reactive protein and incidence of inflammato-
ry conditions has been noted. In the 1999-2000 National
Health and Nutrition Examination Survey, subjects with
the highest fiber intakes had lower C-reactive protein levels,
and this has been shown in subjects following a high-fiber,
vegetarian diet.39,40 A randomized study incorporating 30
g/day of fiber a day, through either diet or supplementation,
was found to decrease C-reactive protein values significant-
ly.41 Additionally, epidemiologic studies have identified an
inverse relationship between high fiber–containing dietary
components (cooked vegetables, cruciferous vegetables, and
fruits) and RA risk.42
Essential Fatty Acids. The use of essential fatty acids for
the treatment of pain is a broad and difficult-to-summa-
rize topic. The variation in research stems from factors in-
cluding the source (e.g., diet or supplements of fish, flax,
borage, or other oils), the concentration of constituents
(i.e., eicosapentaenoic acid, docosahexaenoic acid, gamma
linolenic acid), and background diet. Several trials have
attempted to ascertain the dietary and supplement intake
levels necessary for clinical efficacy. Subjects with RA who
used 1.8 g/day of O3FFA showed long-term improvement
in disease parameters.43 In a another study of RA, 2.6 g/
day of O3FFA was required to decrease symptoms and pain
medication use, which was maintained at 12 months.44
Fifty-one patients with RA were randomly assigned to 3.6
mg of omega-3 fish oils versus capsules containing stan-
dard dietary fats. After 12 weeks, the patients on fish oil
were noted to have small but significant improvements in
morning stiffness, joint tenderness, and C-reactive protein
values.45 A multicenter study using the same protocol as
the previous study found similar findings in morning stiff-
ness and joint tenderness.46 Similar modest but clinically
significant improvements were shown in a double-blind,
placebo-controlled trial of 56 RA patients with use of 2.8
g/day of gamma linolenic acid.47 A survey of the literature
concluded that in RA patients, doses of 2 to 8 g/day of
gamma linolenic acid are required for 12 weeks to exhibit
505
an anti-inflammatory effect that compared with therapy
with NSAIDs.48
Preliminary research using essential fatty acids has been
done in other rheumatologic conditions. A study from the
University of Pittsburgh, Department of Neurosurgery,
looked at 250 patients with nonsurgical back or neck pain.49
The patients used 1200 to 2400 mg of O3FFA for an aver-
age of 75 days; 59% of them reported improvement in joint
pain, and 68% were able to discontinue prescription pain
medications. Side effects were minimal, and 86% of patients
noted they would continue to use the product. In a system-
atic review of 17 trials that examined the use of O3FFAs for
an average of 3 to 4 months in inflammatory states, there
was a significant improvement in patient-reported joint
pain, minutes of morning stiffness, number of painful or
tender joints, and NSAID consumption.50
Avoidance in Combination with Anti-inflammatory Diet.
The effects of background diet have been examined as a fac-
tor affecting the potential benefit of essential fatty acid sup-
plementation. Sixty-eight patients with RA were randomly
assigned to 2 months of a standard Western diet or an anti-
inflammatory diet in which arachidonic acid intake was less
than 90 mg/day. Patients in both groups were additionally
randomly assigned to receive placebo or fish oil supplements
at 30 mg/kg for a third month. With placebo supplementa-
tion, the anti-inflammatory diet decreased swollen joints by
14% compared with the Western diet. With the addition of
fish oil, a significant difference in tender (28% versus 11%)
and swollen (34% versus 22%) joints was noted (P < .01).
Serologic studies also showed that an anti-inflammatory diet,
especially with the combination of fish oils, caused signifi-
cant decreases in leukotriene B4, 11-dehydrothromboxane
B2, and prostaglandin metabolites. The authors concluded
that an anti-inflammatory diet improved symptoms of RA
and augmented the effects of fish oil supplementation.51
Although essential fatty acids have documented changes
with in vitro and in vivo inflammatory markers and may
have a role in clinical management in certain scenarios,
there are several important notes regarding use. There have
been many nonsignificant trials with use of essential fatty
acids, including the use of alpha linoleic acid from flax seeds
as a precursor of eicosapentaenoic acid and docosahexae-
noic acid to treat RA.52 Side effects with use of essential oils
are typically dose dependent and include gastrointestinal
intolerance. The potential for symptom decrease or drug-
sparing benefit seems to be strongly related to the type and
dose of the treatment and the disease state and background
diet of the subjects studied. At this point, the strongest evi-
dence appears in the literature for individuals consuming
2 g or greater of gamma linolenic acid or fish oil supplement
per day, while limiting their intake of arachidonic acid (<90
mg/day) or (n-6) fatty acid intake (<10 g/day).48,53
Obesity and Inflammation
It is now generally accepted that obesity itself has pro-
inflammatory effects. Adipocytes, in particular central
adipose tissue, are endocrinologically active and can
sequester arachidonic acid. This effect eventually leads
to increased production of cytokines, including IL-6 and
TNF, and of proinflammatory adhesion. Although an
 506 bonakdar  |  Integrative Medicine in Rheumatology: An Evidence-Based Approach
anti-inflammatory diet is no guarantee of weight loss, the
decrease in animal fats, the general increase in nutrient-
dense food, and subsequent avoidance of “empty calories”
(e.g., fast foods, high-fat foods, and fried foods) could
logically be expected to result in gradual and permanent
weight loss.
Anti-Inflammatory Diet Recommendations
In recommending an anti-inflammatory diet, several impor-
tant points should be kept in mind. First, initiation of the
diet, similar to other lifestyle change, should be gradual to
allow better tolerability and compliance. Second, for goal-
setting purposes, the patient should not expect immedi-
ate results, allowing at least 12 weeks before re-evaluating
benefit, and should be made aware that this intervention
may have additional nonrheumatologic health benefits in
the setting of cardiovascular diseases, diabetes, and cancer
risk. Third, what is avoided is often as important as what is
added. There should be a strong emphasis on avoidance of
potentially proinflammatory foods while initiating an anti-
inflammatory diet (Table 34-6). Next, consultation with a
dietitian to analyze current dietary patterns and understand-
ing and to provide meal planning suggestions is highly rec-
ommended. Also, the patient should be told that a journal
can be helpful in this process to monitor ongoing diet and
symptom changes.
DIETARY SUPPLEMENTS
Willow Bark (Salix species)
White willow bark has a long history of use in medicine with
records describing Hippocrates recommending its use for pain
relief. In 1898, aspirin (acetylsalicylic acid), as a synthetic
derivative of white willow bark, was discovered and became
a leading analgesic. The mechanism of benefit of white wil-
low bark does not seem to be strictly based on salicylate lev-
els, which are low compared with aspirin products, and may
be linked to other active ingredients, including glycosides,
phenolic glucosides, flavanoids, polyphenols, procyanidins,
and tannins.54 In vitro, white willow bark has shown COX-
2, prostaglandin E2, and leukotriene inhibitory activity.
Clinically, white willow bark has been tested for efficacy
in arthritis and low back pain. A randomized double-blind,
controlled trial of 78 subjects with hip or knee ­osteoarthritis
treated with placebo or white willow bark containing 240 mg
of salicin over a 2-week period showed a statistically
­significant improvement in WOMAC pain scores in the
white willow bark group.55 Similar results were exhibited
in a smaller randomized double-blind, placebo-controlled
trial of 21 subjects taking white willow bark containing
240 mg of salicin over a 2-week period for knee and hip
­osteoarthritis. Results of the study showed a 40% decrease
in WOMAC scores with white willow bark versus 18%
with placebo.56
In the treatment of acute exacerbation of low back pain,
119 subjects were randomly assigned to white willow bark
standardized to 120 mg or 240 mg of salicin versus placebo
over 4 weeks. The study showed complete relief of pain in
39% in the high-dose white willow bark group, 21% in the
low-dose white willow bark group, and 6% in the placebo
Table 34-6  Anti-Inflammatory Diet
Foods to Minimize or Eliminate
Trans fats/partially hydrogenated oils found in margarines, butter
spreads, and shortening
Oils—sunflower, safflower, cottonseed, and corn oils
Refined sugar and high glycemic load foods (products with  
high-fructose corn syrup, candy, fruit juices, and soda)
High saturated fat foods—saturated animal fats, cheeses, egg yolks
Fried foods—fast foods, doughnuts
Highly processed foods—hot dogs, processed meats
Refined starch products—muffins, tortillas, waffles, pasta, rice
Baked and pastry goods
Alcohol and caffeine in excess
Foods to Incorporate
Most of the diet should be derived from the following 4 areas with
additional nutrients for support
  Whole-grain products (whole-wheat breads and pastas)
  Fresh fruits and vegetables
  Legumes (soy, lentil)
  Seeds and nuts (almonds, walnuts)
The above provide rich sources of several anti-inflammatory
­components
  Fiber—sources include whole-grain products, fruits and
­vegetables, legumes, soy
  Isoflavones—found in soybeans and soy products, such as
­tempeh, tofu, and miso; isoflavones include genistein, daidzein,
and others that lower levels of inflammatory mediators
  Carotenoids (alpha carotene, beta carotene, lycopene, and
lutein)—found in orange fruits and vegetables and tomatoes,
spinach, and kale
  Flavonoids—include flavonols such as quercetin,
­epigallocatechin-3-gallate (EGCG), and anthocyanidins; they are
found in green tea, dark berries, citrus fruits, tomatoes, and in
greens
  Plant sterols and unesterified plant sterols (i.e., β-sitosterol)—
found in soy foods and many vegetable-based foods
  Omega-3 free fatty acids (flaxseed, fish oils, green leafy
­vegetables, walnuts, soy, algae, and hemp seeds)
  Monounsaturated oils (olive and canola oil)
  Probiotics-rich foods—yogurt, kefir, tempeh, and miso
group (P < .001). The pain relief with white willow bark was
not immediate, but typically occurred within 1 week of ini-
tiating treatment. Additionally, one patient had an aller-
gic reaction attributed to white willow bark.57 A 4-week,
randomized, open-label study of standardized white willow
bark (Assalix) containing 240 mg of salicin was found to
be noninferior to 12.5 mg of rofecoxib in 228 patients with
acute exacerbation of low back pain.
Ginger (Zingiber species)
Ginger (Zingiber officinale) has been used for more than 2500
years in traditional Chinese medicine, Ayurvedic (Indian)
medicine, and Tibetan medicine for rheumatologic, gastroin-
testinal, and oncologic conditions. Numerous active ingredi-
ents have been isolated from the ginger plant, including the
gingeroles, which exert direct anti-­inflammatory activity,
and galanolactones, which have serotonin receptor activity
and likely modulate gastrointestinal and anti-inflammatory
actions. Additionally, animal studies have shown that some
ginger subspecies have documented COX-2 and prostaglan-
din E2 inhibitory activities.58,59
In clinical pain trials, ginger has been used mostly for
osteoarthritis of the knee. In a 6-week randomized double-
blind, placebo-controlled trial comprising 261 subjects with
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
knee osteoarthritis, a combination ginger extract (EV.EXT
77) containing Z. officinale and Alpinia glanga was evaluated.
In this intent-to-treat analysis, moderate improvement was
noted in knee pain on standing and walking (P < .05). Mild
gastrointestinal side effects in the ginger group were statisti-
cally greater than the side effects in the control group.60 In a
more recent trial, an extract of Zingiberis rhizoma (Zintona EC)
was tested against placebo in 29 patients with knee osteoar-
thritis. Patients were randomly assigned to 1000 mg/day or to
placebo with crossover taking place at 3 months. The results
showed significant improvement over placebo starting only at
the 6-month mark.61 At this point, ginger is known to have
many anti-inflammatory constituents, and future research is
required to elucidate its potential clinical role.
Devil’s Claw (Harpagophytum procumbens)
Devil’s claw is a traditional South African plant used for
arthritis and myalgia and as an external ointment for burns
and sores. Its efficacy has been attributed to numerous active
ingredients, most notably harpagosides, which have shown
in vitro downregulation of the lipoxygenase-mediated and
COX-mediated pathways and inhibition of TNF-α syn-
thesis.62-64 Clinically, it has been used most commonly in
osteoarthritis and low back pain. Two meta-analyses have
found “moderate” evidence from high-quality studies for
use of devil’s claw (standardized to 60 mg of harpagoside)
in the treatment of osteoarthritis of the spine, hip, and knee
and in higher doses (100 mg of harpagoside) in the treat-
ment of acute exacerbations of chronic nonspecific low back
pain.65,66 The 60-mg harpagoside dose was found not to be
inferior to 12.5 mg/day of rofecoxib for chronic nonspecific
low back pain in a well-controlled 6-week trial.67
Glucosamine and Chondroitin
Glucosamine and chondroitin are discussed here jointly
because of their general similarity in structural joint func-
tion and their combination in commonly used preparations.
Both ingredients have been reported to reduce the progres-
sive degradation of articular cartilage and to help stimulate
proteoglycan synthesis in in vitro trials.68,69 In clinical trials,
glucosamine and chondroitin have been evaluated for their
ability to improve pain and functional ability, typically of
the knee and hip. A Cochrane review of glucosamine versus
placebo summarized that 12 of 13 trials were positive.70 Two
additional meta-analyses have examined glucosamine and
chondroitin in the treatment of osteoarthritis.71,72 These
trials examined approximately 15 randomized double-blind,
controlled trials with greater than 1500 patients. The meta-
analysis by Richy and colleagues72 found that glucosamine
at 1500 mg/day was associated with decrease in progression
of joint space narrowing. Additionally, glucosamine at this
dose and chondroitin at varying doses (200 to 1200 mg/day)
were associated with statistically significant pain relief and
improvement in joint mobility at 4 weeks. There were no
significant adverse effects.71,72
Several important clinical notes exist regarding these for-
mulations. The Cochrane systematic review pointed out that
most studies were performed on the Rotta company’s crystal-
line glucosamine formulation (brand name, Dona), and that
results may be difficult to extrapolate to other brands because
507
studies have shown wide variations in glucosamine formula-
tions.66 The meta-analysis by Richy and colleagues72 deemed
the chondroitin trials to be of overall low quality with the
combination of the two products not being evaluated. Prod-
ucts with glucosamine and chondroitin typically state that
the two combined work better than either alone. The results
of the Glucosamine/Chondroitin Arthritis Intervention
Trial (GAIT), although inconclusive, found benefit for the
combination versus celecoxib or placebo in the treatment of
moderate-to-severe knee osteoarthritis. Glucosamine hydro-
chloride or chondroitin alone was not superior in any of the
subgroup analyses. A recent meta-analysis also confirmed
the lack of significant benefit with the use of chondroitin
alone.73 At this point, prudent recommendation includes
the use of single ingredients that have proved effective as
in the previous Cochrane review (i.e., glucosamine sulfate
at 1500 mg/day for 8 to 12 weeks) to assess pain reduction
and functional improvement, with possible use of combina-
tion products based on initial response and patient subgroup
(moderate-to-severe osteoarthritis).74,75
S-adenosyl-l-methionine
S-adenosyl-l-methionine (SAMe) is a sulfur-containing dietary
supplement synthesized from reactions between adenosine
triphosphate and methionine and promoted for treatment
of numerous conditions, including depression, liver disease,
and joint disease. Its role in these conditions is derived from
potential intrinsic anti-inflammatory, serotoninergic, and
proteoglycan stimulatory mechanisms.76-78 A meta-analysis
of 11 randomized controlled trials showed improvement in
functional ability and pain levels compared with placebo
or NSAIDs in the treatment of osteoarthritis.79 Adverse
effects from SAMe were similar to placebo and significantly
decreased compared with NSAIDs.
A more recent randomized controlled trial compared
SAMe (1200 mg) with celecoxib (200 mg) in 61 subjects
with knee osteoarthritis over 16 weeks.80 After 1 month
of intervention, celecoxib showed greater pain reduction
over SAMe (P = .024). From the second month onward,
there was no significant difference between groups on pain
scores and functional joint ability, however. The investiga-
tors concluded that SAMe had a slower onset of action, but
similar efficacy to celecoxib. Individuals considering SAMe
should be monitored for concomitant use of serotoninergic
­medication because of synergistic potential. Typical initial
dosing is 600 mg/day in divided doses with titration up
to 1200 mg/day. Doses of 1600 mg/day have been used in
selected settings with monitoring.
Avocado-Soybean Unsaponifiables
The unsaponifiable portion of vegetable oils has been hypoth-
esized to possess anti-inflammatory activity owing to sterol,
tocopherol, and squalene content.81 A specific mixture
derived from the unsaponifiables of avocado and soybean,
typically in a ratio of 2:1, has been examined since the 1970s
for use in scleroderma, periodontal conditions, and rheuma-
tologic conditions.82 In vitro research has shown the ability
of avocado-soybean unsaponifiables (ASUs) to decrease sev-
eral key agents implicated in joint degradation and inflam-
mation, including IL-1β-induced collagenase, stromelysin,
 508 bonakdar  |  Integrative Medicine in Rheumatology: An Evidence-Based Approach
IL-6, IL-8, and prostaglandin E2 release.83 In addition, there
is in vitro evidence regarding a supportive role for ASUs
in maintaining the joint matrix, including upregulation of
transforming growth factor-β and aggrecan synthesis.75,84
In clinical trials, ASUs have been examined in the
­treatment of knee and hip osteoarthritis. In one multicenter
randomized controlled trial, 300 mg/day or 600 mg/day
of ASU was compared with placebo in patients with knee
osteoarthritis. At 3 months, the number of patients decreas-
ing NSAID or analgesic intake by more than 50% in either
ASU dosage group was 71% versus 36% in the placebo group.
Clinical improvements also were noted (P < .01) in many
parameters, including Lequesne’s index, which decreased
by 3.2 and 2.9 points (with high and low dose) versus 1.6
points in the placebo group.85 A second trial showed a treat-
ment benefit for 8 months, including a 2-month follow-up
while not on the supplement.86
A systematic review of four randomized controlled trials
found three trials showing efficacy with ASUs in osteoar-
thritis.87 The negative trial examined joint space preserva-
tion, which was not significantly noted with ASUs. There
were no significant differences between 300-mg and 600-mg
dosage efficacy, and side effects have been shown to be simi-
lar to placebo and typically gastrointestinal in nature. At
this point, the use of ASUs is promising; long-term efficacy
trials are needed to confirm clinical and potential structural
benefits in practice.
Capsaicin and Other Botanicals
Various topicals that possess counterirritant or substance
P–depleting activity are used in rheumatologic conditions.
Capsaicin is the most commonly used agent in this setting.
Three trials show benefit in the setting of low back pain (ver-
sus placebo and homeopathic cream), and several small trials
find benefit in the setting of localized arthritis.68,88,89 Several
other botanicals, including green tea (Camellia sinensis), tur-
meric (Curcuma longa), cat’s claw (Uncaria tomentosa), and
thunder god vine (Tripterygium wilfordii), are noted to be
candidates in the setting of arthritis-related pain.27,90-92
ACUPUNCTURE
Acupuncture is a component of traditional Chinese medicine
that is typically defined as the therapeutic use of fine needles
at specific body points. Acupuncture traditionally is used in
conjunction with other traditional Chinese medicine treat-
ments (Chinese herbal medicine, acupressure, moxibustion),
which are collectively used to correct physiologic or energetic
dysfunction. Because acupuncture has been the most popular
and translatable component of traditional Chinese medicine,
it has received increasing attention regarding its mechanism
and potential efficacy in rheumatologic conditions. The mech-
anism for acupuncture’s benefits stems from potential local tis-
sue, spinal cord, and cortical effects. The strongest evidence
seems to focus on frequency-dependent increase of endomor-
phin-1, β-endorphin, encephalin, and serotonin levels after
acupuncture, with partial reversal with naloxone.93-95
The clinical efficacy of acupuncture in rheumatology
varies and seems to be based on several factors, such as
the condition being treated and the specific methodology
employed, including treatment protocol and randomization.
The most researched condition seems to be knee osteoar-
thritis, in which a National Institutes of Health–funded, pla-
cebo-controlled trial found that a course of 23 acupuncture
treatments was significantly superior to placebo in decreas-
ing knee osteoarthritis pain and dysfunction as assessed by
WOMAC and patient global assessment.96 A meta-analysis
of 10 randomized controlled trials (N = 329) confirmed the
benefit of acupuncture compared with sham acupuncture in
the setting of peripheral joint osteoarthritis.97 The results of
acupuncture in RA are less certain, limited by the number
of well-controlled trials.98
Acupuncture is a common treatment used by fibromy-
algia patients. Although research findings are inconsistent,
trials have shown benefit for fibromyalgia-related pain.99
Symptomatic improvement was not isolated to pain sever-
ity, but also noted in the level of fatigue and anxiety.100 In
the treatment of low back pain, meta-analyses found acu-
puncture clinically effective and generally cost-effective in
the relief of chronic low back pain.101 Because of its safety
and potential benefit, acupuncture should be considered for
a short-term trial, especially in the setting of refractory knee
osteoarthritis, low back pain, and fibromyalgia.
BIOSTIMULATION
Biostimulation refers to the application of light, electrical,
magnetic, or similar modalities for therapeutic purposes.
Several biostimulation methods, such as ultrasound or
transcutaneous electrical nerve stimulation (TENS) have
become standard practice in the treatment of musculoskel-
etal dysfunction. Most types of biostimulation continue to
be considered CAM.
Low-Level Laser Therapy
Low-level laser therapy refers to the application of low-
intensity monochromatic wavelengths of light typically for
the treatment of pain and inflammatory conditions. The
treatment intensity is below that which is used for laser
in other medical settings, such as surgical or dermatologic
applications, and is also referred to as “cold laser.” Although
low-level laser therapy has been used for several decades
in Europe, the mechanism for low-level laser therapy has
not been fully elucidated. It seems to be a nonthermal,
photochemical cellular reaction, which may provide anti-
­inflammatory and tissue healing effects.
Several more recent controlled trials have attempted to
examine the role of low-level laser therapy in rheumatologic
conditions. A 2005 Cochrane review of 222 RA patients
included in five placebo-controlled trials found a statisti-
cally significant reduction in pain (visual analog scale) and
morning stiffness duration by 27.5 minutes for low-level
laser therapy versus placebo. Other outcomes, such as func-
tional assessment, range of motion, and local swelling, did
not differ between groups.102
Electromagnetic and Magnetic Stimulation
Various types of electrical stimulation have been employed
to treat rheumatologic conditions, especially RA and osteo-
arthritis. These have been used conventionally (i.e., stimu-
lation of the area of dysfunction) and in an acupuncture-like
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
setup (i.e., stimulation to acupuncture points). Although
there is evidence in focused settings (neuromuscular elec-
trical stimulation, TENS, and acupuncture-like TENS in
the setting of knee OA103,104), most evidence regarding the
use of electrostimulation is difficult to interpret.105 Several
other types of biostimulation, including the use of thermo-
therapy (moist heat) or cryotherapy, have shown short-term
benefit as a palliative therapy in RA.106
Magnets, static or pulsed, are often touted as helpful agents
in arthritis. Selected pulsed electromagnetic fields have shown
in vitro effects on bone and cartilage growth and are consid-
ered helpful in delayed bone healing.107 Limited evidence is
available in the setting of knee osteoarthritis for pulsed elec-
tromagnetic fields as noted in a meta-analysis.108 Evidence for
long-term benefit of static or pulsed magnets in other clinical
applications, such as osteoarthritis, is limited, however, by short-
term assessment or contradictory research findings.109,110
MIND/BODY INTERVENTIONS
The importance of stress management and control of depres-
sive disorders in RA cannot be overstated. RA may be exac-
erbated by psychological or physiologic stressors, and these
stressors may be involved to some extent in the actual etiol-
ogy of the disease itself. Depression is a common comorbid
condition that can exacerbate or be a result of RA. Depres-
sion and psychological stressors may account for 20% of the
disability in RA, a substantial amount if one considers that
the actual joint pain and dysfunction account for 14%. Feel-
ings of helplessness also have been shown to have a direct
effect on the disease activity.111-114
The effect of psychological factors on pain severity also
has been noted in several studies. A 5-month study of 188
older women with RA and osteoarthritis found depression to
affect the levels of pain and reactivity to perceived stress.115 A
12-month longitudinal study looking at pain and depression
in RA patients found that physical disability, helplessness,
and passive coping have a significant impact on the levels of
pain and depression experienced by RA patients.116
As interventions, multiple mind/body techniques have
been shown to reduce physiologic stress effectively. In addi-
tion to intrinsic benefits, stress management techniques,
meditation, biofeedback, exercise, and prayer are potential
means for controlling mood and psychological sequelae of
rheumatologic conditions.
Exercise
Multiple studies confirm the benefits of aerobic and weight
resistance types of exercise. The Arthritis, Diet, and Activ-
ity Promotion Trial (ADAPT) showed that the combina-
tion of modest weight loss plus moderate exercise provides
better overall improvements in function and pain and in
performance with knee osteoarthritis compared with either
intervention alone.118 The Fitness and Arthritis in Seniors
Trial (FAST) incorporated regular walking and leg strength-
ening exercises versus no exercise. At 1.5 years, the exer-
cisers had less pain and disability compared with subjects
who did not exercise. Positive effects were noted using
both types of exercise. Notable markers of improvement
were increased walking and stair climbing ability. Knee
radiography confirmed no worsening of the joint.119 In a
509
separate study, Sharma and coworkers120 showed reduced
risk for poor arthritis-related functioning measured over
3 years in individuals performing at least 60 minutes of exer-
cise per week. This study also showed that the aforemen-
tioned exercise regimen resulted in no worsening of knee
osteoarthritis.120
The importance and positive health effects of exercise in
patients with RA are well established.121-123 The evidence-
based guidelines of the American College of Rheumatology
and the European League Against Rheumatism endorse the
early use of exercise in the management of knee osteoar-
thritis.124,125 Feinglass and associates126 concluded that even
modest increases in the rates of physical activity could have
a significant impact on disability-free life expectancy.
Dunlop and colleagues127 reported that elderly adults
with arthritis who participated in Asset and Health Dynam-
ics of the Oldest Old Survey who regularly engaged in vig-
orous activity had the lowest risk for functional decline.
Multiple studies conclude that not only is there no detri-
ment to joint status, but also arthritis patients in structured
programs (which may be home based) have decreased pain,
decreased fatigue, increased strength, and increased qual-
ity of life.117,123,128 Moderate aerobic and weight resistance
exercise should be a central component of treatment plans
for patients with rheumatic conditions. Despite this body of
positive evidence, however, less than half of patients with
rheumatic conditions report receiving exercise advice.129
The role of the physician in encouraging patients to
become physically active is well established, although often
underused.130 Iversen and colleagues131 showed that when
physicians initiated discussion of exercise, the subject was
four times as likely to occur, with strong impact on the sub-
sequent follow-through. The most profound limitation for
exercise as a treatment modality may be physician indiffer-
ence to the actual recommendation of exercise.
Rest and inactivity of an RA joint results in decreased
overall conditioning, stamina, and strength.132,133 Exercise
has been found to decrease joint damage and maintain joint
activity and range of motion while being cost-effective.134
Retrospective studies show that individuals who exercise do
not sustain more rapid joint degeneration (barring severe
joint injury) compared with nonexercisers. The benefits of
exercise are multifactorial and not limited directly to rheu-
matologic conditions.
RA frequently occurs in the setting of obesity, each compound-
ing and exacerbating the other. In addition, RA patients
often have significant comorbidities, such as coronary artery
disease and diabetes, both of which can be improved with
aerobic and anaerobic exercise. The positive benefits of
exercise go beyond the joint itself; of direct benefit to RA
patients is the fact that exercise increases pain tolerance
(psychological and endorphin driven), and aerobic exercise
increases tissue oxygen efficiency.131
Many RA patients get caught in the vicious cycle of pain
limiting ability to exercise resulting in overall worsening of
joints, worsening of pain, and perpetuation of the cycle. The
myriad comorbidities that infiltrate this cycle (e.g., coronary
artery disease, diabetes, obesity) all are worsened by lack of
exercise and serve to drive the downward cycle. It is often
up to the physician to help the patient break from this cycle,
and exercise remains one of the most effective, easy to apply,
and cost-effective methods of breaking this cycle.
 510 bonakdar  |  Integrative Medicine in Rheumatology: An Evidence-Based Approach
The American College of Sports Medicine recommends
avoiding vigorous exercise during acute joint inflammation
or systemic disease that is not controlled. Recommendations
include 48 to 72 hours of rest during acute flares, but some
advocate static strengthening and gentle range of motion.
Recommendations are for gentle stretching exercise of all
major joints once daily held for 6 seconds.
Pain, stiffness, biomechanical deficiencies, and altera-
tions in gait can increase the metabolic demands of RA
patients by 50%, and training should be adjusted accord-
ingly. Several studies confirm that weight resistance training
improves function, improves activities of daily living, and
decreases pain. In addition, these results are found in home
training programs. There were no negative effects on RA
activity or function noted.
RA often limits joint stiffness, and maintenance of range
of motion is crucial. Multiple studies conclude that RA
patients can improve flexibility and retain range of motion
through exercise programs.123 The main goals for exercise
and RA should be (1) improving or stabilizing range of
motion, (2) improving or maintaining strength and endur-
ance leading to improved joint stability, (3) improving
overall aerobic capacity, and (4) slowing or stabilizing the
disabling process (with the corollary of a reduction of over-
all inflammatory burden).
Fibromyalgia. The use of exercise for fibromyalgia has pro-
duced mixed results, with some studies positive and some
not showing a positive response. Exercise likely benefits
certain individuals with fibromyalgia and does not benefit
others. Intensity and type of exercises may be key factors. It
seems warranted to use exercise in motivated and compliant
patients who tolerate it well, and not to force patients into
exercise regimens.
Richards and Scott135 looked at more than 100 patients
with American College of Rheumatology criteria for fibro-
myalgia who were randomly assigned to 12 weeks of either
cardiovascular or relaxation and flexibility exercises, twice
weekly. The primary outcome was patient-rated global
impression of change in symptoms, evaluation of the 18
specific tender points, and three standardized question-
naires addressing different aspects of mental and physical
fatigue and the impact of fibromyalgia. Of the patients ran-
domly assigned to cardiovascular exercise, 24 of 69 (35%)
were much better or very much better at 3 months versus
12 (18%) of the patients randomly assigned to relaxation
(statistically significant). These results were sustained at
1 year. Although tender points decreased significantly in
both groups, at 1 year the difference between groups was
significantly in favor of the cardiovascular exercise group.135
Another study looked at the use of exercise along with
mind/body therapies to evaluate pain and function for fibro-
myalgia. This study showed increased efficacy of exercise
compared with mind/body therapies with both superior to
no treatment.136
A 2001 Cochrane review of exercise and fibromyalgia
examined 16 studies with 724 participants. There was evi-
dence from well-defined and conducted studies that mark-
ers such as pain threshold using pressure, global well-being
(including ratings of general improvement in fibromyalgia
syndrome), and aerobic performance were improved by
short-term aerobic fitness training. Muscle strengthening
also was believed to be of potential benefit, but required fur-
ther investigation. Flexibility training could not be evalu-
ated conclusively because of significant study flaws.
Recommendations for exercise include starting with
motivated individuals and proceeding slowly with gradual
time increase to a goal of 45 minutes daily (minimum 4
times a week). Initial consultation with American College
of Sports Medicine–certified trainers familiar with fibro-
myalgia or pain patients is strongly recommended. It also
is advisable to inform patients that some discomfort after
exercise is normal, but to contact their physician if the pain
does not lessen in 24 hours or involves the major joints.
Medical clearance should be obtained when necessary.
Recommended therapies to begin with include low impact
and low joint stress (elliptical, yoga, swimming) exercises.
All programs should include appropriate warm up and cool
down. Physical therapists or trainers should be consulted for
further advice on taping/bracing sore joints.
Yoga. Most studies done are substandard to draw firm conclu-
sions about efficacy for the use of yoga. A 1998 study using
yoga for carpal tunnel syndrome done over 8 weeks showed
improved grip and decreased pain.137 Wood showed that
a 30-minute breathing technique done daily had “marked
invigorating” effects, improving mood and physical energy,
and was superior to relaxation and visualization.137a Multiple
studies show that yoga relieves anxiety138 and decreases de-
pression,139 both major contributors to RA. A small 1994
study published in the Journal of Rheumatology found yoga
to be beneficial in improving pain, tenderness, and range of
motion (finger) without difference in joint circumference or
grip strength in osteoarthritis of the hands.140 Also in 1994,
a study was published suggesting overall benefit from yoga
in the treatment of RA.141 Yoga instruction for rheumato-
logic patients should be done with a skilled yoga instructor
familiar with rheumatologic conditions and pain-related
issues. Many national certifications are available for yoga
instructors.
OTHER INTERVENTIONS: BELIEF SYSTEMS
AND COPING
A host of additional interventions that are often con-
sidered CAM should be kept in mind because they may
play an important role in patients’ coping strategies with
­rheumatologic issues. Several surveys have shown improved
coping ability with pain and dysfunction when patients
incorporate regular spiritual or reflective practices, such
as prayer and journaling.142 Generally, patients should feel
comfortable discussing interventions they are currently
using for prevention, treatment, and coping with open dis-
cussion regarding the place of these interventions in the
comprehensive management of their condition.
INTEGRATION INTO CLINICAL CARE:
AN EXAMPLE
The guideline for “Treatment of knee pain in older adults
in primary care: development of an evidence-based model
of care,” serves as an example of how complementary thera-
pies can be incorporated into clinical care for rheumatology
patients. This systematic survey completed by the Primary
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES 511

Table 34-7  Stepwise Approach to Treatment   REFERENCES

of Knee Pain in Older Adults   1. Wetzel MS, Eisenberg DM, Kaptchuk TJ: Courses involving comple-
Step 1 mentary and alternative medicine at US medical schools. JAMA
280:784-787, 1998.
Weight loss   2. NIH Center for Complementary and Alternative Medicine
Paracetamol (acetaminophen) (NCCAM): Available at: http://nccam.nih.gov/health/whatiscam/
Exercise #sup2 on 10.23.06.
Written information   3. Wolsko PM, Eisenberg DM, Davis RB, et al: Patterns and percep-
Restorative sleep advice tions of care for treatment of back and neck pain: Results of a na-
Thermotherapy tional survey. Spine 28:292-297, 2003.
Symptomatic slow-acting drugs (e.g., glucosamine, chondroitin,   4. Barnes PM, Powell-Griner E, McFann K, et al: Complementary
diacerein, avocado-soybean unsaponifiables) and alternative medicine use among adults: United States, 2002.
Step 2 Adv Data 343:1-19, 2004.
  5. Astin JA: Why patients use alternative medicine: Results of a na-
Nonselective NSAIDs tional study. JAMA 279:1548-1553, 1998.
Compound opioid analgesics   6. Furnham A, Bhagrath R: A comparison of health beliefs and behav-
Physiotherapy iours of clients of orthodox and complementary medicine. Br J Clin
Wedged insoles Psychol 32:237, 1993.
Selective NSAIDs   7. Astin JA, Pelletier KR, Marie A, et al: Complementary and alter-
Group education native medicine use among elderly persons: One-year analysis of a
Capsaicin Blue Shield Medicare supplement. J Gerontol A Biol Sci Med Sci 55:
Acupuncture M4-M9, 2000.
Appliances   8. Food and Drug Administration Press Release: FDA announces plans
Walking aids to prohibit sales of dietary supplements containing ephedra. Decem-
Step 3 ber 30, 2003. Available at: http://www.fda.gov/oc/initiatives/ephedra/
december2003/. Accessed May 1, 2004.
Intra-articular hyaluronan   9. Food and Drug Administration Medwatch Press Release: 2002
Intra-articular corticosteroids Safety Alert—SPES, PC SPES, June 5, 2002. Available at: http://
Occupational therapy www.fda.gov/medwatch/SAFETY/2002/spes_press2.htm. Accessed
Transcutaneous electrical nerve stimulation May 1, 2004.
Topical NSAIDs 10. FDA issues dietary supplements final rule. Available at: http://www.
Cognitive-behavioral therapy fda.gov/bbs/topics/NEWS/2007/NEW01657.html. Accessed June 22,
Step 4 2007.
11. Ashar BH, Rice TN, Sisson SD: Physicians’ understanding of the
Surgical referral regulation of dietary supplements. Arch Intern Med 167:966-969,
NSAIDs, nonsteroidal anti-inflammatory drugs. 2007.
From Porcheret M, Jordan K, Croft P; Primary Care Rheumatology Society: 12. Kemper KJ, Gardiner P, Gobble J, et al: Expertise about herbs
Treatment of knee pain in older adults in primary care: Development of an and dietary supplements among diverse health professionals. BMC
evidence-based model of care. Rheumatology (Oxf) 46:638-648, 2007. Complement Altern Med 286:15, 2006.
13. Harris Poll. Widespread ignorance of regulation and labeling of vita-
mins, minerals and food supplements. Vol 2, Issue 23, Dec 23, 2002.
Available at: http://www.harrisinteractive.com/news/newsletters_
Care Rheumatology Society outlines a stepwise approach healthcare.asp.
for incorporating conventional, lifestyle, and complemen- 14. Wold RS, Wayne SJ, Waters DL, et al: Behaviors underlying the use
of nonvitamin nonmineral dietary supplements in a healthy elderly
tary options (e.g., glucosamine, acupuncture, and ASUs).143 cohort. J Nutr Health Aging 11:3-7, 2007.
This example is outlined in Table 34-7. As more research is 15. Azaz-Livshits T, Muszkat M, Levy M, et al: Use of complementary
completed in this area, clinicians will have more guidance alternative medicine in patients admitted to internal medicine wards.
on the potential benefit and sequential role of specific com- J Clin Pharmacol Ther 40:539-547, 2002.
16. Adler SR, Fosket JR: Disclosing complementary and alternative med-
plementary therapies in developing an integrative approach icine use in the medical encounter: A qualitative study in women
to rheumatologic care. with breast cancer. J Fam Pract 48:453-458, 1999.
17. Hensrud DD, Engle DD, Scheitel SM, et al: Underreporting the
use of dietary supplements and nonprescription medication among
CONCLUSION patients undergoing a periodic health examination. Mayo Clin Proc
74:443-447, 1999.
Patients with acute and chronic inflammatory conditions 18. Corbin-Winslow L, Shapiro H: Physicians want education about
continue to trend toward the use of nonpharmacologic CAM to enhance communication with their patients. Arch Intern
remedies for rheumatologic conditions. It is imperative that Med 162:1176-1181, 2002.
health care practitioners remain informed on appropriate 19. Schulze MB, Hoffmann K, Manson JE: Dietary pattern, inflamma-
tion, and incidence of type 2 diabetes in women. Am J Clin Nutr
indications for use, risks, and limitations of such treatments. 82:675-684. quiz 714-715, 2005.
Paradoxically, it is even more important now, with wide- 20. Parkinson AJ, Cruz AL, Heyward WL: Elevated concentrations of
spread availability and free consumer access, that the appro- plasma omega-3 polyunsaturated fatty acids among Alaskan Eskimos.
priate use of CAM be overseen by knowledgeable health Am J Clin Nutr 59:384-388, 1994.
care professionals. 21. Linos A, Kaklamani VG, Kaklamani E, et al: Dietary factors in rela-
tion to rheumatoid arthritis: A role for olive oil and cooked vegeta-
An integrative approach using evidence-based CAM bles? Am J Clin Nutr 70:1077-1082, 1999. (Erratum in: Am J Clin
interventions is generally safe and often highly effective in Nutr 2000 71:1010, 2000.)
treatment of acute aspects of rheumatologic conditions and 22. Kremer JM, Lawrence DA, Jubiz W, et al: Dietary fish oil and olive
in management of the chronic disease state. More impor- oil supplementation in patients with rheumatoid arthritis: Clinical
and immunologic effects. Arthritis Rheum 33:810-820, 1990.
tantly, these measures may offer additional preventive and 23. Sardesai VM: The essential fatty acids. Nutr Clin Pract 7:179-186, 1992.
palliative strategies to aid in the optimization of health care 24. Simopoulos AP: Essential fatty acids in health and chronic disease.
for patients with rheumatologic conditions. Am J Clin Nutr 70(3 Suppl):560S-569S, 1999.
 512 bonakdar  |  Integrative Medicine in Rheumatology: An Evidence-Based Approach
25. Davis JN, Kucuk O, Djuric Z, Sarkar FH: Soy isoflavone supplemen-
tation in healthy men prevents NF-kappa B activation by TNF-alpha
in blood lymphocytes. Free Radic Biol Med 30:1293-1302, 2001.
26. Singh AK, Seth P, Anthony P, et al: Green tea constituent epigal-
locatechin-3-gallate inhibits angiogenic differentiation of human
endothelial cells. Arch Biochem Biophys 401(1):29-37, 2002.
27. Ahmed S, Rahman A, Hasnain A: Green tea polyphenol epigallo-
catechin-3-gallate inhibits the IL-1β induced activity and expression
of cyclooxygenase-2 and nitric oxide synthase-2 in human chondro-
cytes. Free Radic Biol Med 33:1097-1105, 2002.
28. Abe Y, Hashimoto S, Horie T: Curcumin inhibition of inflamma-
tory cytokine production by human peripheral blood monocytes and
alveolar macrophages. Pharmacol Res 39:41-47, 1999.
29. Liu S, Manson JE, Buring JE, et al: Relation between a diet with a
high glycemic load and plasma concentrations of high-sensitivity­
C-reactive protein in middle-aged women. Am J Clin Nutr 75:
492-498, 2002.
30. Hafström I, Ringertz B, Gyllenhammar H, et al: Effects of fasting on
disease activity, neutrophil function, fatty acid composition, and leu-
kotriene biosynthesis in patients with rheumatoid arthritis. Arthritis
Rheum 31:585-592, 1988.
31. Salas-Salvadó J, Garcia-Arellano A, Estruch R, et al: Components of
the Mediterranean-type food pattern and serum inflammatory mark-
ers among patients at high risk for cardiovascular disease. Eur J Clin
Nutr, 2007.
32. Tzoulaki I, Murray GD, Lee AJ, et al: C-reactive protein, interleukin-
6, and soluble adhesion molecules as predictors of progressive periph-
eral atherosclerosis in the general population: Edinburgh Artery
Study. Circulation 112:976-983, 2005.
33. Sköldstam L, Hagfors L, Johansson G: An experimental study of a
Mediterranean diet intervention for patients with rheumatoid arthri-
tis. Ann Rheum Dis 62:208-214, 2003.
34. Sköldstam L, Brudin L, Hagfors L, et al: Weight reduction is not a
major reason for improvement in rheumatoid arthritis from lacto-
vegetarian, vegan or Mediterranean diets. Nutr J 44:15, 2005.
35. Kaartinen K, Lammi K, Hypen M, et al: Vegan diet alleviates fibro-
myalgia symptoms. Scand J Rheumatol 29:308-313, 2000.
36. Kjeldsen-Kragh J, Haugen M, Borchgrevink CF, Førre O: Vegetarian
diet for patients with rheumatoid arthritis—status: Two years after
introduction of the diet. Clin Rheumatol 13:475-482, 1994. (Erratum
in: Clin Rheumatol 13:649, 1994.)
37. Kjeldsen-Kragh J, Mellbye OJ, Haugen M, et al: Changes in labora-
tory variables in rheumatoid arthritis patients during a trial of fasting
and one-year vegetarian diet. J Rheumatol 24:85-93, 1995.
38. Müller H, de Toledo FW, Resch KL, et al: Fasting followed by vegetar-
ian diet in patients with rheumatoid arthritis: A systematic review.
Scand J Rheumatol 30:1-10, 2001.
39. King DE, Egan BM, Geesey ME: Relation of dietary fat, fiber to eleva-
tion of C-reactive protein. Am J Cardiol 92:1335-1339, 2003. (Erra-
tum in Am J Cardiol 93:812, 2004.)
40. Jenkins DJ, Kendall CW, Marchie A, et al: Effects of a dietary port-
folio of cholesterol-lowering foods vs. lovastatin on serum lipids and
C-reactive protein. JAMA 290:502-510, 2003.
41. King DE, Egan BM, Woolson RF: Effect of a high-fiber diet vs a
fiber-supplemented diet on C-reactive protein level. Arch Intern
Med 167:502-506, 2007.
42. Cerhan JR, Saag KG, Merlino LA, et al: Antioxidant micronutrients
and risk of rheumatoid arthritis in a cohort of older women. Am J
Epidemiol 157:345-354, 2003.
43. Kremer JM, Bigauoette J, Michalek AV, et al: Effects of manipulation
of dietary fatty acids on clinical manifestations of rheumatoid arthri-
tis. Lancet 26(8422):184-187, 1985.
44. Guesens P, et al: Long-term effect of omega-3 fatty acid supplementa-
tion in active rheumatoid arthritis: A 12-month, double-blind, con-
trolled study. Arthritis Rheum 37:824-829, 1994.
45. Faarvang KL, Nielsen GL, Thomsen BS, et al: [Fish oils and rheuma-
toid arthritis: A randomized and double-blind study]. Ugeskr Laeger
156:3495-3498, 1994.
46. Nielsen GL, Faarvang KL, Thomsen BS, et al: The effects of dietary
supplementation with n-3 polyunsaturated fatty acids in patients
with rheumatoid arthritis: A randomized, double blind trial. Eur J
Clin Invest 22:687-691, 1992.
47. Zurier RB, Rossetti RG, Jacobson EW, et al: Gamma-Linolenic acid
treatment of rheumatoid arthritis: A randomized, placebo-controlled
trial. Arthritis Rheum 39:1808-1817, 1996.
48. MacLean CH, Mojica WA, Morton SC, et al: Effects of omega-3
fatty acids on lipids and glycemic control in type II diabetes and
the metabolic syndrome and on inflammatory bowel disease, rheu-
matoid arthritis, renal disease, systemic lupus erythematosus, and
osteoporosis. Evidence Report/Technology Assessment No. 89
(Prepared by Southern California/RAND Evidence-based Practice
Center, under Contract No. 290-02-0003). AHRQ Publication
No. 04-E012-2. Rockville, Md, Agency for Healthcare Research
and Quality, 2004.
49. Maroon JC, Bost JW: Omega-3 fatty acids (fish oil) as an anti-inflam-
matory: An alternative to nonsteroidal anti-inflammatory drugs for
discogenic pain. Surg Neurol 65:326-331, 2006.
50. Goldberg RJ, Katz J: A meta-analysis of the analgesic effects of
omega-3 polyunsaturated fatty acid supplementation for inflamma-
tory joint pain. Pain 129:210-223, 2007.
51. Adam O, Beringer C, Kless T, et al: Anti-inflammatory effects of a
low arachidonic acid diet and fish oil in patients with rheumatoid
arthritis. Rheumatol Int 23:27-36, 2003.
52. Nordström DC, Honkanen VE, Nasu Y, et al: Alpha-linolenic acid in
the treatment of rheumatoid arthritis: A double-blind, placebo-con-
trolled and randomized study: Flaxseed vs. safflower seed. Rheumatol
Int 14:231-234, 1995.
53. Volker D, Fitzgerald P, Major G, Garg M: Efficacy of fish oil concen-
trate in the treatment of rheumatoid arthritis. J Rheumatol 27:2343-
2346, 2000.
54. Schmid B, Kotter I, Heide L: Pharmacokinetics of salicin after oral
administration of a standardized willow bark extract. Eur J Clin Phar-
macol 57:387-391, 2001.
55. Schmid B, Lüdtke R, Selbmann HK, et al: Efficacy and tolerability
of a standardized willow bark extract in patients with osteoarthritis:
Randomized placebo-controlled, double blind clinical trial. Phyto-
ther Res 15:344-350, 2001.
56. Schaffner W: Eidenrinde-Ein Antiarrheumatikum der modernen
Phytotherapie. In Chrubasik S, Wink M (eds): Rheumatherapie mit
Phytopharmaka. Stuttgart, Hippokrates Verlag, 1997, pp 125-127.
57. Chrubasik S, Eisenberg E, Balan E, et al: Treatment of low back pain
exacerbations with willow bark extract: A randomized double-blind
study. Am J Med 109:9-14, 2000.
58. Murakami A: Zerumbone, a Southeast Asian ginger sesquiterpene,
markedly suppresses free radical generation, proinflammatory protein
production, and cancer cell proliferation accompanied by apoptosis:
The alpha, beta-unsaturated carbonyl group is a prerequisite. Carci-
nogenesis 23:795-802, 2002.
59. Thomson M: The use of ginger (Zingiber officinale Rosc.) as a potential
anti-inflammatory and antithrombotic agent. Prostaglandins Leukot
Essent Fatty Acids 67:475-478, 2002.
60. Altman RD, Marcussen KC: Effects of a ginger extract on knee
pain in patients with osteoarthritis. Arthritis Rheum 44:2531-2538,
2001.
61. Wigler I, Grotto I, Caspi D, Yaron M: The effects of Zintona EC (a
ginger extract) on symptomatic gonarthritis. Osteoarthritis Cartilage
11:783-789, 2003.
62. Jang MH, Lim S, Han SM, et al: Harpagophytum procumbens sup-
presses lipopolysaccharide-stimulated expressions of cyclooxygenase-2
and inducible nitric oxide synthase in fibroblast cell line L929. J Phar-
macol Sci 93:367-371, 2003.
63. Loew D, Möllerfeld J, Schrödter A, et al: Investigations on the phar-
macokinetic properties of Harpagophytum extracts and their effects on
eicosanoid biosynthesis in vitro and ex vivo. Clin Pharmacol Ther
69:356-364, 2001.
64. Fiebich BL, Heinrich M, Hiller KO, et al: Inhibition of TNFα synthe-
sis in LPS-stimulated primary human monocytes by Harpagophytum
extract SteiHap 69. Phytomedicine 8:28-30, 2001.
65. Gagnier JJ, Chrubasik S, Manheimer E: Harpagophytum procumbens
for osteoarthritis and low back pain: A systematic review. BMC Com-
plement Altern Med 4:13, 2004.
66. Gagnier JJ, van Tulder M, Berman B, et al: Herbal medicine for
low pack pain. Cochrane Database Syst Rev 2:CD004504, 2006.
67. Chrubasik S, Model A, Black A, Pollak S: A randomized double-
blind pilot study comparing Doloteffin and Vioxx in the treatment of
low back pain. Rheumatology (Oxf) 42:141-148, 2003.
68. Bassleer C, Rovati L, Franchimont P: Stimulation of proteoglycan
production by glucosamine sulfate in chondrocytes isolated from
human osteoarthritic articular cartilage in vitro. Osteoarthritis Carti-
lage 6:427-434, 1998.
 PART 4  |  BROAD ISSUES IN THE APPROACH TO RHEUMATIC DISEASES
69. Uelbelhart D, Thonar EJ, Zhang J, Williams JM: Protective effect of
exogenous chondroitin 4,6-sulfate in the acute degradation of articular
cartilage in the rabbit. Osteoarthritis Cartilage 6(Suppl A):6-13, 1998.
70. Towheed TE, Anastassiades TP, Shea B, et al: Glucosamine ther-
apy for treating osteoarthritis. Cochrane Database Syst Rev 1:
CD002946, 2001.
71. McAlindon T, Lavalley MP, Gulin JP, Felson DT: Glucosamine
and chondroitin for the treatment of osteoarthritis. JAMA 283:
1469-1475, 2000.
72. Richy F, Bruyere O, Ethgen O, et al: Structural and symptomatic
efficacy of glucosamine and chondroitin in knee osteoarthritis. Arch
Intern Med 163:1514-1522, 2003.
73. Reichenbach R, Sterchi R: Martin Scherer M: Meta-analysis: Chon-
droitin for osteoarthritis of the knee or hip. Ann Intern Med 146:580-
590, 2007.
74. Clegg DO, Reda DJ, Harris CL, et al: Glucosamine, chondroitin
­sulfate, and the two in combination for painful knee osteoarthritis.
N Engl J Med 354:795-808, 2006.
75. Hochberg MC: Nutritional supplements for knee osteoarthritis—still
no resolution. N Engl J Med 354:858-860, 2006.
76. Stramentinoli G: Pharmacologic aspects of S-adenosylmethionine.
Am J Med 83(Suppl 5A):35-42, 1987.
77. Harmand MF, Vilamitjana J, Maloche E, et al: Effects of S-adenosyl-
methionine on human articular chondrocyte differentiation: An in
vitro study. Am J Med 83:48-54, 1987.
78. Parcell S: Sulfur in human nutrition and applications in medicine.
Altern Med Rev 7:22-44, 2002.
79. Soeken KL, Lee WL, Bausell RB, et al: Safety and efficacy of S-
adenosylmethionine (SAMe) for osteoarthritis. J Fam Pract 51:
425-430, 2002.
80. Najm WI, Reinsch S, Hoehler F, et al: S-Adenosyl methionine
(SAMe) versus celecoxib for the treatment of osteoarthritis symptoms:
A double-blind cross-over trial. BMC Musculoskelet Disord 5:6, 2004.
81. Rosenblat G, et al: Chemical characteristics of lysyl oxidase inhibitor
from avocado seed oil. JAOCS 72:225-229, 1995.
82. Reginster JY, Gillot V, Bruyere O, et al: Evidence of neutraceutical
effectiveness in the treatment of osteoarthritis. Curr Rheumatol Rep
2:472-477, 2000.
83. Henrotin YE, Sanchez C, Deberg MA, et al: Avocado/soybean unsa-
ponifiables increase aggrecan synthesis and reduce catabolic and
proinflammatory mediator production by human osteoarthritic chon-
drocytes. J Rheumatol 30:1825-1834, 2003.
84. Boumediene K, Felisaz N, Bogdanowicz P, et al: Avocado-soya unsa-
ponifiables enhance the expression of transforming growth factor
beta1 and beta2 in cultured articular chondrocytes. Arthritis Rheum
42:148-156, 1999.
85. Appelboom T, Schuermans J, Verbruggen G, et al: Symptoms modi-
fying effect of avocado-soybean unsaponifiables (ASU) in knee
osteoarthritis: A double blind, prospective, placebo-controlled study.
Scand J Rheumatol 30:242-247, 2001.
86. Maheu E, Mazières B, Valat JP, et al: Symptomatic efficacy of avo-
cado-soybean unsaponifiables in the treatment of osteoarthritis of the
knee: A prospective, randomized, double-blind, placebo-controlled,
multicenter trial with a six-month treatment period and a two-month
followup demonstrating a persistent effect. Arthritis Rheum 41:
81-91, 1998.
87. Ernst E: Avocado-soybean unsaponifiables (ASU) for osteoarthri-
tis—a systematic review. Clin Rheumatol 22(4-5):285-288, 2003.
88. Deal CL, Schnitzer TJ, Lipstein E: Treatment of arthritis with topical
capsaicin: A double-blind trial. Clin Ther 13:383-395, 1991.
89. McCleane G: The analgesic efficacy of topical capsaicin is enhanced
by glyceryl trinitrate in painful osteoarthritis: A randomized, double
blind, placebo controlled study. Eur J Pain 4:355-360, 2000.
90. Singh R, Ahmed S, Malemud CJ: Epigallocatechin-3-gallate selec-
tively inhibits interleukin-1β-induced activation of mitogen acti-
vated protein kinase subgroup c-jun N-terminal kinase (JNK) in
human osteoarthritis chondrocytes. J Orthop Res 21:102-109, 2003.
91. Liacini A, Sylvester J, Li WQ, et al: Inhibition of interleukin-1-stim-
ulated MAP kinases, activating protein-1 (AP-1) and nuclear factor
kappa B (NF-κB) transcription factors down-regulates matrix metal-
loproteinase gene expression in articular chondrocytes. Matrix Biol
21:251-262, 2002.
92. Ahmed S, Anuntiyo J, Malemud CJ, et al: Biological basis for the
use of botanicals in osteoarthritis and rheumatoid arthritis: A review.
Evid Based Complement Alternat Med 2:301-308, 2005.
513
93. Cabyoglu MT, Ergene N, Tan U: The mechanism of acupuncture and
clinical applications. Int J Neurosci 116:115-125, 2006.
94. White P: A background to acupuncture and its use in chronic painful
musculoskeletal conditions. J R Soc Health 126:219-227, 2006.
95. Staud R, Price DD: Mechanisms of acupuncture analgesia for clinical
and experimental pain. Expert Rev Neurother 6:661-667, 2006.
96. Berman BM, Lao L, Langenberg P: Effectiveness of acupuncture as
adjunctive therapy in osteoarthritis of the knee: A randomized, con-
trolled trial. Ann Intern Med 141:901-910, 2004.
97. Kwon YD, Pittler MH, Ernst E: Acupuncture for peripheral joint
osteoarthritis: A systematic review and meta-analysis. Rheumatology
(Oxf) 45:1331-1337, 2006.
98. Casimiro L, Barnsley L, Brosseau L, et al: Acupuncture and elec-
troacupuncture for the treatment of rheumatoid arthritis. Cochrane
Database Syst Rev 4:CD003788, 2005.
99. Singh BB, Wu WS, Hwang SH: Effectiveness of acupuncture in the
treatment of fibromyalgia. Altern Ther Health Med 12:34-41, 2006.
100. Martin DP, Sletten CD, Williams BA, et al: Improvement in fibromy-
algia symptoms with acupuncture: Results of a randomized controlled
trial. Mayo Clin Proc 81:749-757, 2006.
101. Manheimer E, White A, Berman B: Meta-analysis: Acupuncture for
low back pain. Ann Intern Med 142:651-663, 2005.
102. Brosseau L, Robinson V, Wells G, et al: Low level laser therapy
(classes I, II and III) for treating rheumatoid arthritis. Cochrane
Database Syst Rev 4:CD002049, 2005.
103. Talbot LA, Gaines JM, Ling SM, et al: A home-based protocol of
electrical muscle stimulation for quadriceps muscle strength in older
adults with osteoarthritis of the knee. J Rheumatol 30:1571-1578,
2003.
104. Osiri M, Welch V, Brosseau L, et al: Transcutaneous electrical nerve
stimulation for knee osteoarthritis. Cochrane Database Syst Rev 4:
CD002823, 2000.
105. Brosseau L, Judd MG, Marchand S, et al: Transcutaneous electrical
nerve stimulation (TENS) for the treatment of rheumatoid arthritis
in the hand. Cochrane Database Syst Rev 3:CD004377, 2003.
106. Robinson V, Brosseau L, Casimiro L, et al: Thermotherapy for treat-
ing rheumatoid arthritis. Cochrane Database Syst Rev 2:CD002826,
2002.
107. Trock DH: Electromagnetic fields and magnets: Investigational treat-
ment for musculoskeletal disorders. Rheum Dis Clin N Am 26:51-62,
2000.
108. Hulme J, Robinson V, DeBie R: Electromagnetic fields for the treat-
ment of osteoarthritis. Cochrane Database Syst Rev 1:CD003523,
2002.
109. Wolsko PM, Eisenberg DM, Simon LS: Double-blind placebo-con-
trolled trial of static magnets for the treatment of osteoarthritis of
the knee: Results of a pilot study. Altern Ther Health Med 10:36-43,
2004.
110. Kroeling P, Gross A, Houghton PE; Cervical Overview Group:
Electrotherapy for neck disorders. Cochrane Database Syst Rev 2:
CD004251, 2005.
111. Huyser B, Parker JC: Stress and rheumatoid arthritis: An integrative
review. Arthritis Care Res 11:135-145, 1998.
112. Escalante A, del Rincon I: How much disability in rheumatoid
arthritis is explained by rheumatoid arthritis? Arthritis Rheum 42:
1712-1721, 1999.
113. Parker JC, Smarr KL, Angelone EO, et al: Psychological factors,
immunologic activation, and disease activity in rheumatoid arthritis.
Arthritis Care Res 5:196-201, 1992.
114. Parker J, Smarr K, Anderson S, et al: Relationship of changes in help-
lessness and depression to disease activity in rheumatoid arthritis.
J Rheumatol 19:1901-1905, 1992.
115. Zautra AJ, Smith BW: Depression and reactivity to stress in older
women with rheumatoid arthritis and osteoarthritis. Psychosom Med
63:687-696, 2001.
116. Covic T, Adamson B, Spencer D, et al: A biopsychosocial model of
pain and depression in rheumatoid arthritis: A 12-month longitudi-
nal study. Rheumatology (Oxf) 42:1287-1294, 2003.
117. Deleted in press.
118. Messier SP, Loesser RF, Miller GD, et al: Exercise and dietary weight
loss in overweight and obese older adults with knee osteoarthritis.
The Arthritis, Diet, and Activity Promotion Trial (ADAPT). Arthri-
tis Rheum 50:1501-1510, 2004.
119. Ettinger WH Jr, Barns R, Messier SP, et al: The Fitness and Arthritis
in Seniors Trial (FAST). JAMA 277:25-31, 1997.
 514 bonakdar  |  Integrative Medicine in Rheumatology: An Evidence-Based Approach
120. Sharma L, Cahue S, Song J, et al: Physical functioning over three
years in knee osteoarthritis: Roles of psychosocial, local mechanical,
and neuromuscular factors. Arthritis Rheum 48:3359-3370, 2003.
121. Hurley MV: The role of muscle weakness in the pathogenesis of
osteoarthritis. Rheum Dis Clin N Am 25:283-298, 1999.
122. van Baar ME, Assendelft WJJ, Dekker J, et al: Effectiveness of exer-
cise therapy in patients with osteoarthritis of the hip and knee:
A systematic review of randomized clinical trials. Arthritis Rheum
42:1361-1369, 1999.
123. Minor MA: Exercise in the treatment of osteoarthritis. Rheum Dis
Clin N Am 25:397-415, 1999.
124. Pendleton A, Arden N, Dougados M, et al: EULAR recommenda-
tions for the management of knee osteoarthritis: Report of a task
force of the Standing Committee for International Clinical Studies
Including Therapeutic Trials (ESCISIT). Ann Rheum Dis 59:936-
944, 2000.
125. Altman RD, Hochberg MC, Moskowitz RW, et al: Recommenda-
tions for the medical management of osteoarthritis of the hip and
knee. Arthritis Rheum 43:1905-1915, 2000.
126. Feinglass J, Thompson JA, He XZ, et al: Effects of physical activ-
ity on functional status among older middle age adults with arthritis.
Arthritis Rheum 53:879-885, 2005.
127. Dunlop DD, Manheim LM, Yelin EH, et al: The costs of arthritis.
Arthritis Rheum 49:101-113, 2003.
128. Hopman-Rock M, Westhoff MH: The effects of a health educational
and exercise program for older adults with osteoarthritis of the hip or
knee. J Rheumatol 27:1947-1954, 2000.
129. Dexter PA: Joint exercises in elderly persons with symptomatic osteo-
arthritis of the hip or knee: Performance patterns, medical support,
and the relationship between exercising and medical care. Arthritis
Care Res 5:36-41, 1992.
130. Andersen RE, Blair SN, Cheskin LJ, Bartlett SJ: Encouraging patients
to become more physically active: The physician’s role. Ann Intern
Med 127:395-400, 1997.
131. Iversen MD, Eaton HM, Daltroy LH: How rheumatologists and
patients with rheumatoid arthritis discuss exercise and the influence
of discussions on exercise prescriptions. Arthritis Rheum 51:63-72,
2004.
132. Häkkinen A, Sokka T, Kotaniemi A, et al: A randomized two-year
study of the effects of dynamic strength training on muscle strength,
disease activity, functional capacity, and bone mineral density in
early rheumatoid arthritis. Arthritis Rheum 44:515-522, 2001.
133. Munneke M, de Jong Z, Zwinderman AH: Effect of a high-intensity
weight-bearing exercise program on radiologic damage progression
of the large joints in subgroups of patients with rheumatoid arthritis.
Arthritis Rheum 53:410-417, 2005.
134. van den Hout WB, de Jong Z, Munneke M: Cost-utility and cost-
effectiveness analyses of a long-term, high-intensity exercise pro-
gram compared with conventional physical therapy in patients with
rheumatoid arthritis. Arthritis Rheum 53:39-47, 2005.
135. Richards SC, Scott DL: Prescribed exercise in people with fibromy-
algia: Parallel group randomized controlled trial. BMJ 325:185-187,
2002.
136. Hadhazy VA, Ezzo J, Creamer P, Berman BM: Mind-body therapies
for the treatment of fibromyalgia: A systematic review. J Rheumatol
27:2911-2918, 2000.
137. Garfinkel MS, Singhal A, Katz WA, et al: Yoga-based intervention
for carpal tunnel syndrome. JAMA 280:1601-1603, 1998.
137a. Wood C: Mood change and perceptions of vitality: A comparison of
the effects of relaxation, visualization and yoga. J R Soc Med 86(5):
254-258, 1993.
138. Smith C, Hancock H, Blake-Mortimer J, et al: A randomised com-
parative trial of yoga and relaxation to reduce stress and anxiety.
Complement Ther Med 15:77-83, 2007.
139. Pilkington K, Kirkwood G, Rampes H, et al: Yoga for depression:
The research evidence. J Affect Disord 89(1-3):13-24, 2005.
140. Garfinkel MS, Schumacher HR, Husain A, et al: Evaluation of a
yoga based regimen for treatment of osteoarthritis of the hands. J
Rheumatol 21:2341-2343, 1994.
141. Haslock I, Monro R, Nagarathna R, et al: Measuring the effects of
yoga in rheumatoid arthritis. Br J Rheumatol 33:787-788, 1994.
142. Keefe FJ, Affleck G, Lefebvre J, et al: Living with rheumatoid arthri-
tis: The role of daily spirituality and daily religious and spiritual cop-
ing. J Pain 2:101-110, 2001.
143. Porcheret M, Jordan K, Croft P: Primary Care Rhumatology Soci-
ety: Treatment of knee pain in older adults in primary care: Devel-
opment of an evidence-based model of care. Rheumatology (Oxf)
46:638-648, 2007.
 Part EVALUATION OF GENERALIZED
5 AND LOCALIZED SYMPTOMS
35
KEY POINTS
Taking a detailed and accurate history is crucial for ­making
the correct diagnosis for patients with musculoskeletal
­diseases.
The cardinal symptoms of musculoskeletal disease are pain,
stiffness, swelling, limitation of motion, weakness, and
fatigue.
Understanding the anatomy, the planes of motion, and
­particularly the configuration of the synovial lining is impera-
tive for proper physical diagnosis of musculoskeletal diseases.
It is important to record qualitative and quantitative aspects
of the joint examination to monitor disease activity in patients
with inflammatory arthropathies.
Early recognition of how patients’ psychosocial factors
­affect their musculoskeletal symptoms and musculoskeletal
­examination enhances clinical assessment.
HISTORY IN A PATIENT WITH
MUSCULOSKELETAL DISEASE
Taking an accurate and comprehensive history of a patient’s
musculoskeletal symptoms is crucial for making the correct
diagnosis. This history must include a precise understanding
of what the patient means by the description of symptoms.
The physician must obtain a detailed account of symptom
onset, location, patterns of progression, severity, exacerbat-
ing and alleviating factors, and associated symptoms. The
relationship of the symptoms to psychosocial stressors is
important and should be determined. The impact of the
symptoms on all aspects of the patient’s functioning must be
assessed to guide therapy.
The effects of current or previous therapy on the course
of the illness are helpful in understanding the current
­symptoms. Response to anti-inflammatory or glucocorti-
coid medications may suggest an inflammatory etiology.
Such responses are not specific to inflammatory rheumatic
© 2006 Mayo Foundation for Medical Education and Research.
  Video available on the Expert Consult Premium Edition website.
History and Physical
Examination of the
Musculoskeletal System
John M. Davis iii  •  Kevin G. Moder  • 
Gene G. Hunder
d­ iseases, however, and must be considered in light of the
entire history and physical examination. The physician
must assess compliance with therapies for musculoskeletal
diseases. Noncompliance with the recommended treatment
must be differentiated from treatment failure as the explana-
tion for the patient’s lack of improvement.
While the physician is taking the patient’s history, the
patient provides verbal and nonverbal clues to the nature of
the illness and how the patient has responded to it. Patients
with early rheumatoid arthritis may hold their hands in a
flexed posture to minimize intra-articular pressure and pain.
Patients with fibromyalgia or chronic pain syndromes often
dramatize in describing their symptoms. Some patients may
be overly concerned, whereas others may seem inappropri-
ately indifferent to their symptoms. The physician must
appreciate the patient’s understanding of the illness and
attitudes toward it to begin effective treatment.
PAIN
Pain is the most common symptom that brings a patient
with musculoskeletal diseases to the physician. Pain is a
­subjective hurting sensation or experience described in
­various terms, often of actual or perceived physical dam-
age. Pain is a complex sensation that is difficult to define,
qualify, and measure. The patient’s pain may be modified by
­emotional factors and previous experiences.
The character of the pain usually is best defined early
in the interview because this can be helpful in ­categorizing
the patient’s complaints. Aching in a joint area suggests an
arthritic disorder, whereas “burning” or “numbness” in an
extremity may indicate a neuropathy. Descriptions of pain as
“excruciating” or “intolerable” when the patient is ­otherwise
able to function provide a clue that emotional or psychoso-
cial factors are contributing to or amplifying the symptoms.
The physician must elicit the distribution of the patient’s
pain and determine if this fits with anatomic structures.
Patients describe their pain location in terms of body part
names, but frequently the terms are used in a nonanatomic
manner. Patients frequently complain of “hip” pain when
actually referring to pain in the low back, buttock, or thigh.
The interviewer must attempt to clarify this complaint by
515
 516 DAVIS  |  History and Physical Examination of the Musculoskeletal System
asking the patient to point to the area of pain with one
finger. Pain localized in the distribution of a joint or joints
likely reflects an articular disorder. Pain may localize to
bursae, tendons, ligaments, or nerves, implying disorders of
these structures. In contrast to superficial structures, deep
structures often give rise to poorly localizing pain. Simi-
larly, pain arising from small, peripheral joints is often more
focal than pain arising from proximal, large joints, such as
the shoulders and hips. Pain that is widespread, vaguely
described, and not respecting anatomic distributions gener-
ally suggests a chronic pain syndrome, such as fibromyalgia
or psychiatric disease.
The severity of pain should be assessed. A common
ap­proach is to ask the patient to describe the level of pain
on a numeric scale of intensity from 0 (no pain) to 10
(very severe pain). For monitoring of disease activity of
inflammatory arthritis, measuring pain on a visual analog
scale by having the patient mark the severity of pain over
the past week on a 100-mm line can be helpful. Similar
scales are used in validated instruments such as the Health
­Assessment Questionnaire.
The physician must determine what exacerbates and
alleviates the pain. Joint pain present at rest but worse with
movement suggests an inflammatory process, whereas pain
occurring primarily with activity and relieved by rest gener-
ally indicates a mechanical disorder, such as degenerative
arthritis. Timing of the pain symptoms during the day and
night also provides important information, as discussed in
the next section.
STIFFNESS
Stiffness is a common complaint among patients with arthri-
tis. What is meant by stiffness varies from patient to patient,
however. Some patients may use the term stiffness to refer to
pain, soreness, weakness, fatigue, or limitation of motion.1
Rheumatologists generally use the term stiffness to describe
discomfort and limitation on attempted movement of joints
after a period of inactivity. This “gel” phenomenon occurs
usually after an hour or more of inactivity. The duration of
stiffness related to inactivity varies, with mild stiffness last-
ing minutes to severe stiffness lasting hours.
Morning stiffness is an early feature of inflammatory
arthropathies and is particularly noted in rheumatoid arthri-
tis and polymyalgia rheumatica, in which morning stiffness
may last for several hours. The absence of morning stiffness
does not exclude inflammatory arthritis, but its absence is
uncommon. A useful question to assess morning stiffness is:
“In the morning, how long does it take for your joints to
limber up as good as they are going to get for the day?” Morn-
ing stiffness associated with noninflammatory joint diseases,
such as degenerative arthritis, is generally of short duration
(usually <30 minutes) and less severe than stiffness of inflam-
matory joint disease. Additionally, the degree of stiffness in
noninflammatory joint diseases is related to the extent of use
of the damaged joint—stiffness is worse after excessive use,
generally improving within several days to the baseline level.
Morning stiffness is not specific for inflammatory arthropa-
thies and may be described by patients with fibromyalgia
or chronic idiopathic pain syndromes, neurologic disorders
such as Parkinson’s disease (although ­generally without lim-
bering up), and sleep-related ­breathing ­disorders.
LIMITATION OF MOTION
Limitation of motion is a common complaint among patients
with articular disorders. This complaint must be differenti-
ated from stiffness, which is usually transient and variable,
whereas limitation of motion secondary to joint disease is
generally fixed and varies less over time. The interviewer
should determine the extent of disability resulting from the
restriction in joint motion. The duration of the restriction in
joint motion frequently predicts the likelihood of improve-
ment with interventions such as oral and intra-articular
­glucocorticoids or physical therapy. Determining the rapid-
ity of onset of the limitation of motion may be helpful in the
differential diagnosis; the abrupt onset of the limitation of
motion suggests a structural derangement, such as a tendon
rupture or torn knee cartilage, whereas an insidious onset of
restricted joint motion is more common with inflammatory
joint disease.
SWELLING
Joint swelling is an important symptom in patients with
rheumatic diseases. The presence of true joint swelling nar-
rows the differential diagnosis in a patient with arthralgia.
To determine if the swelling is related to joint synovitis as
opposed to soft tissue conditions, clarifying the anatomic
location and distribution of the swelling is key. Diffuse soft
tissue swelling can occur because of venous or lymphatic
obstruction, soft tissue injury, or obesity. The description
of swelling in patients with such conditions usually is ill-
defined or not in a distribution of particular joints, bursae, or
tendons. Obese patients may interpret normal adipose tissue
over the medial aspect of the elbow, the knee, or the lateral
aspect of the ankle as joint swelling. In contrast, patients
with inflammatory arthritis may describe swelling of joints
in a distribution typical of a specific disease—symmetric
swelling of the metacarpophalangeal joints and wrists in
rheumatoid arthritis or swelling of several toes and a knee
in psoriatic arthritis.
It is useful to delineate the onset and progression of
swelling and the factors that influence it. Swelling of a joint
resulting from synovitis or bursitis frequently is ­ associated
with discomfort with motion because of tension on the
inflamed tissues. If the swollen tissues are periarticular,
however, no discomfort may be present with joint motion
because the inflamed tissues are not stressed. Swelling of a
confined structure, such as a synovial cavity or bursa, is most
painful when it has developed acutely, whereas a similar
degree of swelling that has developed slowly is often much
more tolerable.
WEAKNESS
Weakness is another common complaint that can be
­associated with myriad different subjective meanings. True
weakness is the loss of muscle power. When present, it is
demonstrable on physical examination.
The temporal course of weakness is important to the dif-
ferential diagnosis. Weakness of sudden onset without trauma
often indicates a neurologic disorder, such as an acute cere-
brovascular event, which generally results in a fixed, non-
progressive deficit. Weakness of insidious onset more often
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
suggests a muscle disease, such as an ­inflammatory myopathy
(i.e., polymyositis). The latter tends to be ongoing and pro-
gressive. Weakness that is intermittent suggests a disorder
of the neuromuscular junction, such as myasthenia gravis.
Patients with this disease may describe muscle fatigue with
activity as opposed to true weakness.
The physician should determine the distribution of the
patient’s weakness. Proximal weakness that is bilateral and
symmetric suggests an inflammatory myopathy. In contrast,
inclusion body myositis causes an asymmetric and more
­distal weakness. The presence of a unilateral or isolated
deficit generally indicates a neurogenic etiology. Distal
weakness, in the absence of joint findings, generally indi-
cates neurologic disorders, such as peripheral neuropathy.
Patients with peripheral neuropathies also complain of pain
and sensory symptoms, such as paresthesias. In contrast,
patients with inflammatory myopathy typically present with
painless weakness.
Inquiring about the patient’s family history may provide
valuable information. A history of other family members
with similar symptoms may increase the likelihood that the
patient has a hereditary disorder, such as muscular dystrophy
or familial neuropathy.
It also is important to review medication taken recently
or currently. Many medications, including corticosteroids
and lipid-lowering agents, can cause muscle injury. Less
commonly, environmental exposures can lead to symptoms
of weakness. Heavy metal poisoning causes a peripheral
neuropathy. Dietary exposures also should be investigated
such as eating undercooked pork as a source of trichi-
nosis. ­ Excessive alcohol intake has been associated with
­neuropathy and myopathy.
Taking a complete review of systems is helpful in evaluat-
ing a patient with weakness. Constitutional symptoms, such
as weight loss and night sweats, may indicate the presence
of a malignancy as the cause of generalized weakness. Rash,
arthralgia, or Raynaud’s phenomenon may prompt further
testing for a connective tissue disease.
FATIGUE
Patients with musculoskeletal disorders frequently complain
of fatigue. Fatigue can be defined as an inclination to rest
even though pain and weakness are not limiting factors.
Fatigue after varying degrees of activity that is relieved by
rest is normal. Patients with rheumatic diseases experience
fatigue even without activity. Fatigue generally improves
as the systemic rheumatic disease improves. Malaise fre-
quently occurs with, but is not synonymous with, fatigue.
Malaise indicates the lack of well-being that often occurs
at the onset of an illness. Fatigue and malaise may occur in
the absence of identifiable disease, and psychosocial factors,
anxiety, or depression may account for the symptoms.
SYSTEMATIC METHOD OF EXAMINATION
The musculoskeletal examination should be a systematic,
thorough assessment of the status of the joints, periarticu-
lar soft tissues, tendons, ligaments, bursae, and muscles.
Rheumatologists commonly begin by examining the upper
extremities followed by the trunk and lower extremities, but
many routines may be effective, provided that a systematic,
517
consistent approach is used. Gentle handling of the tender
and painful joints enhances cooperation by the patient and
allows an accurate evaluation of the joints.
The general aim of the examination of the joints is to
detect abnormalities in structure and function. The key
signs of articular disease are swelling, tenderness, limitation
of motion, crepitation, deformity, and instability.
SWELLING
Swelling around a joint may be caused by intra-articular
effusion, synovial proliferation, periarticular subcutaneous
tissue inflammation, bursitis, tendinitis, bony enlargement,
or extra-articular fat pads. A keen understanding of the ana-
tomic configuration of each joint’s synovial membrane is
crucial in differentiating soft tissue swelling secondary to a
joint effusion from swelling of periarticular tissues. First, the
examiner should inspect the joints for visible evidence of
swelling, such as loss of normal landmarks or contours. It is
frequently helpful to compare the same joints on both sides
of the body visually to detect subtle evidence of swelling
and to appreciate symmetry.
Second, the examiner should palpate each joint. The
normal synovial membrane is too thin to palpate, whereas
the thickened synovial membrane in many chronic inflam-
matory arthritides, such as rheumatoid arthritis, may have a
“doughy” or “boggy” consistency. In some joints, such as the
knee, the extent of the synovial cavity can be delineated on
physical examination by compressing the fluid into one of
the extreme synovial recesses. The edge of the resulting bulge
may be palpated more easily. If this palpable edge is within
the anatomic confines of the synovial membrane and disap-
pears on release of the compression, the distention usually
represents synovial effusion; if it persists, it is an indication
of a thickened synovial membrane. Reliable differentiation
between synovial membrane thickening and effusion is not
always possible by physical examination, however. Ultraso-
nography is being used increasingly as an extension of the
physical examination, allowing the examiner to differenti-
ate between synovial proliferation and effusion.
TENDERNESS
In the musculoskeletal examination, tenderness indicates
unusual discomfort on palpating and putting pressure on
articular and periarticular tissues. Localizing the tender-
ness to palpation may assist the examiner in determining
whether the pathology is in an articular or a periarticular
structure, such as a fat pad, tendon attachment, ligament,
bursa, muscle, or skin. It can be useful to palpate structures
that are not involved to assess the importance of tenderness.
Finding tender joints in a patient who also has numerous
other myofascial tender points is less concerning for arthri-
tis than finding tender joints in a patient with no extra-
­articular tenderness.
LIMITATION OF MOTION
Limitation of motion is a common manifestation of articu-
lar disease; the examiner must know the normal type and
range of motion of each joint. Comparison of the affected
joint with an unaffected joint of the opposite extremity is
 518 DAVIS  |  History and Physical Examination of the Musculoskeletal System
invaluable to evaluate individual variation. The restricted
joint motion may be caused by changes in the joint itself or
in periarticular structures. To distinguish these possibilities,
it is crucial to compare the passive range of motion with
the active range of motion. If the passive range of motion is
greater than the active range of motion, the restriction may
be the result of pain, weakness, or the state of the articular
and periarticular structures. It also is important to distin-
guish muscle tension from a true limitation of joint motion,
emphasizing the importance of ensuring relaxation of the
patient. Pain that occurs with attempting to move a joint
passively to the limit of range of motion in one plane is
referred to as stress pain. Pain in the joint with attempted
active or passive range of motion usually indicates an abnor-
mality in the joint.
CREPITATION
Crepitation is a palpable or audible grating or crunching
sensation produced by motion. This sensation may or may
not be accompanied by discomfort. Crepitation occurs when
roughened articular or extra-articular surfaces are rubbed
together by active motion or by manual compression. Fine
crepitation is often palpable over joints involved by chronic
inflammatory arthritis and usually indicates roughening of
the opposing cartilage surfaces as a result of erosion or the
presence of granulation tissue. Coarse crepitation may be
caused by inflammatory or noninflammatory arthritis. Bone-
on-bone crepitus produces a higher frequency, palpable, and
audible squeak. Crepitation from within a joint should be
differentiated from cracking or popping sounds caused by
the slipping of ligaments or tendons over bony surfaces dur-
ing motion. The latter phenomena are usually less contribu-
tory to the diagnosis of joint disease and may be heard over
normal joints. In scleroderma, a distinct, coarse, creaking,
leathery crepitation may be palpable or audible over tendon
sheaths.
DEFORMITY
Deformity of the joints may manifest as a bony enlarge-
ment, articular subluxation, contracture, or ankylosis in
­nonanatomic positions. Deformed joints usually do not
function normally, frequently restrict activities, and may be
associated with pain, especially with overuse. Occasionally, a
deformed joint may function well, but is a cosmetic concern.
Joint deformities may be reversible or irreversible. Multiple
swan neck deformities of the fingers that can be corrected
with manipulation may indicate Jaccoud’s arthropathy of
lupus. In contrast, hand deformities in rheumatoid arthritis
generally are not correctable.
INSTABILITY
Joint instability is present when the joint has greater than
normal movement in any plane. Subluxation refers to a
joint in which there is partial displacement of the ­articular
surfaces, but still some joint surface-to-surface contact. A
dislocated joint has lost all cartilage surface-to-surface con-
tact. Instability is best determined by supporting the joint
between the examiner’s hands and stressing the adjacent
bones in directions in which the normal joint does not
move. The patient must be relaxed during the examination
because muscle tension may stabilize an otherwise ­unstable
joint. A knee with a deficient ligament might appear ­stable
if the patient contracts the quadriceps muscles during
­evaluation.
OTHER ASPECTS OF THE EXAMINATION
Examinations of the cervical spine and low back are discus­
sed in Chapters 39 and 41.
RECORDING THE JOINT EXAMINATION
Documentation of the joint examination is important in
making decisions about therapy, monitoring the activity
of arthritis, and determining the efficacy of interventions.
Many different recording methods have been described.
Abbreviations for each joint can be used, such as PIP for
the proximal interphalangeal joints. The S-T-L system has
been used historically to record the degree of swelling (S),
tenderness (T), and limitation of motion (L) of each joint
on the basis of a quantitative estimate of gradation.2 This
method remains useful, but is used less commonly today
because of the increasing reliance on electronic medical
records. It is easier to describe the joint findings in nar-
rative form; for example, “there is 2+ swelling of the sec-
ond and third MCP joints,” where grade 0 indicates no
swelling and grade 3 indicates maximal swelling for the
metacarpophalangeal joint. An alternative method is to
record the joint examination findings using a schematic
skeleton or homunculus. When accuracy is necessary, the
range of motion of individual joints may be measured using
a goniometer.
Joint counts are being used increasingly to monitor the
activity of inflammatory arthritides in practice and in clini-
cal trials.3 For monitoring disease activity of rheumatoid
arthritis, a 28-joint count for tenderness and swelling has
been recommended. To assess the tender joint count, the
examiner documents which joints the patient indicates
are painful on palpation with enough pressure to blanch
the nail bed of the examiner’s thumb and index fingers. To
assess the swollen joint count, the examiner documents
which joints have palpable soft tissue swelling or fluctu-
ance, excluding joints affected only by deformity or bony
hypertrophy. The 28-joint count4 includes the shoulders,
elbows, wrists, first to fifth metacarpophalangeal joints, first
to fifth proximal interphalangeal joints, and knees on both
sides of the body. Compared to more extensive joint counts,
the 28-joint count has the advantage of being quick and
easy to perform; however, it is limited by the fact that the
ankles and metatarsophalangeal joints are not included,
so active disease in the feet may be underestimated. The
28-joint count is used to calculate the disease activity score
28 (DAS28),5 which is a validated instrument to monitor
disease activity.
The function of the joints in normal use is not ­captured
by the assessments of tenderness, swelling, or range of
motion, so other examination techniques are necessary.
The patient’s grip strength can be measured by asking the
patient to squeeze a partially inflated (20 mm Hg) sphyg-
momanometer or by using a dynamometer. Other tests
are available that attempt to measure joint function by
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
assessing the patient’s ability to perform a coordinated
task (i.e., measuring the 50-foot walk time). The results of
such functional tests may vary, however. For observations
such as joint tenderness or grip strength, interobserver
variability is often greater than intraobserver variabil-
ity. There may be considerable intraobserver variability
in observations of the same patient, even over a short
interval. Biologic factors contribute to variability, such as
circadian changes in joint size and grip strength among
rheumatoid patients observed during a 24-hour interval.
These tests are best suited for clinical trials and tend to be
less useful in observing individual patients.
EXAMINATION OF SPECIFIC JOINTS
TEMPOROMANDIBULAR JOINT
The temporomandibular joint is formed by the condyle of
the mandible and the fossa of the temporal bone just ante-
rior to the external auditory canal. It is difficult to visualize
swelling of this joint. The examiner may palpate the joint by
placing a finger just anterior to the external auditory canal
and asking the patient to open and close the mouth and to
move the mandible from side to side.6 The presence of syno-
vial thickness or swelling of minimal or moderate degree
can be detected most easily if the synovitis is unilateral or
asymmetric compared with the other side. To assess vertical
movement of the temporomandibular joint, the examiner
should ask the patient to open the mouth maximally and
then measure the distance between the upper and lower
incisor teeth, normally 3 to 6 cm. Lateral movement can be
determined by using incisor teeth as landmarks. Audible or
palpable crepitus or clicking may be present in patients with
and without evidence of severe arthritis.
Many arthritides can affect the temporomandibular
joints, including juvenile and adult rheumatoid arthritis.
Children in whom these joints are affected may develop
micrognathia, resulting from arrested bone growth of the
mandible. Patients without inflammatory arthritis may
develop arthralgias of the temporomandibular joint, con-
sistent with the temporomandibular joint syndrome (see
Chapter 45). This syndrome is thought by some investi-
gators to result from bruxism and is likely to be a form of
­myofascial pain, similar to fibromyalgia.
CRICOARYTENOID JOINTS
The paired cricoarytenoid joints are formed by the articu-
lation of the base of the small pyramidal arytenoid car-
tilage and the upper posterolateral border of the cricoid
cartilage. The vocal ligaments (true vocal cords) are
attached to the arytenoid cartilages. The cricoarytenoid
joints are diarthrodial joints that normally move medially
and laterally and rotate during the opening and closing
of the vocal cords. Examination of these joints is done by
direct or indirect laryngoscopy. Erythema, swelling, and
lack of mobility during phonation may result from inflam-
mation of the joints. The cricoarytenoid joints may be
involved in rheumatoid arthritis, trauma, and infection.
Involvement in rheumatoid arthritis is more common than
clinically apparent. ­Symptoms may include hoarseness or a
sense of fullness or discomfort in the throat, which is worse
519
on speaking or swallowing. Severe airway obstruction may
rarely occur.
STERNOCLAVICULAR, MANUBRIOSTERNAL,
AND STERNOCOSTAL JOINTS
The medial ends of the clavicles articulate on each side of
the sternum at its upper end to form the sternoclavicular
joints. The articulations of the first ribs and the sternum
(sternocostal joints) are immediately caudal. The articu-
lation of the manubrium and body of the sternum is at
the level of the attachment of the second costal cartilage
to the sternum. The third through seventh sternocostal
joints articulate distally along the lateral borders of the
sternum. The sternoclavicular joints are the only articula-
tions in this group that are always diarthrodial; the others
are amphiarthroses or synchondroses. The sternoclavicular
joints are the only true points of articulation of the shoul-
der girdle with the trunk. These joints are just beneath the
skin; synovitis is usually visible and palpable. These joints
have only slight movement, which cannot be accurately
measured.
The sternoclavicular joints are commonly involved by
ankylosing spondylitis, rheumatoid arthritis, and degenera-
tive arthritis, although this involvement is often subclinical.
The sternoclavicular joint may be the site of septic ­arthritis,
especially in injection drug users. These joints should be
examined for tenderness, swelling, and bony abnormali-
ties. Tenderness of the manubriosternal or sternocostal
joints is much more frequent than actual swelling. Tender-
ness of these joints without actual swelling has been termed
­costochondritis; if actual swelling is present, Tietze’s syndrome
may be the term used.
ACROMIOCLAVICULAR JOINT
The acromioclavicular joint is formed by the lateral end
of the clavicle and medial margin of the acromion process
of the scapula. Arthritis of the acromioclavicular joint is
most commonly attributable to trauma leading to degen-
erative arthritis. Bony enlargement of this joint is ­typically
observed, but soft tissue swelling is not usually visible or
­palpable. Tenderness or pain with adduction of the arm
across the chest indicates pathology of the acromiocla-
vicular joint. Movement of this joint occurs with shoulder
motion, but is difficult to measure accurately. The acromio-
clavicular joint may be involved by rheumatoid arthritis or
spondyloarthropathies, although these are often not severe
enough to come to clinical attention.
SHOULDER
See Chapter 40.
ELBOW
The elbow joint is composed of three bony articulations
(Fig. 35-1). The principal articulation is the humeroulnar
joint, which is a hinge joint. The radiohumeral and ­proximal
radioulnar articulations allow rotation of the forearm.
To examine the elbow joint, the examiner places the
thumb between the lateral epicondyle and the olecranon
 520 DAVIS  |  History and Physical Examination of the Musculoskeletal System

Humerus

Medial
epicondyle

Lateral
epicondyle

Olecranon

Annular ligament
Coronoid
process

Radius Ulna

Figure 35-1  Diagram of the elbow. Posterior aspect of the elbow

joint showing radius and ulna in extension and distribution of sy-
novial membrane in distention. (From Polley HF, Hunder GG: Rheu-
matologic Interviewing and Physical Examination of the Joints, 2nd
ed. Philadelphia, WB Saunders, 1978. Used with permission of Mayo
Foundation for Medical Education and Research.)

process in the lateral paraolecranon groove and places one
or two fingers in the corresponding groove medial to the
olecranon. The examiner relaxes and passively moves the
elbow through flexion, extension, and rotation. One should
examine the skin around the elbow joint carefully, noting
abnormalities such as psoriatic plaques, rheumatoid nod-
ules, or tophi. It is useful to palpate the olecranon bursa
carefully to exclude the presence of small nodules or tophi.
Limitation of motion and crepitus should be noted. Syno-
vial swelling is most easily palpated as it bulges under the
examiner’s thumb when the elbow is passively extended.
Synovial membrane sometimes can be palpated over the
posterior aspect of the joint between the olecranon process
and distal humerus. Synovitis or effusion generally results in
limitation of elbow extension.
The olecranon bursa overlies the olecranon process of
the ulna. Olecranon bursitis is common after chronic local
trauma and in rheumatic diseases, including rheumatoid
arthritis and gout. A septic olecranon bursitis may occur.
A patient who has olecranon bursitis usually presents with
a swelling over the olecranon process, which is often ten-
der and may be erythematous. Sometimes a large collection
of fluid over the area is palpable as a cystic mass and often
requires aspiration and drainage. There is generally no pain
with elbow movement.
The medial and lateral epicondyles of the humerus are
the sites of attachment of the common flexor and extensor
tendons, controlling hand and wrist motion. Tenderness at
the epicondyles without swelling or other signs of inflam-
mation may indicate overuse tendinopathy, termed lateral
epicondylitis (tennis elbow) and medial epicondylitis (golfer’s
elbow). In lateral epicondylitis, discomfort can be elicited
by resisted supination of the forearm or resisted extension of
the pronated wrist.7 In medial epicondylitis, discomfort can
be elicited by resisted flexion of the supinated wrist.
To assess motor function of the elbow, flexion and
­extension can be assessed. The principal flexors of the elbow
are the biceps brachii (nerve roots C5 and C6), brachialis
(C5 and C6), and brachioradialis (C5 and C6) muscles. The
principal extensor of the elbow is the triceps brachii mus-
cle (C7 and C8). Occasionally, a patient may rupture the
attachment site of one of the heads of the biceps, ­resulting
in visible and palpable muscle swelling on the anterior
upper arm.
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
WRIST AND CARPAL JOINTS
The wrist is a complex joint formed by many articulations
between the radius, ulna, and carpal bones. The true wrist
or radiocarpal articulation is a biaxial ellipsoid joint formed
proximally by the distal end of the radius and the triangular
fibrocartilage and distally by a row of three carpal bones:
scaphoid (navicular), lunate, and triquetrum (triangular).
The distal radioulnar joint is a uniaxial pivot joint. The
midcarpal joints are formed by the junction of the proxi-
mal and distal rows of the carpal bones. The midcarpal and
carpometacarpal articular cavities often communicate. The
intercarpal joints refer to the articulations between the indi-
vidual carpal bones.
Movements of the wrist include flexion (palmar flexion),
extension (dorsiflexion), radial deviation, ulnar deviation,
and circumduction. Pronation and supination of the hand
and forearm occur primarily at the proximal and distal radio-
ulnar joints. The only carpometacarpal joint that moves to
a notable degree is the carpometacarpal joint of the thumb.
This joint is saddle-shaped and moves in three planes. Crep-
itus at this joint is common because it is frequently involved
in degenerative arthritis.
The wrist normally can be extended to 70 to 80 degrees
and flexed to 80 to 90 degrees. Ulnar and radial deviation
should allow 50 degrees (ulnar) and 20 to 30 degrees (radial)
of movement. Loss of extension is the most incapacitating
functional impairment of wrist motion.
The long flexor tendons of the forearm musculature cross
the volar aspect of the wrist and are enclosed in the flexor
tendon sheath under the flexor retinaculum (transverse
carpal ligament). The flexor retinaculum and the underly-
ing carpal bones form the carpal tunnel. The median nerve
passes through the carpal tunnel superficial to the flexor
tendons. The extensor tendons of the forearm musculature
are enclosed by six synovial-lined compartments.
The palmar aponeurosis (fascia) spreads out into the
palm from the flexor retinaculum. Dupuytren’s contracture
is a fibrosing condition affecting the palmar aponeurosis,
which becomes thickened and contracted and may draw
one or more fingers into flexion at the metacarpophalangeal
joint. The fourth finger is frequently affected first.
Swelling of the wrist may be caused by effusion or synovial
proliferation, or both, of the tendon sheaths (tenosynovitis),
the wrist joint, or a combination thereof. When swelling is
attributable to tenosynovitis, the swelling is localized to the
distribution of a particular tendon sheath or compartment
(i.e., ulnar swelling attributable to tenosynovitis of the
flexor carpi ulnaris tendon), tends to be more localized, and
moves with flexion and extension of the fingers. Articular
swelling tends to be more diffuse and protrudes anteriorly
and posteriorly from under the tendons.
Synovitis of the wrist is best detected by palpation of the
dorsal aspect of the joint. Accurate localization of the syno-
vial margins is difficult because of structures overlying the
volar and the dorsal aspects of the wrist. To examine the
wrist, the examiner should palpate the joint gently between
the thumbs dorsally and the fingers on the volar aspect.
Thickening or frank synovial proliferation of the synovium
should be noted. When this thickening or proliferation is
severe, the range of motion of the wrist joint is frequently
limited and associated with stress pain.
521
Figure 35-2  Subluxation of the wrist. Side view of the wrist of a patient
with rheumatoid arthritis. Note the prominence of the ulna.
A ganglion is a cystic enlargement arising from a joint
capsule. Ganglions characteristically occur at the volar or
dorsal aspects of the wrist between the tendons.
Subluxation of the ulna may develop as a result of severe
chronic inflammatory arthritis (Fig. 35-2). The subluxated
ulna appears as a prominence on the dorsomedial wrist.
Chronic irritation of the extensor tendons, primarily the
fourth and fifth finger extensor tendons, may cause these
tendons to rupture.
“Trigger fingers” secondary to stenosing tenosynovitis
can be detected by palpating crepitus or nodules along the
tendons in the palm while the patient slowly flexes and
extends the fingers.8 The patient usually gives a history of
the affected finger catching or locking with movement.
Tenosynovitis of the first extensor compartment, which
encloses the abductor pollicis longus and extensor pollicis
brevis muscles of the thumb, is known as de Quervain’s teno-
synovitis. Patients complain of pain at the radial aspect of
the wrist. Tenderness may be elicited by palpating near the
radial styloid process. The Finkelstein test for de Quervain’s
tenosynovitis is performed by asking the patient to make a
fist with the thumb enclosed in the palm of the hand, then
moving the wrist into ulnar deviation. Severe pain over the
radial styloid is a positive finding, often indicating stretch-
ing of the thumb tendons in a stenosed tendon sheath.
Carpal tunnel syndrome results from pressure on the
median nerve in the carpal tunnel. Carpal tunnel syndrome
is discussed in detail in Chapter 44.
Muscle function of the wrist may be measured by test-
ing flexion and extension and supination and pronation of
the forearm. The principal flexors of the wrist are the flexor
carpi radialis (nerve roots C6 and C7) and flexor carpi ulna-
ris (C8 and T1) muscles. Each of these muscles can be tested
separately. This testing can be accomplished if the exam-
iner provides resistance to flexion at the base of the second
metacarpal bone in the direction of extension and ulnar
deviation in the case of the flexor carpi radialis muscle and
resistance at the base of the fifth metacarpal in the direction
of extension and radial deviation in the case of the flexor
carpi ulnaris muscle. The principal extensors of the wrist
are the extensor carpi radialis longus (C6 and C7), extensor
carpi radialis brevis (C6 and C7), and extensor carpi ulnaris
(C7 and C8) muscles. The radial and ulnar extensor mus-
cles can be tested separately. The principal supinators of the
forearm are the biceps brachii (C5 and C6) and supinator
(C6) muscles. The principal pronators of the forearm are
 522 DAVIS  |  History and Physical Examination of the Musculoskeletal System
the pronator teres (C6 and C7) and pronator quadratus (C8
and T1) muscles.
METACARPOPHALANGEAL AND PROXIMAL
AND DISTAL INTERPHALANGEAL JOINTS
The metacarpophalangeal joints are hinge joints. Lateral
collateral ligaments that are loose in extension tighten
in flexion, preventing lateral movement of the digits.
The extensor tendons that cross the dorsum of each joint
strengthen the articular capsule. When the extensor tendon
of the digit reaches the distal end of the metacarpal head, it
is joined by fibers of the interossei and lumbricales muscles
and expands over the entire dorsum of the metacarpopha-
langeal joint and onto the dorsum of the adjacent phalanx.
This expansion of the extensor mechanism is known as the
extensor hood.
The proximal and distal interphalangeal joints also are
hinge joints. The ligaments of the interphalangeal joints
resemble those of the metacarpophalangeal joints. When
the fingers are flexed, the bases of the proximal phalanges
slide toward the palmar side of the heads of the metacarpal
bones. The metacarpal heads form the rounded prominences
of the knuckles, with the metacarpal joint spaces situated
about 1 cm distal to the apex of the prominences.
To examine the metacarpophalangeal joints, the exam-
iner should palpate the dorsal and volar aspects of each
joint in 20 to 30 degrees of flexion (Fig. 35-3). The skin on
the palmar surface of the hand is thick and covers a fat pad
between it and the metacarpophalangeal joint. This makes
palpation of the palmar surface of the joint difficult. It is
especially helpful in examining the small joints to compare
one with another to detect subtle synovitis. Gentle lateral
compression with force applied at the base of the second and
fifth metacarpophalangeal joints often elicits tenderness if
synovitis is present (squeeze test).
The proximal and distal interphalangeal joints are best
examined by palpating gently over the lateral and medial
aspects of the joint where the flexor and extensor tendons
do not interfere with assessment of the synovial membrane.
Figure 35-3  Palpation of the metacarpophalangeal joints is done with
the examiner’s thumbs palpating the dorsal aspect of the joint, while the
forefingers palpate the volar aspect of the metacarpal head. The joints
should be examined while the examiner holds the patient’s hand in a
relaxed position of partial flexion.
Alternatively, the joint can be compressed anteroposteri-
orly by the thumb and index finger of one of the examiner’s
hands, while the other thumb and index finger palpate for
synovial distention medially and laterally. The Bunnell test
is useful to differentiate synovitis of the proximal interpha-
langeal joints from tightening of the intrinsic muscles (see
Chapter 44).
Swelling of the fingers may result from articular or
periarticular causes. Synovial swelling usually produces
­symmetric enlargement of the joint itself, whereas extra-
articular swelling may be diffuse and extend beyond the
joint space. Asymmetric enlargement, involving only one
side of the digit or joint, is less common and usually indi-
cates an extra-articular process. Diffuse swelling of an entire
digit, known by the terms dactylitis and sausage digit, may
result from tenosynovitis and is seen most commonly in the
spondyloarthropathies, such as reactive arthritis or psoriatic
arthritis. Rheumatoid nodules are firm periarticular swell-
ings that ­frequently overlie the joints or bony prominences
in patients with chronic rheumatoid disease (Fig. 35-4).
Chronic swelling and ­ distention of the metacarpophalan-
geal joints tends to produce stretching and laxity of the
articular capsule and ligaments. This laxity, combined with
muscle imbalance and other forces, eventually results in the
extensor tendons of the digits slipping off the metacarpal
heads to the ulnar sides of the joints. The abnormal pull of
the displaced ­tendons is one of the factors that causes ulnar
deviation of the fingers in chronic inflammatory arthritis.
Swan neck deformity describes a finger with a flexion con-
tracture of the metacarpophalangeal joint, ­hyperextension
of the proximal interphalangeal joint, and flexion of the
distal interphalangeal joint. These changes are produced
by contraction of the interossei and other muscles that flex
the metacarpophalangeal joints and extend the ­ proximal
interphalangeal joints. This deformity is characteristic
of rheumatoid arthritis, but may be seen in other chronic
arthritides (Fig. 35-5).
Boutonnière deformity describes a finger with a flexion
contracture of the proximal interphalangeal joint associated
with hyperextension of the distal interphalangeal joint.
Figure 35-4  Chronic nodular rheumatoid arthritis. Swelling of both
wrists is present. Rheumatoid nodules are present on the right thumb,
right second proximal interphalangeal joint, right second and fourth
metacarpophalangeal joints, and left second distal interphalangeal joint.
The last-mentioned is notable for central purulence resulting from infec-
tion. A mild boutonnière deformity is present in the right third finger.
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS 523

Figure 35-5  Swan neck deformity in a patient with psoriatic arthritis.

Note hyperextension of the proximal interphalangeal joint and hyper-
flexion of the distal interphalangeal joint of the second digit. Also note
the psoriatic changes of the third and fourth fingernails.
Figure 35-6  Gout. Note the enlargement and deformity of the left sec-
ond proximal interphalangeal joint. This patient has gout, and aspiration
The deformity is common in rheumatoid arthritis and results of this joint confirmed the presence of uric acid crystals.
when the central slip of the extensor tendon of the proxi-
mal interphalangeal joint becomes detached from the base
of the middle phalanx, allowing palmar dislocation of the The patient’s fingernails should be inspected for ­evidence
lateral bands. The dislocated bands cross the fulcrum of the of clubbing or other abnormalities. Often in patients with
joint and act as flexors instead of extensors of the joint. psoriatic arthritis, ridging, onycholysis, or nail pitting is
Another abnormality is telescoping or shortening of the present. Occasionally, patients with osteoarthritis develop
digits produced by resorption of the ends of the phalanges a groove deformity of the nail on a digit with a Heberden
secondary to destructive arthropathy. This may be seen in node. (This nail deformity has been called a Heberden node
the arthritis mutilans form of psoriatic arthritis. Shortening nail.) The abnormality is believed to occur secondary to the
of the fingers is associated with wrinkling of the skin over synovial cyst encroachment on the nail bed by the evolv-
involved joints and is also called opera-glass hand or la main ing osteoarthritis process. With time, the nail may return
en lorgnette. to normal.
A mallet finger results from avulsion or rupture of the A crude but sometimes useful assessment of hand func-
extensor tendon at the level of the distal interphalangeal tion can be made by asking the patient to make a fist.
joint. With this deformity, the patient is unable to extend An estimate of the patient’s ability to form a full fist can
the distal phalanx, which remains in a flexed position. This be recorded as a percentage fist, with 100% being a com-
deformity frequently results from traumatic injuries. plete fist. A fist of 75% indicates that the patient can touch
The Murphy sign is a test for lunate dislocation. The the palm with the fingertips. The ability to oppose fingers,
patient is asked to make a fist. The third metacarpal head is
usually more prominent than the second and fourth. If the
third metacarpal is level with the second and fourth, the
finding is positive for lunate dislocation.
Involvement of the distal interphalangeal joints in rheu-
matoid arthritis is uncommon. Bony hypertrophy and osteo-
phyte formation are commonly seen, however, at the distal
and the proximal interphalangeal joints in patients with
osteoarthritis. Enlarged, bony, hypertrophic distal interpha-
langeal joints are called Heberden nodes, whereas similar
changes at the proximal interphalangeal joints are called
Bouchard nodes. These are usually easily differentiated from
the synovitis of inflammatory arthritis because, on palpa-
tion, the enlargement is hard or bony. In addition, signs of
inflammation are minimal. Heberden and Bouchard nodes
should be easily distinguished from rheumatoid ­ nodules,
but patients occasionally confuse these when describing
­swellings over joints. The examiner should be aware of
other causes of nodules on the hands, including tophaceous
Figure 35-7  Osteoarthritis of the hands. Note the advanced hypertro-
gout (Fig. 35-6) and, rarely, multicentric reticulohistiocyto- phic enlargement at the base of both thumbs. Both second distal inter-
sis. The first carpometacarpal joint also is often affected in phalangeal joints are notable for advanced bony enlargement, and more
osteoarthritis (Fig. 35-7). moderate changes are seen in the other interphalangeal joints.
 524 DAVIS  |  History and Physical Examination of the Musculoskeletal System
e­ specially the thumb, is crucial to hand function because
of the necessity to grasp or at least pinch for objects. If the
patient is unable to form a full fist, a demonstration of the
ability or inability to pinch or oppose fingers can be made by
asking the patient to pick up a small object.
Strength of the hands can be assessed crudely by ­asking
the patient to grip firmly two or more of the examiner’s
­fingers. More accurate measures of grip strength can be
made by using a dynamometer or having the patient squeeze
a partially inflated sphygmomanometer (at 20 mm Hg). It
is sometimes useful to test the strength of the fingers sepa-
rately. The prime movers of flexion of the second through
fifth metacarpophalangeal joints are the dorsal and palmar
interossei muscles (nerve roots C8 and T1). The lumbrica-
les muscles (C6, C7, and C8) flex the metacarpophalangeal
joints when the proximal phalangeal joints are extended.
The flexors of the proximal interphalangeal joints are the
flexor digitorum superficialis muscles (C7, C8, and T1), and
the flexor of the distal interphalangeal joints is the flexor
digitorum profundus muscle (C7, C8, and T1).
The prime extensors of the metacarpophalangeal joints
and interphalangeal joints of the second through fifth
­fingers are the extensor digitorum communis (nerve roots
C6, C7, and C8), the extensor indicis proprius (C6, C7,
and C8), and the extensor digiti minimi (C7) muscles. The
interossei and lumbricale muscles simultaneously flex the
metacarpophalangeal joints and extend the ­interphalangeal
joints. The dorsal interossei (C8 and T1) and abductor digiti
minimi (C8) muscles abduct the fingers, whereas the palmar
interosseous muscles adduct the fingers.
The thumb is moved by several muscles. The prime flexor
of the first metacarpophalangeal joint is the flexor pollicis
brevis muscle (nerve roots C6, C7, C8, and T1). The prime
flexor of the interphalangeal joint is the flexor pollicis ­longus
muscle (C8 and T1). The metacarpophalangeal joint of the
thumb is extended by the extensor pollicis brevis muscle,
and the prime extensor of the interphalangeal joint is the
extensor pollicis longus muscle (C6, C7, C8, and C9).
The principal abductors of the thumb are the abductor
pollicis longus (nerve roots C6 and C7) and the abductor
pollicis brevis (C6 and C7) muscles. Motion occurs ­primarily
at the carpometacarpal joint. The principal adductor of the
thumb is the adductor pollicis muscle (C8 and T1). Motion
occurs primarily at the carpometacarpal joint. The principal
movers in opposition of the thumb and fifth fingers are the
opponens pollicis (C6 and C7) and opponens digiti minimi
(C8 and T1) muscles.
Sensation and nerve injuries in the upper extremity are
discussed in Chapters 39 and 44.
HIP
The hip is a spheroidal or ball-and-socket joint formed by the
rounded head of the femur and the cup-shaped ­acetabulum
(see Chapter 42). Stability of the joint is ensured by the
fibrocartilaginous rim of the glenoid labrum and the dense
articular capsule and surrounding ligaments, including the
iliofemoral, pubofemoral, and ischiocapsular ligaments that
reinforce the capsule. Support also is provided by the power-
ful muscle groups that surround the hip. The principal hip
flexor is the iliopsoas muscle assisted by the sartorius and
the rectus femoris muscles. Hip adduction is accomplished
by the three adductors (longus, brevis, and magnus) plus the
gracilis and pectineus muscles. The gluteus medius is the
major hip abductor, whereas the gluteus maximus and ham-
strings extend the hip. There are several clinically ­important
bursae around the hip joint. Anteriorly, the iliopsoas bursa
lies between the psoas muscle and the joint surface. The tro-
chanteric bursa lies between the gluteus maximus muscle and
the posterolateral greater trochanter, and the ­ischiogluteal
bursa overlies the ischial tuberosity.
Examination of the hip should begin by observing the
patient’s stance and gait. The patient should stand in front
of the examiner so that the anterior iliac spines are ­visible.
Pelvic tilt or obliquity may be present and related to a
structural scoliosis, anatomic leg-length discrepancy, or hip
­disease.
Hip contractures may result in abduction or adduction
deformities. To compensate for an adduction contracture,
the pelvis is tilted upward on the side of the contracture.
This allows the legs to be parallel during walking and
weight bearing. With a fixed abduction deformity, the ­pelvis
becomes elevated on the normal side during standing or
walking. This elevation causes an apparent shortening of
the normal leg and forces the patient to stand or walk on the
toes of the normal side or to flex the knee on the abnormal
leg. Viewed from behind with the legs parallel, the patient
with hip disease and an adducted hip contracture may have
asymmetric gluteal folds secondary to pelvic tilt, with the
diseased side elevated. In this situation, the patient is unable
to stand with the foot of the involved leg flat on the floor. In
abduction contracture, the findings are reversed; with both
legs extended and parallel, the uninvolved side is elevated.
A hip flexion deformity commonly occurs in diseases of
the hip. Unilateral flexion of the hip in the standing position
reduces weight bearing on the involved side and relaxes the
joint capsule, causing less pain. This posture is best noted by
observing the patient from the side. There is a hyperlordotic
curve of the lumbar spine to compensate for lack of full hip
extension.
Gait should be assessed in the patient with possible
hip joint disease. With a normal gait, the abductors of
the weight-bearing leg contract to hold the pelvis level or
­elevate the non–weight-bearing side slightly. Two abnor-
malities of gait may be commonly observed in patients with
hip ­ disease. The most common abnormality seen with a
painful hip is the antalgic (limping) gait. In this gait, the
individual leans over the diseased hip during the phase of
weight bearing on that hip, placing the body weight directly
over the joint to avoid painful contraction of the hip abduc-
tors. In a Trendelenburg gait, with weight bearing on the
affected side, the pelvis drops and the trunk shifts to the
normal side. Although the antalgic gait is frequently seen
with painful hips and the Trendelenburg gait is seen in
patients with weak hip abductors, these gaits are not spe-
cific, and either may occur as a result of hip pain from one
of several causes. A mild Trendelenburg gait is seen often in
normal individuals.
The Trendelenburg test assesses the stability of the hip
together with the hip abductor muscle’s ability to stabilize
the pelvis on the femur.9 It is a measure of the gluteus medius
hip abductor strength. The patient is asked to stand bearing
weight on only one leg. Normally, the abductors hold the
pelvis level or the nonsupported side slightly elevated. If the
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
non–weight-bearing side drops, the test is positive for weak-
ness of the weight-bearing side hip abductors, especially the
gluteus medius muscle. This test is nonspecific and may be
observed in primary neurologic or muscle disorders and in
hip diseases that lead to weakness of the hip abductors.
The motion of the hip should be assessed with the
patient in the supine position. The range of motion of the
hip includes flexion, extension, abduction, adduction, inter-
nal and external rotation, and circumduction. The degree
of flexion permitted varies with the manner with which it is
assessed. When the knee is held flexed at 90 degrees, the hip
normally flexes to an angle of 120 degrees between the thigh
and long axis of the body. If the knee is held in extension,
the hamstrings limit the hip flexion to about 90 degrees.
The presence of a hip flexion contracture is ­ suggested by
persistence of lumbar lordosis and pelvic tilt masking the
­contracture by allowing the involved leg to remain in
­contact with the examination table. The Thomas test shows
the flexion contracture. In this test, the opposite hip is fully
flexed to flatten the lumbar lordosis and fix the pelvis. The
involved leg should be extended toward the table as far as
possible. The diseased hip’s flexion contracture becomes
more obvious and can be estimated in degrees from full
extension. Measurement for leg-length discrepancy is per-
formed with the patient supine and the legs fully extended.
Each leg is measured from the anterior superior iliac spine to
the medial malleolus. A difference of 1 cm or less is unlikely
to cause any abnormality of gait and may be considered
normal. In addition to true leg-length asymmetries, appar-
ent leg-length discrepancies may result from pelvic tilt or
abduction or adduction contractures of the hip.
Abduction is measured with the patient supine and the
leg in an extended position perpendicular to the pelvis.
­Pelvic stabilization is achieved by placing an arm across the
pelvis with the hand on the opposite anterior iliac spine.
With the other hand, the examiner grasps the patient’s
ankle and abducts the leg until the pelvis begins to move.
Abduction to about 45 degrees is normal. It is helpful to
compare one side with the other because the normal range
of motion may vary. Alternatively, the examiner could stand
at the foot of the table, grasp both the patient’s ankles, and
simultaneously abduct both legs. Abduction is commonly
limited in hip joint disease. Adduction is assessed by grasp-
ing the patient’s ankle and raising the leg off the examina-
tion table by flexing the hip enough to allow the tested leg
to cross over the opposite leg. Normal adduction is about 20
to 30 degrees. Hip rotation may be tested with the hip and
knee flexed to 90 degrees or with the leg extended. Nor-
mal hip external rotation and internal rotation are to 45
degrees and 40 degrees. The difference in rotation between
the flexed and extended hip is attributable to the increased
stabilization of the joint by the surrounding ligaments in
the extended position. Rotation decreases with extension.
To test hip rotation, the extended leg is grasped above the
ankle and rotated externally and internally from the ­neutral
­position. Limitation of internal rotation of the hip is a
­sensitive indicator of hip joint disease.
Extension is tested with the patient in the prone position.
Estimating hip extension can be difficult because some of
the apparent motion arises from hyperextension of the lum-
bar spine, pelvis rotation, motion of the buttock soft tissue,
and flexion of the opposite hip. The pelvis and lumbar spine
525
can be partially immobilized by placing an arm across the
posterior iliac crest and lower lumbar spine. The examiner
places the other hand under the thigh with the knee flexed
and hyperextends the thigh. Normal extension ranges from
10 to 20 degrees. Limitation of extension is often secondary
to a hip flexion contracture.
Swelling around the hip can only rarely be discerned
on examination. The Patrick test or FABERE maneuver is
a commonly used test to screen for pathologic conditions
of the hip. (FABERE is a mnemonic for the movements
required to elicit the response—flexion, abduction, external
rotation, and extension.) In this test, the patient lies supine,
and the examiner flexes, abducts, and externally rotates the
patient’s test leg so that the foot of the test leg is on top of
the opposite knee. The examiner then slowly lowers the test
leg toward the examining table. For a negative test result, the
test leg falls at least parallel to the opposite leg. A positive
test result occurs when the test leg remains above the oppo-
site leg. A positive result of the Patrick test may indicate hip
disease, iliopsoas tightness, or sacroiliac abnormality.
In children, two useful screening tests for congenital hip
disease are the Ortolani maneuver and the Galeazzi sign.
In the Ortolani maneuver, the examiner flexes the hips
and grasps the legs of a supine infant so that the examiner’s
thumbs are against the inner thighs and fingers are draped
over the outer (lateral) side of the thighs. With gentle
­traction, the hips are abducted and laterally rotated. Resis-
tance is usually felt at 30 to 40 degrees of lateral rotation
and abduction. In a positive result of the Ortolani test, a
click is felt before abduction to the normal 70 degrees can
be attained. The Ortolani test should not be done repeat-
edly because it can lead to damage of the articular cartilage
on the femoral head. The Galeazzi sign is useful for assessing
unilateral congenital hip dislocation in children younger
than 18 months. The child is placed supine with the knees
and hips flexed to 90 degrees. Both knees should normally
reside at the same level, and a positive test result is indicated
if one knee is higher than the other.
The iliotibial band is a part of the fascia lata extending
from the iliac crest, sacrum, and ischium over the greater
trochanter to the lateral femoral condyle, tibial condyle, and
fibular head and along the lateral intermuscular system, sep-
arating the hamstrings from the vastus lateralis muscle. The
tensor fasciae latae muscle may produce an audible snap as
it slips over the greater trochanter if the weight-bearing leg
moves from hip flexion and adduction to a neutral position,
as in climbing stairs. Most commonly observed in young
women, the snapping hip usually does not cause severe pain.
The Ober test evaluates the iliotibial band for contracture.
The patient lies on the side with the lower leg flexed at the
hip and knee. The examiner abducts and extends the upper
leg with the knee flexed at 90 degrees. The hips should be
slightly extended to allow the iliotibial band to pass over
the greater trochanter. The examiner slowly lowers the limb
with the muscles relaxed. A positive test result indicative of
an iliotibial band contracture occurs if the leg does not fall
back to the level of the table top.
A common cause of lateral hip pain is trochanteric bur-
sitis. Patients with this condition often complain of pain
and tenderness when they attempt to lie on the affected side
or climb stairs. The greater trochanter should be palpated
for tenderness and compared with the opposite side. In
 526 DAVIS  |  History and Physical Examination of the Musculoskeletal System
trochanteric bursitis, this area is usually exquisitely tender.
The pain of trochanteric bursitis is aggravated by actively
resisted abduction of the hip. Aching and tenderness over
the buttock area may be secondary to an ischial bursitis.
Other causes of lateral and posterior hip (buttock) discom-
fort include pain at muscle and tendon insertion sites.
Anterior hip and groin pain may be secondary to
hip abnormality, most commonly degenerative arthri-
tis. Decreased range of motion should be noted in these
patients. Other causes include iliopsoas bursitis, in which
swelling and tenderness may be noted in the middle third of
the inguinal ligament lateral to the femoral pulse. This pain
is aggravated by hip extension and reduced by flexion. The
bursitis may be a localized problem or represent extension of
hip synovitis. It is usually impossible to distinguish between
a localized bursitis and an extension of hip synovitis on the
basis of the physical examination. If the patient is tender
in the region of the iliopsoas bursa, but no swelling is pal-
pable, the examiner also should consider tendinitis of the
iliopsoas muscle. The inguinal region should be palpated
for other abnormalities, such as hernias, femoral aneurysms,
­adenopathy, tumor, and psoas abscess or masses.
Muscle strength testing should include the hip flexors,
extensors, abductors, and adductors. The primary hip flexor
is the iliopsoas muscle (nerve roots L2 and L3). Flexion may
be tested with the patient sitting at the edge of a table. The
examiner exerts downward pressure against the thigh proxi-
mal to the knee while the patient attempts to flex the hip.
The pelvis may be stabilized by the examiner’s other hand
placed on the ipsilateral iliac crest. Alternatively, with the
patient supine and holding the leg in 90 degrees of flexion at
the hip, the examiner may attempt to straighten the hip.
Hip extension is tested with the patient lying prone. The
primary hip extensor is the gluteus maximus muscle (L5
and S1). With the knee flexed to remove hamstring action,
the patient is instructed to extend the hip and thigh off the
­surface of the table as the examiner places a forearm across
the posterior iliac crest to stabilize the pelvis and applies
downward pressure to prevent the lateral trunk muscles
from elevating the pelvis and leg off the table.
Abduction may be tested with the patient prone or
supine. The patient should abduct the thigh and leg against
resistance from the examiner applied at the midthigh level.
The primary adductor is the adductor longus muscle
(nerve roots L3 and L4). The examiner holds the upper leg
proximal to the knee in slight abduction while the patient
resists and attempts to adduct the leg. Testing for abduction
and adduction also may be done in the two legs simultane-
ously. The patient lies supine with the legs fully extended
and the hips moderately abducted. To test abduction, the
patient actively pushes out against the examiner’s resistance
against the lateral malleoli. Adduction is tested by move-
ment against resistance at the medial malleoli.
KNEE
The knee is a compound condylar joint with three articu-
lations: the patellofemoral and the lateral and medial tib-
iofemoral condyles with their fibrocartilaginous menisci.10
The knee is stabilized by its articular capsule, the patel-
lar ligament, medial and lateral collateral ligaments, and
anterior and posterior cruciate ligaments. The collateral
ligaments provide medial and lateral stability, whereas the
cruciates provide anteroposterior and rotatory stability.
Normal knee motion is a combination of flexion or exten-
sion and rotation. With flexion, the tibia internally rotates,
and with extension, it externally rotates on the femur. The
surrounding synovial membrane is the largest of the body’s
joints; it extends 6 cm proximal to the joint as the suprapa-
tellar pouch beneath the quadriceps femoris muscle. There
are several important bursae around the knee, including
the superficial prepatellar bursa, the superficial and deep
infrapatellar bursae, the pes anserine bursa distal to the
medial tibial plateau, and the posterior medial semimembra-
nous and posterolateral gastrocnemius bursae. Knee exten-
sion is primarily mediated by the quadriceps femoris muscle;
knee flexion is mediated by the hamstrings. The biceps fem-
oris muscle externally rotates the lower leg on the femur,
whereas the popliteus and semitendinous muscles mediate
internal ­rotation.
In taking the history of a patient with knee complaints,
the patient should be asked about symptoms of locking,
catching, or giving way. Locking is the sudden loss of ability
to extend the knee; it is usually painful and may be associated
with an audible noise, such as a click or pop. It often implies
extensive intra-articular abnormality, including loose bod-
ies or cartilaginous tears. Catching refers to a subjective sen-
sation of the patient that the knee might lock; the patient
may experience a momentary interruption in the smooth
range of motion of the joint, but is able to continue with
normal motion after this brief hesitation. Catching usually
implies less abnormality than true locking and may occur in
various pathologic conditions. True give-way indicates that
the knee actually buckles and gives out in certain positions
or with certain activities. It is important to elicit the details
of the history to verify this common subjective complaint.
Patients often experience a sensation that the knee will
give out when it actually does not. Other patients say their
knees are “giving out” to describe severe pain that neces-
sitates stopping an activity. True give-way implies severe
intra-articular abnormality, such as an unstable joint from
ligamentous injury or incompetence.
Examination of the knees should always include observa-
tion of the patient while standing and walking. Deviation
of the knees, including genu varum (lateral deviation of the
knee joint with medial deviation of the lower leg), genu val-
gum (medial deviation of the knee with lateral deviation of
the lower leg), and genu recurvatum, is most easily appreci-
ated with the patient standing. The patient also should be
observed ambulating for evidence of gait abnormalities.
Inspection should be done with the patient standing and
supine. It is essential to compare side to side, noting any
asymmetry that may be caused by swelling or muscle atro-
phy. Suprapatellar swelling with fullness of the distal ante-
rior thigh that obliterates the normal depressed contours
along the sides of the patella usually indicates knee joint
effusion or synovitis. Localized swelling over the surface of
the patella is generally secondary to prepatellar bursitis (Fig.
35-8). Patellar alignment should be noted, including high-
riding or laterally displaced patellae. The examiner also
should inspect the knee from behind to identify popliteal
swelling owing to a popliteal or Baker cyst, most ­commonly
caused by medial semimembranous bursal swelling. If the
calves appear asymmetric, calf circumference should be
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
A B
Figure 35-8  Prepatellar bursitis. A, Note the cystic swelling overlying the right patella with the appearance of two separate compartments of bursal
swelling. B, High-resolution ultrasound image of the same patient showing collection of anechoic fluid with an internal septation.
measured and compared bilaterally. Popliteal cysts may
rupture and dissect down into the calf muscles, resulting in
enlargement and palpable fullness. Edema may be present if
the cyst causes secondary venous or lymphatic obstruction.
Acute rupture and dissection of a popliteal cyst can mimic
thrombophlebitis, with local pain, heat, redness, and swell-
ing. This is probably a more common cause of unilateral calf
swelling in patients with rheumatoid arthritis than is deep
venous thrombosis. The two conditions may be difficult to
distinguish on physical examination alone.
Quadriceps femoris muscle atrophy usually develops in
chronic arthritis of the knee. Atrophy of the vastus media-
lis muscle is the earliest change and may be appreciated by
comparing the two thighs for medial asymmetry and circum-
ference. Measurement of the thigh circumference should be
performed at 15 cm above the knee to avoid spurious results
owing to suprapatellar effusions.
Palpation of the knee should be performed with the joint
relaxed. This is usually best accomplished with the patient
supine and the knees fully extended and not touching. Pal-
pation should begin over the anterior thigh approximately
10 cm above the patella. To identify the superior margin of
the suprapatellar pouch, which is an extension of the knee
joint cavity, the examiner should palpate the anterior thigh,
moving distally toward the knee. Swelling, thickening, nod-
ules, loose bodies, tenderness, and warmth should be noted.
A thickened synovial membrane has a boggy, doughy con-
sistency, which differs from the surrounding soft tissue and
muscle. It is usually palpated earlier over the medial aspect
of the suprapatellar pouch and medial tibiofemoral joint. To
enhance detection of knee fluid, any fluid in the suprapatel-
lar pouch is compressed with the palm of the hand placed
just proximal to the patella. The synovial fluid forced into
the inferior distal articular cavity is palpated with the oppo-
site thumb and index finger laterally and medially to the
527
Right knee prepatellar bursa longitudinal
patella. If the examiner alternates compression and release
of the suprapatellar pouch, the synovial thickening can be
differentiated from a synovial effusion. An effusion inter-
mittently distends the joint capsule under the thumb and
index finger of the opposite hand, whereas synovial thicken-
ing does not.
The examiner should not compress the suprapatellar
pouch too firmly or push the tissues distally because the
patella or normal soft tissue, including the fat pads, fill the
palpated space and could be misinterpreted as synovitis or
joint swelling. With a large effusion, the patella can be bal-
loted by pushing it posteriorly against the femur with the
right forefinger, while maintaining suprapatellar compres-
sion with the left hand.
At the other extreme, effusions 4 to 8 mL can be detected
by eliciting the bulge sign. This test is performed with the
knee extended and relaxed. The examiner strokes or com-
presses the medial aspect of the knee proximally and later-
ally with the palm of a hand to move the fluid from the
area. The lateral aspect of the knee is tapped or stroked,
and a fluid wave or bulge appears medially (Fig. 35-9). A
so-called spontaneous bulge sign occurs if, on compression
along the medial side of the joint space, fluid reaccumulates
without any pressure or compression along the lateral side
of the joint.
The medial and lateral tibiofemoral joint margins are pal-
pated for tenderness and bony lipping or exostosis as can be
seen in degenerative joint disease. Palpating the joint mar-
gins can be done easily with the hip flexed to 45 degrees,
the knee flexed to 90 degrees, and the foot resting on the
examining table. Tenderness localized over the medial or lat-
eral joint margins may represent articular cartilage disease,
medial or lateral meniscal abnormality, or medial or lateral
collateral ligament injury. Other causes of tenderness include
pathologic conditions in the underlying bony structures.
 528 DAVIS  |  History and Physical Examination of the Musculoskeletal System
A riceps relaxed, and the knee in 30 degrees of flexion. The
B extension, a palpable or audible snapping may be heard, and
Figure 35-9  A and B, Demonstration of the bulge sign for a small sy-
novial knee effusion. The medial aspect of the knee has been stroked
to move the synovial fluid from this area (shaded depressed area in A). 
B shows a bulge in the previously depressed area after the lateral aspect
of the knee has been tapped.
Bursitis is another cause of localized tenderness around
the knee, and the two most common sites are the pes anser-
ine and prepatellar bursae. Exquisite local tenderness usu-
ally can be elicited if bursitis is present. Mild swelling also
may be appreciated. Occasionally, the prepatellar bursa can
become quite swollen. It is important not to interpret this
swelling mistakenly as knee joint synovitis. The two can be
differentiated because the bursal margins can be outlined by
palpation; other features of true joint effusion, such as the
bulge sign, are absent.
Patellofemoral malalignment is another common cause
of knee pain. It is more common in female patients because
of the wider Q angle caused by the broader female pelvis.
The Q angle is the angle formed between the quadriceps
and the patellar tendon. Patients with patellofemoral dis-
ease may complain of stiffness in the knee after a period
of flexion (the moviegoer sign) or have particular difficulty
with stair climbing. Some patients may experience a sensa-
tion of catching as the patella moves over the distal femur.
Patellar palpation is best performed with the knee extended
and relaxed. The patella is compressed and moved so that its
entire articular surface comes into contact with the underly-
ing femur. Slight crepitation may be observed in many nor-
mally functioning knees. Pain with crepitation may suggest
patellofemoral degenerative arthritis or chondromalacia
patellae.
Retropatellar pain occurring with active knee flexion
and extension and secondary to patellofemoral disease may
be differentiated from tibiofemoral articular pain. To test
this, the examiner should attempt to lift the patella away
from the knee, while passively moving the knee through
the range of motion. Painless motion during this maneuver
indicates that the patellofemoral joint is the likely source.
In addition, the “patellar grind” test is useful in patients
with extensive patellofemoral abnormality. In this test, the
examiner compresses the patella distally away from the fem-
oral condyles, while instructing the patient to contract the
quadriceps isometrically. Sudden patellar pain and quadri-
ceps relaxation indicate a positive test result. This test has
frequent false-positive results, however.
The patellar stability should be assessed. The Fairbanks
apprehension test is done with the patient supine, the quad-
examiner slowly pushes the patella laterally. A sudden con-
traction of the quadriceps and a distressed reaction from
the patient constitute a positive apprehension test result. A
patient who has had previous patella dislocations usually has
a positive apprehension test result. The patella also can be
examined for subluxation while the knee is moved through
a range of motion from full flexion to extension.
The plica syndrome also occasionally causes symptoms
that suggest patellofemoral disease. Plicae are bands of syno-
vial tissue, most often located on the medial side of the knee.
If present, a tender bandlike structure may be palpated par-
allel to the medial border of the patella. During flexion and
the patient may experience symptoms of catching. Many
plicae are asymptomatic, however, and are common, so they
may be considered a normal variant.
The normal knee range of motion should be from full
extension (0 degrees) to full flexion of 120 to 150 degrees.
Some normal individuals may be able to hyperextend to 15
degrees. Loss of full extension that is generally reversible fre-
quently occurs with a knee joint effusion, synovitis, or both.
A frequently permanent loss of extension owing to flexion
contracture is a common finding, however, that accompa-
nies chronic arthritis of the knee. In advanced arthritis,
such as in some cases of rheumatoid arthritis, a posterior
subluxation of the tibia on the femur may be observed.
Ligamentous instability is tested by applying valgus and
varus stress to the knee and by using the drawer test. The
knee should be extended and relaxed. The examiner per-
forms the abduction or valgus test by stabilizing the lower
femur, while placing a valgus stress on the knee by abduct-
ing the lower leg with the other hand placed proximal to
the ankle. A medial joint line separation with the knee fully
extended indicates a tear of the medial collateral ligament
plus the posterior cruciate ligament. The test is performed
with the knee in 30 degrees of flexion. If the test is nega-
tive at 0 degrees, but positive at 30 degrees, the instability
represents a tear of the medial collateral ligament with the
posterior cruciate ligament remaining intact. The adduction
or varus test is performed with the knee extended and again
at 30 degrees of flexion. Separation of the lateral joint line
indicates a lateral collateral ligament tear—either associ-
ated or not with a posterior cruciate ligament tear.
The degree of ligamentous laxity observed during testing
can be graded on a scale of 1 to 3. A mild or grade 1 instabil-
ity indicates that the joint surfaces separate 5 mm or less; for
moderate or grade 2 instability, a separation of 5 to 10 mm
is seen. Grade 3 instability is a separation greater than 10
mm. In cases of trauma, opening of the joint space indicates
ligamentous instability secondary to rupture or stretching of
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
the ligaments. In cases of chronic arthritis of the tibiofemo-
ral compartment, there may be apparent medial or lateral
separation as a result of the “pseudolaxity” created by loss of
cartilage and bone. If the ligaments are intact, the resulting
degree of valgus or varus displacement with stressing is not
any greater than in the normal knee.
The drawer test is performed with the hip flexed to 45
degrees and the knee flexed to 90 degrees. To stabilize the
knee, the examiner either sits on the foot while grasping
the posterior calf with both hands or supports the lower leg
between his or her lateral chest wall and forearm. The ante-
rior drawer test is performed by pulling the tibia forward.
More than 6 mm of movement is abnormal and may indi-
cate an anterior cruciate tear or laxity. Anterior subluxation
may represent more complex instability, however. Rotatory
instability of the knee also may exist. A positive result of
the anterior drawer test, in which the lateral tibial plateau
subluxates forward while the medial stays in normal posi-
tion, can represent anterolateral rotatory instability. If both
plateaus subluxate, tears of the middle third of the medial
lateral capsular ligaments may be present. If the subluxation
is not present with the tibia internally rotated, the posterior
cruciate ligament is intact. A positive result of the anterior
drawer test with the leg in external rotation represents a tear
of the medial capsular ligament.
The Lachman test is a modification of the anterior drawer
sign and tests for one-plane anterior instability. At least six
variations of this test have been described. In the test as
originally described, the patient lies supine with the tested
knee between full extension and 30 degrees of flexion. The
femur is stabilized with a hand of the examiner while the
hand pulls the proximal aspect of the tibia forward. A posi-
tive test result is indicated by a soft feel rather than a firm
end point when the tibia moves forward on the femur. A
positive result of the Lachman test may indicate an anterior
cruciate injury or abnormality in the posterior oblique liga-
ment or arcuate popliteus complex. A posterior drawer test
may be done with the patient positioned as for an anterior
drawer test, but the examiner pushes the tibia toward the
patient. A positive test result suggests damage to the poste-
rior cruciate ligament.
During the complete joint examination, tests for menis-
cal injury should be included. Symptoms suggesting a
meniscal tear include locking during joint extension,
clicking or popping during motion, and localized tender-
ness along the medial or lateral joint line. To examine the
menisci, the medial and lateral joint line should be pal-
pated with the lower leg internally rotated and the knee
flexed to 90 degrees. Localized tenderness over the medial
or lateral joint line suggests involvement of the medial or
lateral meniscus. The McMurray test12 evaluates for evi-
dence of meniscal tear, especially in the posterior half of
the menisci. The patient’s knee is placed in full flexion,
and the examiner places a hand over the knee with the
fingers along the side of the knee over the joint line and
the thumb along the other side. The other hand holds the
leg at the ankle and is used to rotate the lower leg medially
and to apply varus stress. This test can be done repeatedly,
with the knee in gradually decreasing degrees of flexion.
A palpable or audible snap suggests a tear of the medial
meniscus. The test can be done in a similar fashion by lat-
erally rotating the tibia and applying valgus stress to test for
529
a lateral meniscal injury. A positive result of a lateral test
also may represent a tear of the popliteus tendon, which
can accompany a lateral meniscal tear.
The Apley grind test also evaluates for a torn menis-
cus.11 With the patient lying prone and the knee flexed to
90 degrees, the examiner places downward compression on
the foot, while medially and then laterally rotating the tibia
on the femur. Pain elicited during this maneuver suggests a
meniscal tear.
The distraction test is performed by the examiner’s plac-
ing his or her knee on the patient’s posterior thigh to stabi-
lize the leg while applying an upward distractive force on the
foot. Pain from rotating the tibia suggests ligament damage.
A simple hyperflexion test is a useful screening test for
meniscal damage. If the examiner is able to hyperflex the
knee to more than 135 degrees without eliciting pain, it is
unlikely that serious cartilaginous injury is present. If pain
occurs with hyperflexion, the patient sometimes is able to
localize it medially or laterally, which often correlates with
the location of the meniscal injury. Although helpful, none
of these tests for meniscal injury is completely reliable when
verified by arthroscopy. Tenderness along the joint line is
most sensitive, but is less specific than manipulative tests
such as the McMurray test.
Muscle strength testing includes testing flexion supplied
by the hamstrings (i.e., the biceps femoris, semitendinosus,
and semimembranosus) (nerve roots L5 to S3) and exten-
sion supplied by the quadriceps femori (L2, L3, and L4).
The hamstrings are tested best with the patient prone and
attempting to move the knee from 90 degrees to maximal
flexion. The ankle should be kept in neutral position or dor-
siflexed to remove gastrocnemius action. With the leg exter-
nally rotated, the biceps femoris, which inserts on the fibula
and lateral tibia, is primarily tested, whereas flexion with
internal rotation tests the semitendinosus and semimembra-
nosus muscles, which insert on the medial side of the tibia.
Extension is tested with the patient sitting upright with the
knee fully extended. The examiner stabilizes the thigh with
downward pressure just proximal to the knee and places
downward pressure at the ankle to test the knee extensors.
ANKLE
The true ankle is a hinged joint, and movement is limited
to plantar flexion and dorsiflexion. It is formed by the distal
ends of the tibia and fibula and proximal aspect of the body
of the talus. Inversion and eversion occur at the subtalar
joint (see Chapter 43). The tibia forms the weight-bearing
portion of the ankle joint, whereas the fibula articulates on
the side of the tibia. The malleoli of the tibia and fibula
extend downward beyond the weight-bearing part of the
joint and articulate with the sides of the talus. The malleoli
provide medial and lateral stability by enveloping the talus
in a mortise-like fashion. The articular capsule of the ankle
is lax on the anterior and posterior aspects of the joint,
allowing extension and flexion, but it is tightly bound bilat-
erally by ligaments. The synovial membrane of the ankle on
the inside of the capsule usually does not communicate with
any other joints, bursae, or tendon sheaths.
The medial and lateral ligaments surrounding the ankle
contribute to medial and lateral stability of the joint. The
deltoid ligament, the only ligament on the medial side of the
 530 DAVIS  |  History and Physical Examination of the Musculoskeletal System

Tibialis anterior muscle Flexor digitorum longus muscle

Soleus muscle
Tibialis posterior muscle
Flexor hallucis longus muscle
Tibia
Posterior tibial artery
Inferior extensor retinaculum
Posterior tibial nerve

Tendo calcaneus
Deltoid ligament
Flexor retinaculum
Extensor hallucis longus
tendon sheath
Abductor hallucis
Figure 35-10  Diagram of the ankle. ­Medial brevis muscle (cut)
aspect of the ankle shows the ­ relationship
between the tendons, ligaments, artery,
and nerve. (From Polley HF, Hunder GG: Tibialis posterior tendon
Rheumatologic Interviewing and Physical Ex-
amination of the Joints, 2nd ed. Philadelphia, Flexor digitorum longus tendon
WB Saunders, 1978. Used with permission of Tibialis anterior tendon
Mayo Foundation for Medical Education and
Research.) Flexor hallucis longus tendon

ankle, is a triangle-shaped fibrous band that resists eversion

of the foot. It may be torn in eversion sprains of the ankle.
The lateral ligaments of the foot consist of three distinct
bands forming the posterior talofibular, the calcaneofibular,
and the anterior talofibular ligaments. These ligaments may
be injured in inversion sprains of the ankle.
All tendons crossing the ankle joint lie superficial to the
articular capsule and are enclosed in synovial sheaths for
part of their course across the ankle. On the anterior aspect
of the ankle, the tendons and synovial tendon sheaths of the
tibialis anterior, extensor digitorum longus, peroneus tertius,
and extensor hallucis longus muscles overlie the articular
capsule and synovial membrane. On the medial side of the
ankle, posterior and inferior to the medial malleolus, lie the
flexor tendons and tendon sheaths of the tibialis posterior,
flexor digitorum longus, and flexor hallucis longus muscles
(Fig. 35-10). All three of these muscles plantar flex and
supinate the foot. The tendon of the flexor hallucis longus is
located more posteriorly than the other flexor tendons and Figure 35-11  Achilles tendinitis in a patient with reactive arthritis. Note
lies beneath the Achilles tendon for part of its course. The swelling of the left Achilles tendon insertion caused by active enthesitis
calcaneus tendon (Achilles tendon) is the common tendon and milder swelling of the right Achilles tendon insertion.
of the gastrocnemius and soleus muscles and inserts into
the posterior surface of the calcaneus, where it is subject to side of the ankle, the peroneal retinaculum forms a superior
external trauma, various inflammatory reactions, and irrita- and an inferior fibrous band. These bands bind down ten-
tions from bone spurs beneath it. On the lateral aspect of dons of the peroneus longus and peroneus brevis muscles as
the ankle, posterior and inferior to the lateral malleolus, a they cross the lateral aspect of the ankle.
synovial sheath encloses the tendons of the peroneus lon- Synovial swelling of the ankle joint is most likely to cause
gus and peroneus brevis. These muscles extend the ankle fullness over the anterior or anterolateral aspect of the joint
(plantar flex) and evert (pronate) the foot. Each of the ten- because the capsule is more lax in this area. Mild swelling
dons adjacent to the ankle may be involved separately in of the joint may not be apparent on inspection because of
­traumatic or disease processes. the many structures crossing the joint superficially. Efforts
Three sets of fibrous bands or retinacula hold down the should be made to differentiate superficial linear swelling
tendons that cross the ankle in their passage to the foot. localized to the distribution of the tendon sheaths from more
The extensor retinaculum consists of a superior part (trans- diffuse fullness and swelling attributable to involvement of
verse crural ligament) in the anterior and inferior portions the ankle joint. Swelling of the heels may be observed from
of the leg and an inferior part in the proximal portion of the behind the standing patient and may be caused by enthesitis
dorsum of the foot. The flexor retinaculum is a thickened of the Achilles tendon insertion, which can occur in spon-
fibrous band on the medial side of the ankle. On the lateral dyloarthropathies (Fig. 35-11).
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
It is difficult to observe synovitis of the intertarsal joints.
Intertarsal joint synovitis may produce an erythematous
puffiness or fullness over the dorsum of the foot.
From the normal position of rest in which there is a right
angle between the leg and foot, labeled 0 degrees, the ankle
normally allows about 20 degrees of dorsiflexion and about
45 degrees of plantar flexion. Inversion and eversion of the
foot occur mainly at the subtalar and other intertarsal joints.
From the normal position of the foot, the subtalar joint nor-
mally permits about 20 degrees of eversion and 30 degrees of
inversion. To test the subtalar joint, the examiner grasps the
calcaneus with a hand and attempts to invert and evert it,
holding the ankle motionless.
A general assessment of muscular strength of the ankle
can be obtained by asking the patient to walk on toes and
on heels. If the patient can walk satisfactorily on the toes
and on the heels, the muscle strength of the flexors and
extensors of the ankle can be considered normal. If this
cannot be accomplished, it is desirable to test the muscles
individually.
The principal flexors of the ankle are the gastrocnemius
(nerve roots S1 and S2) and the soleus (S1 and S2) muscles.
The principal extensor (dorsiflexors) of the ankle is the tibi-
alis anterior muscle (L4, L5, and S1). The tibialis posterior
muscle (L5 and S1) is the principal inverter. To test the
tibialis posterior muscle, the foot should be in plantar flex-
ion. The examiner applies graded resistance on the medial
border of the forefoot while the patient attempts to invert
the foot. The principal everters of the foot are the peroneus
longus (L4, L5, and S1) and peroneus brevis (L4, L5, and
S1) muscles.
FOOT
See Chapter 43.
MUSCLE EXAMINATION
A general examination of the patient should be done, look-
ing for any signs of systemic illness. This should include an
examination of the skin, in which signs of pallor (sugges-
tive of anemia), and rashes that suggest lupus, vasculitis, and
dermatomyositis are noted. The patient should be appropri-
ately disrobed for muscle examination. The patient should
be assessed for appearance of the muscles, including bulk,
tone, and tenderness.
Muscle bulk should be compared on one side of the body
with the other to look for any asymmetry, hypertrophy, or atro-
phy. The distribution of the atrophy should be noted because
this may indicate the underlying cause. Distal atrophy can
531
be seen in patients with motor neuron disease. Fasciculations
also are seen more commonly in this condition. Muscle tone
also should be evaluated. Increased muscle tone and spasticity
can be seen with demyelinating conditions. Muscle rigidity
may be an indication of Parkinson’s disease. Muscle tender-
ness may be noted in patients with true weakness, such as
infectious myopathies. Other conditions that do not cause
true weakness, such as fibromyalgia, also can be associated
with tender muscles.
Strength testing should be done on distal and proximal
muscle groups. Commonly tested areas include plantar flex-
ion and dorsiflexion of the foot; hip abduction, adduction,
and flexion; finger abduction; arm flexion; shoulder abduc-
tion; and neck flexion and extension. The patient should be
seated, and both sides of the body should be compared for
asymmetric weakness. Typically, resistance is noted against a
force applied by the examiner. The examiner should attempt
to confirm whether or not true weakness is present and, if it
is, in what distribution.
A neurologic examination also should be done. The
examination should include reflex testing and testing for
sensory loss. Typically, patients with inflammatory myopa-
thies should not have severe abnormalities on examination
of nerve function.
REFERENCES
  1. Woolf AD: How to assess musculoskeletal conditions: History and
physical examination. Best Pract Res Clin Rheumatol 17:381-402,
2003.
  2. Polley HF, Hunder GG: Rheumatologic Interviewing and Physical
Examination of the Joints. 2nd ed. Philadelphia, WB Saunders, 1978.
  3. Sokka T, Pincus T: Quantitative joint assessment in rheumatoid
arthritis. Clin Exp Rheumatol 23:S58-S62, 2005.
  4. Fuchs HA, Brooks RH, Callahan LF, et al: A simplified twenty-eight-
joint quantitative articular index in rheumatoid arthritis. Arthritis
Rheum 32:531-537, 1989.
  5. Prevoo ML, van’t Hof MA, Kuper HH, et al: Modified disease ac-
tivity scores that include twenty-eight-joint counts: Development
and validation in a prospective longitudinal study of patients with
rheumatoid arthritis. Arthritis Rheum 38:44-48, 1995.
  6. Doherty M, Hazleman BL, Hutton CW, et al: Rheumatology Exami-
nation and Injection Techniques, 2nd ed. London, WB Saunders,
1999.
  7. Malanga GA, Nadler SF: Musculoskeletal Physical Examination: An
Evidence-Based Approach. Philadelphia, Mosby, 2006.
  8. Moore G: Atlas of the Musculoskeletal Examination. Philadelphia,
American College of Physicians, 2003.
  9. Waldman SD: Physical Diagnosis of Pain: An Atlas of Signs and
Symptoms. Philadelphia, WB Saunders, 2006.
10. Press JM, Casazza BA, Young JL: Knee ligament injuries: Making the
diagnosis, restoring use. J Musculoskel Med 13:14-28, 1996.
11. Gross J, Fetto J, Rosen E: Musculoskeletal Examination, 2nd ed.
Malden, Mass, Blackwell Scientific, 2002.
 36
KEY POINTS
The most common causes of acute monarthritis are infection,
crystal-induced arthritis, or the onset of a chronic inflamma-
tory arthropathy.
Arthritis causes stiffness, reduced range of motion, and pain
during normal use.
Inflammatory arthritis is characterized by morning stiffness
that improves with motion.
Mechanical joint pain usually worsens with activity and
­improves with rest.
An extremely rapid onset of pain (seconds to minutes)
­suggests an internal derangement, fracture, or trauma.
An acute onset over hours to 2 days is typical of most forms
of inflammatory arthritis.
The most important laboratory test for acute monarthritis
is synovial fluid analysis.
Acute monarticular gout is defined by intracellular, negatively
birefringent, needle-shaped crystals (sodium urate).
With rare exceptions, any joint disorder is capable of
­presenting initially as monarthritis. Monarthritis therefore
represents a diagnostic challenge to even the most experi-
enced clinician. The disorder often remains incompletely
understood after the initial evaluation. Nonetheless, it
is almost always possible to identify patients who require
­vigorous evaluation and treatment to prevent rapid disease
progression, such as those with suspected septic arthritis.
The physician can proceed in a measured and systematic
manner with the remainder of patients, in whom the short-
term clinical course and response to simple therapeutic
measures may provide additional useful information. This
chapter is intended to aid the clinician in distinguishing
true arthritis from syndromes that also manifest with pain
in the ­surrounding joint structures, in narrowing the list of
diagnostic possibilities based on the clinical presentation,
and in using diagnostic tests effectively. Special attention is
given to common entities that present as acute ­inflammatory
arthritis suggesting joint sepsis.
DIFFERENTIAL DIAGNOSIS
Confronted with a patient complaining of pain or ­swelling
in the region of a single joint, the physician must first
attempt to localize the anatomic site of the abnormality
(Fig. 36-1). Joint pain can be the result of abnormalities in
the joint itself, adjacent bone, surrounding ligaments, ten-
dons, bursae, or soft tissues. It can also be referred, resulting
Monarticular Arthritis
Joseph Golbus
from nerve root impingement, an entrapment neuropathy,
or even pathology in another joint. Pain from hip arthri-
tis, for example, can be referred entirely or partially to the
knee. It is also important to remember that the differential
diagnosis of an acute monarthritis includes most causes of
polyarticular arthritis, which can also present with a single
painful or swollen joint.
Arthritis involving a diarthrodial joint causes stiffness,
reduced range of motion, and pain during normal use. With
few exceptions (e.g., the patellofemoral joint in chondroma-
lacia1), intra-articular abnormalities can be detected during
passive and active range of motion. The patient history and
physical examination are essential in determining the cause
of the arthritis. Inflammatory forms of arthritis are charac-
terized by stiffness of the joint that is most noticeable in the
morning (i.e., morning stiffness) or after a period of inactiv-
ity (i.e., gelling) and that improves with motion. Inflam-
matory arthritis is often associated with systemic symptoms
such as fever or malaise. Joint pain due to mechanical fac-
tors usually worsens with activity, improves with rest, and is
not associated with systemic symptoms (Table 36-1).
During physical examination, it is important to compare
the abnormalities (e.g., swelling, warmth, redness) with
findings in the contralateral joint. Although most inflamed
joints show signs of inflammation, deeper-seated articu-
lations, such as the shoulders, hips, and sacroiliac joints,
may not. Effusions almost always result from intra-­articular
pathology, although they may accompany osteomyelitis,
fractures, or tumors.2 The presence of excess synovial fluid
does not specifically indicate joint inflammation unless
the white blood cell count is elevated. The disorders caus-
ing monarthritis are listed in Table 36-2. The conditions
discussed in the following sections may be confused with
arthritis (Table 36-3).
INTERNAL DERANGEMENT
Torn menisci or ligaments or loose bodies may ­episodically
wedge into the joint, producing clicking, locking, or a
sensation of “giving way.” These conditions may pre-
cede or accompany degenerative arthritis and can be a
consequence of inflammatory arthritis when fronds of
proliferative synovium become lodged within the joint.
Symptoms are frequently intermittent. They can often
be elicited on physical examination by repeatedly flex-
ing and extending the joint in various degrees of inter-
nal and external rotation.3 Internal derangements of the
hip, particularly tears of the acetabular labrum, are easily
overlooked. Such tears may be treated arthroscopically
by an orthopedic surgeon with sufficient experience in
hip arthroscopy.
533
 534 GOLBUS  |  Monarticular Arthritis

“Acute monarticular
arthritis”

Careful exam
Periarticular Complete history and reveals
syndrome physical exam polyarticular
arthritis

Tendinitis,
bursitis, strain, True monarticular
sprain, arthritis
osteomyelitis, soft
tissue infection

Acute Fracture,
Yes changes avulsion
Significant
trauma or focal Radiograph
bone pain? Chronic OA, CPPD
changes

No Severe
CBC, ESR, and symptoms Ultrasound-guided
Effusion or
physical aspiration or
inflammation?
exam CT/MRI

Yes

Synovial fluid Synovial fluid

WBC > 5000 Arthrocentesis bloody

Acute
inflammatory Synovial –
MRI Arthroscopy
arthritis fluid WBC
< 1000
+
Figure 36-1  Initial approach to acute
monarticular arthritis. CBC, complete
blood count; CPPD, calcium pyrophos- Non- Occult fracture, Internal
phate deposition disease; CT, computed inflammatory tumor, internal derangement
tomography; ESR, erythrocyte sedimen- arthritis derangement
tation rate; MRI, magnetic resonance (OA, internal
imaging; OA, osteoarthritis; WBC, white derangement)
blood cell count.

BONE PAIN
Bone pain usually results from a disease process involving
the periosteum or the marrow space as a result of sensory
nerves located in these areas. Bone pain is commonly caused
by fractures, osteomyelitis, or periostitis, such as occurs in
pulmonary hypertrophic osteoarthropathy, hemoglobinopa-
thies, hematologic malignancy,4 primary or metastatic bone
tumors, and occasionally with infiltrative processes such as
Paget’s disease. Characteristically, bone pain is accompanied
by tenderness to pressure over the involved periosteum or
pain on weight bearing. When symptoms are long standing,
radiographic findings are typically abnormal. Earlier dis-
ease may be identified by radionuclide scanning, computed
tomography (CT), or magnetic resonance ­imaging (MRI).
Paget’s disease may be associated with both bone pain and
arthritic symptoms as a result of the expansion and defor-
mity of subchondral bone and cartilage, particularly in the
hips and knees.5
TENDINITIS OR BURSITIS
Findings of tendinitis or bursitis are usually localized to one
area around the joint.6 Local tenderness and pain often
increase more with active motion than with passive motion
of the joint, because active motion places more stress on
the involved periarticular structures. An exception to this
rule is supraspinatus tendinitis of the shoulder, in which
passive and active motion may produce similar pain, and
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS 535

Table 36-1  Useful Clinical Features in the Initial Evaluation of Acute Monarticular Pain
Noninflammatory Joint Pain
(Osteoarthritis, Internal
Feature Tendinitis Derangement) Systemic Rheumatic Disease
Symptoms
Morning stiffness Localized, brief Localized, brief Often greater than 15 min
Constitutional symptoms None None Present (fever, malaise)
Peak period of discomfort With use After prolonged use After prolonged inactivity
Locking or instability Unusual, except with rotator   Suggests internal derangement  None
cuff tears or trigger fingers or loose body
Signs
Tenderness Localized, periarticular Mild, over joint line Diffuse over exposed joint space
Inflammation Over tendon or bursa Unusual Common
Instability Uncommon Occasional Uncommon
Multisystem disease No (occasionally with gonococcal No Often
infection)
Modified from American College of Rheumatology Ad Hoc Committee on Clinical Guidelines: Guidelines for the initial evaluation of the adult patient with
acute musculoskeletal symptoms. Arthritis Rheum 39:1-8, 1996.

Table 36-2  Differential Diagnosis of Monarticular ­Arthritis

Usually Monarticular Often Polyarticular
Septic arthritis Common
Bacterial Rheumatoid arthritis
Tuberculous Osteoarthritis
Fungal Psoriatic arthritis
Lyme disease Reiter’s syndrome (idiopathic and human immunodeficiency virus)
Crystal disease Calcium pyrophosphate deposition disease
Gout Chronic articular hemorrhage
Pseudogout Most juvenile rheumatoid arthritis and juvenile spondylitis
Internal derangement Erythema nodosum, sarcoid
Ischemic necrosis Serum sickness
Hemarthrosis Acute hepatitis B
Coagulopathy Rubella
Warfarin (Coumadin) Henoch-Schönlein purpura
Trauma or overuse Systemic lupus erythematosus
Pauciarticular juvenile rheumatoid arthritis Lyme disease
Neuropathic Parvovirus
Congenital hip dysplasia Dialysis arthropathy
Osteochondritis dissecans Other crystal-induced arthropathies
Reflex sympathetic dystrophy Undifferentiated connective tissue disease
Hydroxyapatite deposition Rare
Hemoglobinopathies Relapsing polychondritis
Loose body Enteropathic disease
Palindromic rheumatism Ulcerative colitis
Paget’s disease involving joint Regional enteritis
Stress fracture Bypass arthritis
Osteomyelitis Whipple’s disease
Osteogenic sarcoma Chronic sarcoidosis
Metastatic tumor Hyperlipidemia types II and IV
Synovial osteochondromatosis Still’s disease
Pigmented villonodular synovitis Pyoderma gangrenosum
Plant thorn synovitis Pulmonary hypertrophic osteoarthropathy
Familial Mediterranean fever Chondrocalcinosis-like syndromes, polymyalgia rheumatica due to ochronosis,
Synovioma hemochromatosis
Synovial metastasis Wilson’s disease
Intermittent hydrarthrosis Rheumatic fever
Pancreatic fat necrosis Paraneoplastic syndromes
Gaucher’s disease
Behçet’s disease
Regional migratory osteoporosis
Giant cell arteritis or Sea urchin spine
Amyloidosis (myeloma)
 536 GOLBUS  |  Monarticular Arthritis

Table 36-3  Regional Periarticular Syndromes

Periarticular Monarticular
Region Syndrome Syndrome
Jaw Temporomandibular joint dysfunction   Temporal arteritis
(myofascial pain syndrome) Molar dental problems
Preauricular lymphadenitis Parotid swelling
Shoulder Subacromial bursitis Pancoast’s tumor
Long-head bicipital tendinitis Brachial plexopathy
Rotator cuff tear Cervical nerve root injury
Elbow Olecranon bursitis Ulnar nerve entrapment
Epicondylitis
Wrist Extensor tendinitis   Carpal tunnel syndrome
(including de Quervain’s tenosynovitis)
Gonococcal tenosynovitis
Hand Palmar fasciitis  
(Dupuytren’s contracture)
Ligamentous or capsular injury
Hip Greater trochanteric bursitis Meralgia paresthetica
Adductor syndrome Deep infection
Ischial bursitis Paget’s disease
Fascia lata syndrome Neoplasm
Knee Anserine bursitis Neoplasm
Prepatellar bursitis Osteomyelitis
Meniscal injury
Ligamentous tear, laxity
Baker’s cyst
Ankle Peroneal tendinitis Hypertrophic pulmonary
Achilles tendinitis ­osteoarthropathy
Retrocalcaneal bursitis Tarsal tunnel syndrome
Calcaneal fasciitis
Sprain
Erythema nodosum
Foot Plantar fasciitis Morton’s neuroma
Pes planus (“fallen arches”) Vascular insufficiency
Cellulitis

localized tenderness may be absent. The clinical findings
seen with tendinitis or bursitis can usually be reduced or
eliminated by local instillation of lidocaine. The presence
of a puncture site; a history of glucocorticoid injection; an
adjacent source of infection, such as an ulcerated rheuma-
toid nodule7; or severe inflammation may signify infectious
bursitis.8 Isolated tendinitis less commonly results from
hematogenous spread of infection, except in the case of
disseminated gonococcal disease, which commonly mani-
fests with dorsal tenosynovitis of the wrists9; a similar septic
tenosynovitis may complicate brucellosis.10 Infected olecra-
non or prepatellar bursae commonly mimic septic arthritis.
These conditions can often be identified by high-resolution
ultrasonography.11
NEUROPATHIC PAIN
Compression or irritation of peripheral nerves may
p­ roduce pain referred to the region of joints, such as pain
radiating from the wrist to the palmar surface of the first
four digits in carpal tunnel syndrome,12 pain in the hip
region in lumbosacral radiculopathies, or shoulder pain
with brachial plexopathies. Such symptoms are usually in
the distribution of a peripheral nerve and tend to follow
an irregular time course, with sudden exacerbations, par-
ticularly at night. Maneuvers that compress the affected
nerve at the site of injury, such as straight leg raising or
percussion of the median nerve at the wrist, are help-
ful when they exactly reproduce the patient’s pain in
the ­ distribution of a peripheral nerve. Diffuse polyneu-
ropathies may produce pain that is poorly localized and
superficially resembles joint pain in a stocking-glove dis-
tribution. Pain that localizes exactly to a joint in the set-
ting of a polyneuropathy may be related to neuropathic
joint disease or reflex sympathetic dystrophy, or it may
have an unrelated cause.13
SOFT TISSUE INFECTION
Soft tissue infections may simulate arthritis, particularly
when they occur in the region of deeply buried joints that
are difficult to examine. Hip pain may result from cellulitis,
pyomyositis, psoas or retroperitoneal abscess, or intrapelvic
pathology such as diverticulitis. Fever and the acute onset
of hip pain and stiffness with normal radiographic and syno-
vial fluid findings suggest soft tissue or bone infection. Pain
referred to the sacroiliac joint may result from a variety of
soft tissue infections, including perirectal abscesses. Infec-
tious processes in these locations manifest with unremitting
severe pain, marked elevation of the erythrocyte sedimenta-
tion rate, and variably severe sys­temic toxicity.14,15 Physical
examination may reveal muscular rigidity and guarding, local
tenderness, increased girth of the affected limb, or drain-
ing sinuses. Imaging studies such as radionuclide ­scanning,
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
u­ ltrasonography, CT, or MRI may be essential in identifying
deep infections.
MUSCULAR PAIN SYNDROMES
Muscular pain syndromes (e.g., myofascial pain,
fi­ bromyalgia) consist of local or diffuse muscle tenderness
that causes pain in characteristic regional zones. These
can result in a variety of complaints, including headache;
jaw, neck, or low back pain; and occasionally joint pain,
particularly in the shoulder. They tend to be diffuse and
symmetric and are often associated with complaints out-
side of the musculoskeletal system, such as fatigue, inter-
mittent diffuse paresthesias, and irritable bowel syndrome
(see Chapter 38). Trigger points represent specific sites of
exaggerated sensitivity to pressure and are often seen in
patients with fibromyalgia. Myofascial syndromes rarely
cause monarticular pain but may be associated with exag-
gerated distress associated with benign causes of monar-
ticular pain.
PATIENT HISTORY
IS THE ARTHRITIS ACUTE OR CHRONIC?
An extremely rapid onset of pain (over seconds or
­ inutes) suggests an internal derangement, fracture,
m Although the differential diagnosis of acute inflammatory
trauma, or loose body. An acute onset over several hours
to 2 days is typical of most forms of inflammatory arthri-
tis, particularly bacterial infection and crystal-induced
synovitis. When a careful history reveals marked
­worsening of long-standing symptoms in a joint, it is
important to distinguish exacerbations of ­ preexisting
disease (e.g., worsening of degenerative joint disease
with excessive use) from a second superimposed process
(e.g., ­infection).16
ARE THERE ASSOCIATED SYSTEMIC
OR EXTRA-ARTICULAR SYMPTOMS?
Associated systemic or extra-articular symptoms can often
help establish a diagnosis. Signs or symptoms or other organ
involvement, such as constitutional symptoms, rash, alope-
cia, Raynaud’s phenomenon, sicca symptoms, or pleuritic
chest pain can suggest a systemic autoimmune disease, such
as Sjögren’s syndrome or systemic lupus erythematosus.
Bowel symptoms or genitourinary complaints may provide
a clue to the diagnosis of a seronegative spondyloarthopa-
thy or possible infection, such as gonorrhea or an enteric
infection. Constitutional symptoms, fever, chills, or malaise
also raise the possibility of infection, either intra-articular
or remote.
IS THE ARTHRITIS MECHANICAL
OR INFLAMMATORY?
The question of an inflammatory versus a mechanical pro-
cess is most reliably answered by the synovial fluid white
blood cell count (Table 36-4). Waxing and waning of dis-
ease activity unrelated to patterns of use, including fluctua-
tions of pain and swelling, protracted morning stiffness, and
gelling, suggest inflammation. Pain that occurs only after
537
use, improves with rest, and involves weight-bearing joints
suggests mechanical disease.
IS THE ARTHRITIS TRULY MONARTICULAR?
Careful inquiry may elicit evidence of antecedent or
coincident involvement of additional joints. A his-
tory of inflammatory symptoms in multiple joints for
more than 1 month suggests a chronic, noninfectious
inflammatory condition. Diffuse arthralgias of shorter
duration may accompany the onset of many illnesses,
especially systemic infections. Truly migratory disease,
in which there is only one inflamed joint but a clear-cut
history of recent inflammatory arthritis of other joints
occurring sequentially, suggests gonococcal arthritis or
rheumatic fever. Coexistence of symptoms in the axial
skeleton may provide a clue to the presence of a spon-
dyloarthropathy.16-18 A careful physical examination may
reveal involvement of additional joints at the time of
presentation.
ACUTE INFLAMMATORY MONARTHRITIS
Evaluation of an acute inflammatory monarthritis is vital,
because immediate benefit may result from the identifica-
tion and treatment of the underlying disease (Fig. 36-2).
arthritis is long, the three most common considerations
are infection, crystal-induced arthritis, and the onset of a
potentially chronic inflammatory arthropathy. Each of these
disorders is capable of manifesting with an explosive onset
of inflammation over a few hours, but the hyperacute pre-
sentation is most typical of infection or crystal-induced dis-
ease. As a rule, the most important diagnostic study in the
evaluation of an acute monarthritis is synovial fluid analy-
sis, especially when infection or crystal disease is suspected
(see Chapter 48).
In most cases, the diagnosis of gout can be made
immediately by aspirating joint fluid and examining it by
polarizing microscopy. Calcium pyrophosphate crystals
may be more difficult to identify. The diagnosis of pseu-
dogout is often uncertain until repeated aspirations are
performed and culture results are negative. The finding of
chondrocalcinosis on radiography, however, can increase
the examiner’s clinical suspicion. Infectious arthritis can
be diagnosed by Gram stain in some patients and can be
proved by culture in most cases within 48 hours. The
presence of other conditions may be suggested by extra-
articular disease features, but laboratory testing and con-
tinued observation are often needed before the diagnosis
can be made with certainty. The following observations
are intended to aid in the differential diagnosis of these
entities.
INFECTIOUS ARTHRITIS
In evaluating an acutely inflamed joint, the physician
must first ask whether the likelihood of septic arthritis is
sufficient to hospitalize the patient and intervene imme-
diately.19 Joint sepsis produces dramatic inflammation
followed quickly by ­ irreversible destruction of cartilage
and bone (see ­ Chapter 99). It may also be the initial
Table 36-4  Synovial Fluid and Associated Laboratory Findings in Monarticular Arthritis
538

Synovial Fluid
White Blood Cell Predominant Micro­ Cartilage
Count (cells/mm3) Cell Appearance Viscosity organisms Crystals RBCs Glucose Protein Complement Debris other
0-200 M Clear ↑ − − − 90% 1.5-2 − − Small amount not demonstrable
Normal on physical examination
GOLBUS 

0-2000 M Clear ↑ − +/− − − −/↑ − + Radiographs positive in advanced

| 

Osteoarthritis Occasional disease; synovial fluid findings

CPPD variable
Structural M Clear ↑ − − +/− − −/↑ +
Internal   +/− MR scan, arthrogram (knee),
derangement arthroscopy
Neuropathic +++/− Marked radiographic changes
Osteochondritis   − MR scan, CT scan
dissecans
Ischemic   − MR scan, bone scan, radiograph in
necrosis advanced cases
Traumatic RBC Cloudy, bloody − − +++ − Radiograph
Monarticular Arthritis

↑ ↑/↑↑↑
2000-10,000
Pigmented   RBC Brown, bloody ↓ − − +− ↓ ↑↑↑ − Synovial biopsy
villonodular  
synovitis
Amyloid M Slightly turbid − − Congo red: synovial fluid;
­monoclonal gammopathy
Enteropathic   M/P Slightly turbid − − − Positive stool occult blood; LE cells;
arthritis serum autoantibodies
Systemic lupus M Slightly turbid ↑ − − − ↓
­erythematosus
5000-50,000
Juvenile   P Slightly turbid ↓ − − − ↓ ↑/↑↑ −/↓ Synovial fluid leukocytes may be
rheumatoid ≥100,000
arthritis Serum: positive ANA (50%) +
­rheumatoid factor (<20%)
Sarcoidosis Slightly turbid ↓ − − − Chest radiographs; slit-lamp
Reiter’s syndrome P Slightly turbid ↓ − − − ↓ ↑/↑↑ ↑ examination
Psoriatic   P Slightly turbid ↓ − − − ↓ Negative rheumatoid factor,
arthritis ­positive ANA
Rheumatoid   P Turbid ↓↓ − − − ↓↓ ↑/↑↑↑ ↓ Serum: positive rheumatoid factor  
arthritis (50%-80%) + ANA
Tuberculous   M Turbid ↓↓ +/− − − ↓↓↓ ↑↑/↑↑  PPD usually positive unless ­anergic;
arthritis ↑ synovial biopsy ­essential
10,000-150,000
CPPD   P Turbid ↑↓ − CPPD (approxi- − ↓ ↑↑ +/− Repeated crystal examinations; 
pseudogout mately 60%) radiographs: chondrocalcinosis
Gout P Turbid ↑ − Mono­sodium − ↓ ↑↑ Serum uric acid unreliable
urate (>90%)
Gonococcal   P Turbid to pus ↓↓ +/− − ↓↓ ↑↑↑ Synovial fluid culture 20%-50%
infection positive; culture portals of entry;
urogenital Gram stain
Nongonococcal P Turbid to pus ↓↓↓ + − ↓↓↓ ↑↑↑ Gram stain—gram-positive
bacteria organisms; synovial fluid, blood
cultures
ANA, antinuclear antibody; CPPD, calcium pyrophosphate dihydrate; CT, computed tomography; LE, lupus erythematosus; M, mononuclear; MR, magnetic resonance; P, polynuclear; PPD, purified protein derivative;
RBC, red blood cell.
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS 539

Acute inflammatory arthritis

(synovial fluid WBC > 5000/cm3)

+
Gram stain Infection

Young: GC > staph. >strep.

Old: staph. > strep. > GC
Immunocompromised: staph.,
Gout, CPPD, + Crystal gram-negative;
r/o superinfection exam other unusual organisms

(cultures pending)

CBC, ESR, RF,

ANA, Lyme titer +

Empiric antibiotics
Systemic + × 24 hours, awaiting
toxicity?
cultures

CPPD, Lyme disease,

reactive arthritis, systemic
rheumatic disease

Figure 36-2  Approach to acute inflammatory arthritis.

ANA, antinuclear antibody test; CBC, complete blood count;
CPPD, calcium pyrophosphate deposition disease; ESR,
Anti-inflammatory medication; erythrocyte sedimentation rate; F/U, follow-up; GC, gono-
F/U in 24-48 hours; coccal infection; RF, rheumatoid factor; r/o, rule out; staph.,
Reaspirate joint if worsens staphylococcal infection; strep., streptococcal infection;
WBC, white blood count.

sign of a life-threatening systemic infection. In healthy
adults, the signs are usually obvious. The patient com-
plains of intense local pain and may resist attempts to
examine the affected joint. Infected peripheral joints are
swollen, warm, very tender, and sometimes red, and they
have a markedly restricted range of motion. As a gen-
eral rule, large joints are more frequently affected than
small ones in the absence of local trauma or peripheral
vascular disease.8 Unfortunately, persons at highest risk
for joint sepsis are those in whom confounding factors
obscure symptoms or blunt the inflammatory response.
Infection should be strongly suspected in less sick-appear-
ing patients when systemic risk factors (e.g., corticoste-
roid therapy; immunodeficiency or immunosuppression;
diabetes; intravenous drug abuse; pulmonary, cardiac,
or genitourinary focus of infection) and local pathol-
ogy (e.g., inflammatory arthritis, effusions, penetrating
trauma, previous injection of corticosteroids, prosthetic
joint) are present.
Although the clinical picture is never diagnostic of a
p­ articular infectious agent, certain presentations are char-
acteristic. Gonococcal infection rarely escapes attention
because it tends to manifest as an inordinately painful
monarthritis or polyarthritis and a painful, diffuse tenosyn­
ovitis in an otherwise healthy individual.20 Skin lesions
ranging from macules to pustules and vesicles are well
described15,21 but may be subtle. Meningococcal arthritis
occasionally presents with clinical and Gram stain find-
ings identical to those of gonococcal disease. The diagno-
sis of gonococcal arthritis is typically based on the history,
physical examination, synovial fluid cultures, and cultures
of any skin lesions as well as the pharynx and anogenital
area.
The large increase in intravenous drug abuse in the past
few decades has resulted in a dramatic increase in associated
septic arthritis. In this situation, infectious material is intro-
duced into the intravascular space, with subsequent hema-
togenous spread to the joints. Under unusual ­ conditions,
 540 GOLBUS  |  Monarticular Arthritis
direct inoculation can also occur. These infections are
most commonly caused by Staphylococcus aureus and gram-
­negative organisms, especially Pseudomonas. They commonly
occur in unusual locations compared with septic arthritis
in other populations. There is a marked predilection for
infections in the fibrocartilaginous joints of the skeleton,
such as the sternoclavicular joints, sacroiliac joints, and
intervertebral disk spaces.
Lyme arthritis manifests as a true inflammatory arthri-
tis—most commonly in the knee—weeks to months after
initial exposure and after the development of the early
syndrome of fever, arthralgias, lymphadenopathy, and rash.
This curable infection should be suspected in patients with
compatible symptoms, a history of travel to endemic areas,
or coexistent neurologic or cardiac abnormalities. The
diagnosis can be difficult, however, because many affected
individuals do not recall a tick bite or antecedent rash. If
infection is suspected, serum antibodies to Borrelia burg-
dorferi should be obtained. Viral illnesses, including hepa-
titis B, infectious mononucleosis, parvovirus, rubella, and
rubella vaccination, rarely present as monarticular synovi-
tis (see Chapter 104).
A number of arthritic syndromes that closely mimic
known rheumatic diseases have now been described in
association with human immunodeficiency virus (HIV)
infection.22 Most of these are polyarticular and appear as
arthralgias rather than true arthritis. Opportunistic infec-
tions in individuals infected with HIV can develop within
one joint and in contiguous bone. In addition to the more
typical organisms, such as S. aureus, less common microor-
ganisms have been cultured from fluids of septic joints in
patients with HIV infection, including Sporothrix schenckii,
Cryptococcus neoformans, several Salmonella species, Urea-
plasma urealyticum, and Campylobacter fetus. HIV-infected
individuals can also experience an extremely painful, non-
inflammatory monarthritis. Although the mechanism of
this syndrome is unknown, it usually resolves spontaneously
in 24 to 28 hours (see Chapter 103).
CRYSTAL-INDUCED ARTHRITIS
Crystal-induced arthritis commonly manifests as acute
monarticular arthritis. It is particularly likely when there
is a ­ history of recurrent, self-limited attacks of inflamma-
tion of the same joint. Fortunately, the most fulminant
­arthropathy—gout—is also the most easily and reliably
diagnosed. Monosodium urate crystals can be identified in
at least 95% of acute joint effusions by polarized microscopy
and even in some asymptomatic joints.23 Calcium pyrophos-
phate crystals are often not identified initially in patients
who are later diagnosed with pseudogout.24 Hydroxyapatite
crystals present a particular problem in diagnosis, because
they are reliably identified only by electron microscopy or
alizarin red stain.25 Occasionally, in patients with renal fail-
ure and an acute monarthritis, calcium oxalate crystals can
be identified in joint fluid aspirates. As a general rule, iden-
tification of crystals in joint fluid does not prove the absence
of coexistent infection. Crystal-induced arthropathies are
particularly common and difficult to manage in patients
who are uremic or undergoing dialysis.
Gouty arthritis is definitely present when intracellu-
lar birefringent, needle-shaped crystals (sodium urate) are
identified in synovial fluid or confirmed by documentation
of urate crystals in a tophus; it is probably present if the
crystals are extracellular. The most characteristic clinical
features are extremely rapid onset of severe pain and inflam-
mation, with extension of the inflammatory process into the
surrounding tissues, producing the appearance of cellulitis.
Desquamation of overlying skin may occur as the attack
subsides. In the ankle, the initial phases visually resemble
the periarthritis of erythema nodosum, but the pain is much
more severe.26
Podagra is characteristic but not pathognomonic of gout.27
First attacks of gout can occur in other large joints or in the
small joints of the upper extremities (see Chapter 87).
Calcium pyrophosphate dihydrate (CPPD) deposition is
associated with acute or chronic inflammatory arthritis and
may be superimposed on osteoarthritis.24 Pseudogout can
be diagnosed by the finding of weakly positive, rhomboid-
shaped birefringent crystals in the white blood cells of syno-
vial fluid aspirates (see Chapter 88). These are identified in
half the effusions from patients presenting with monarthritis
due to CPPD. Their presence does not exclude infection in
a patient with a first episode of monarticular arthritis. The
physician can also suspect the diagnosis in an elderly person
with an acute monarthritis of the knee or wrist and chon-
drocalcinosis on radiographs if there are no obvious reasons
to suspect infection. Less common forms of crystal-induced
arthritis may result from deposition of hydroxyapatite (often
in the shoulder) or calcium oxalate (especially in patients
with renal failure).
OTHER CAUSES OF ACUTE INFLAMMATORY
ARTHRITIS
Patients without Systemic Manifestations
If a patient does not have one of the previously mentioned
disorders, there is a significant likelihood that the cause will
remain elusive for a time, and the physician will be obliged
to make a practical decision about how aggressively to pur-
sue a diagnosis. The initial workup under these circum-
stances should be as focused as possible so that a satisfactory
result can be achieved most economically and with the least
patient discomfort.
Juvenile Rheumatoid Arthritis. Although most children
with juvenile rheumatoid arthritis presenting with monar-
ticular disease eventually exhibit involvement of additional
joints, about 25% have isolated monarthritis that may recur
intermittently into adulthood. Antinuclear antibodies are
more common than rheumatoid factor; monarticular disease
and antinuclear antibodies correlate with the development
of iritis28 (see Chapter 97).
Rheumatoid Arthritis. Rheumatoid arthritis may manifest
with an acute or insidious onset of monarthritis. A detailed
history often reveals the gradual onset of fatigue or arthralgias.
Physical examination may reveal unsuspected involvement
of other joints, particularly the metatarsophalangeal joints.
Seronegative Spondyloarthropathies. The spondyloar-
thropathies were mentioned previously in the context of
monarthritis accompanied by axial skeleton stiffness or pain.
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
Joints of the lower extremity are typically involved. There
may also be extra-articular clues to the diagnosis, such as
urethritis, diarrhea, inflammatory eye disease, nail pitting,
or a psoriasiform rash. Joint swelling may be fusiform, creat-
ing “sausage digits.”
Neuropathic Arthropathy. Neuropathic arthropathy
(Charcot’s joints) should be suspected in patients with dia-
betes who present with a subacute or chronic monarthritis
of the knee, ankle, or foot, usually with swelling and effu-
sion but with little pain. The arthritis develops most com-
monly in those with peripheral neuropathy and sensory loss.
The radiographic picture is usually pathognomonic.
Hemarthroses. Hemarthroses may result from trauma, syno-
vial hemangiomas, pigmented villonodular synovitis, exces-
sive anticoagulation, or inherited coagulopathies.
Lyme Arthritis. Lyme arthritis should be suspected in
­endemic areas, especially if there is a history of rash, tick
bite, or exposure.
Patients with Signs of Systemic Illness
Enteropathic Arthritis. Whipple’s disease, which is rare,
can appear with a monarticular arthritis when the bowel dis-
ease is not evident. Regional enteritis and ulcerative ­colitis
are more likely to be symptomatic when arthritis develops29
(see Chapter 73). Celiac disease (sprue) is ­associated with
rheumatic diseases, including rheumatoid arthritis and
­lupus.
Systemic Autoimmune Disease. Systemic lupus erythe-
matosus occasionally manifests with monarticular arthritis,
although small joint polyarthritis is more common. Marked
swelling of the wrist and tenosynovitis may also be accom-
panied by fever and severe systemic signs. Monarticular or
large joint pain in a steroid-treated lupus patient suggests
avascular necrosis30 or infection. Sarcoidosis often manifests
as bilateral arthritis of the ankles, wrists, or knees, which
may be associated with erythema nodosum or hilar adenop-
athy.31 Henoch-Schönlein purpura,32 Takayasu’s disease,33
overlap syndromes,34 polymyalgia rheumatica,35 and giant
cell arteritis36 can all manifest with monarticular or polyar-
ticular arthritis, although polyarthritis is the rule in many of
these disorders.
Familial Mediterranean fever typically causes exquisitely
painful monarthritis with moderately impressive physical
findings.37,38 The combination of fever and evanescent rash
suggests Still’s disease39 or rheumatic fever.40
CHRONIC INFLAMMATORY
MONARTICULAR ARTHRITIS
Many disorders that manifest as acute monarticular inflam-
mation progress to polyarticular involvement, remit and
relapse, or spontaneously resolve. Persistent monarticular
inflammation raises the concern that a more narrow spec-
trum of disorders is present, particularly chronic infections
or tumors. For these at times difficult-to-diagnose condi-
tions, intravenous drug use, travel to certain areas, and
immunosuppression should raise one’s index of suspicion.
541
Synovial biopsy and arthroscopy may be useful in identify-
ing the cause of chronic monarthritis (Fig. 36-3; also see
Chapter 48).
Chronic infections result from slow-growing organisms
or the presence of foreign bodies in the joint. Typically,
there are persistent signs of inflammation, including stiff-
ness, pain, and warmth; characteristically, there is synovial
thickening, regardless of whether an effusion is present.
Tuberculosis, which is undergoing a resurgence in the
United States, almost always affects a single diarthrodial
joint41 (see Chapter 101). A positive tuberculin test may be
the only clue. Infection with atypical mycobacteria or Can-
dida, coccidioidomycosis, histoplasmosis, and blastomycosis
can produce similar syndromes.
Chronic infections may also result from penetrating
wounds or the introduction of foreign bodies. Superficially
located joints on the hands and feet are most likely to be
penetrated during normal activity, often without awareness
by the individual. Sporotrichosis should be suspected in a
gardener with involvement of a hand joint, especially if
there is a surrounding soft tissue reaction.42
Tumors, particularly pigmented villonodular synovitis,43
should be suspected when there is chronic monarticular
inflammation in conjunction with a bloody effusion. Metas-
tasis to synovium from solid tumors or joint involvement by
hematologic malignancies is rare. Tumors involving periar-
ticular structures can mimic arthritis.
NONINFLAMMATORY MONARTICULAR
ARTHRITIS
Structural joint disease should be suspected when there is
little synovial inflammation in proportion to the degree of
destruction of bone and cartilage and when the synovial
fluid white blood cell count is less than 1000 to 2000 cells/
mm3. Truly noninflammatory fluid, however, contains fewer
than 200 cells/mm3. An internal derangement of the knee
is suggested by a history of trauma, episodes of joint locking
or “giving way,” and tenderness at the joint margin; physical
examination reveals locking or episodic pain during range of
motion. It may be confirmed by MRI or arthroscopy, which
demonstrates meniscal or ligamentous tears.
Osteoarthritis frequently manifests as monarthritis, particu-
larly in the knee, hip, acromioclavicular joint, first radiocarpal
joint, or first metatarsophalangeal joint (i.e., hallux rigidus).
Elderly patients with osteoarthritis may have inflammatory
joint effusions that contain CPPD crystals.26 In monarticular
disease, a predisposing factor should be sought, such as con-
genital dysplasia of the hip, trauma to or prior surgical removal
of ligaments or fibrocartilage from the knee, prior inflamma-
tory arthritis or infection, occupational stress, or obesity.
Hip symptoms in a young patient suggest congenital
dysplasia of the hip or a slipped capital femoral epiphy-
sis.44,45 Spontaneous osteonecrosis may occur in the hip
(Legg-Calvé-Perthes disease), metatarsal bones (Freiberg’s
disease), capitellum of the humerus, or carpal lunate. Osteo-
chondritis dissecans should be suspected in a child or teen-
ager who, after minor trauma, has relatively severe knee
pain followed by mechanical dysfunction.46 There is now
considerable experience, especially in pediatric patients,
using ultrasonography to identify hip effusions and periar-
ticular abnormalities.47
 542 GOLBUS  |  Monarticular Arthritis

Chronic monarticular
arthritis

Osteoarthritis

+ Inflammatory
Radiograph arthritis

CPPD, other

Synovial WBC Aspirate

< 1000 mm3/mL joint
Synovial
WBC >5000

Internal derangement,
osteoarthritis +
Crystal
exam Gout, CPPD

Systemic rheumatic RF, ANA, PPD, Occult

disease ESR, cultures CPPD

Figure 36-3  Approach to chronic

monarticular arthritis. ANA, antinuclear
antibody test; CPPD, calcium pyrophos-
phate deposition disease; ESR, erythro-
cyte sedimentation rate; PPD, purified
protein derivative test for ­tuberculosis; Occult
RF, rheumatoid factor test; WBC, white infection
blood cell count.

Rapid development of “osteoarthritis” should lead
to a consideration of the possibility of fracture related to
­osteopenia, an adjacent destructive process such as meta-
static tumor, or avascular necrosis.
Osteonecrosis is a common cause of monarthritis of the
hip, shoulder, and knee in young people with systemic dis-
eases who require corticosteroid therapy. It also occurs in a
variety of other conditions such as alcoholism, barotrauma,
hemoglobinopathies, diabetes, hyperlipidemia, hyperurice-
mia, and systemic lupus erythematosus.
DIAGNOSTIC STUDIES
SYNOVIAL FLUID ANALYSIS
The most important laboratory test in the evaluation of acute
monarthritis is synovial fluid analysis.25 The primary purpose
of synovial fluid analysis is to answer the following questions:
Is the effusion inflammatory?
Is it infected?
Does it contain intracellular or extracellular crystals?
Even a few drops of fluid can be sufficient to obtain a
white blood cell count and differential cell count, ­crystal
analysis, and culture. No other tests are needed (see ­Chapter
48). If fluid is difficult to obtain, ultrasound-guided aspira-
tion may yield fluid even from proximal interphalangeal
joints.
CULTURES
If septic arthritis is a possibility, blood, synovial fluid, and
urine cultures are indicated. If gonococcal arthritis is a con-
sideration, cervicourethral, rectal, and pharyngeal samples
should be placed on Thayer-Martin medium. Culture speci-
mens from normally sterile sites, including the synovial cav-
ity, tenosynovial space, and intracutaneous lesions, should
be placed on chocolate agar without added preservative.
Synovial fluid cultures are usually negative in gonococcal
arthritis (see Chapter 99).
LABORATORY STUDIES
In the initial evaluation of monarthritis, diagnostic ­laboratory
testing for rheumatic diseases should be ­ undertaken only
after a careful history and physical examination.48 A complete
blood count with differential, Westergren sedimentation rate,
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
C-reactive protein, tests of renal and liver function, and uri-
nalysis can be useful if infection or a multisystem disease is sus-
pected. In the appropriate clinical setting, studies that may be
of value include a uric acid level, rheumatoid factor, anticyclic
citrullinated peptide (anti-CCP), and certain serologic studies
such as antinuclear antibody, antineutrophil cytoplasmic anti-
bodies, Lyme disease, hepatitis, and parvovirus serologies. In
the wrong clinical setting, routinely ordered serology studies,
especially if bundled into “arthritis panels,” may cause confu-
sion and lead to further unnecessary testing.
RADIOGRAPHY
Plain radiographs of the affected and contralateral joints
should almost always be obtained in patients with symp-
toms of more than several weeks’ duration. They may also
be helpful for patients with an acute arthritis in whom
infection or crystal disease is suspected. For those with
no prior joint complaints, common findings are soft tis-
sue calcification and evidence of intra-articular pathology
unknown to the patient, such as osteoarthritis, chondro-
calcinosis, or loose bodies. Occasionally, an unsuspected
bony lesion (e.g., fracture) or evidence of osseous or
hematologic malignancy, osteomyelitis, or Paget’s dis-
ease may be detected. Care should be taken to include
enough surrounding bone in the radiograph to identify
such lesions.
NUCLEAR MEDICINE
Radionuclide scans are useful primarily because of their
­sensitivity. They are used when it is important to search for
a site of infection that cannot be detected or localized by
other means, such as in deeply buried joints that are ­difficult
to examine, fibrocartilaginous joints in which range of
motion is poorly tested, and the spine (see Chapter 53).
MAGNETIC RESONANCE IMAGING
MRI is superior to other imaging modalities in the diag-
nosis of ischemic necrosis of bone49 and possibly Legg-
­Calvé-Perthes disease,50 particularly in early cases. In
addition, MRI is the study of choice to detect radiographi-
cally occult fractures, especially in the hip and pelvis.51 In
the knee, MRI provides a more accurate and noninvasive
alternative to arthrography for the detection of meniscal
and cruciate ligament injuries.52 Because of their superior
definition of soft tissue pathology, both MRI and ultraso-
nography are useful in investigating deep infections about
the hip, such as psoas abscesses, which may mimic arthri-
tis.53,54 Although superior in most cases to CT in detecting
subchondral bone and marrow involvement by tumor or
osteomyelitis, MRI is unable to provide the whole-body
survey that is characteristic of radionuclide scans.
ULTRASONOGRAPHY
Musculoskeletal ultrasonography has advanced rapidly since
the 1990s. It is possible to aspirate almost any joint under
ultrasonic control, no matter how inaccessible or small. This
facilitates the workup for infection. Evaluation for the pres-
ence of fluid in joints, bursae, and periarticular ­structures is
543
greatly facilitated by ultrasonography. It is possible to use
“power Doppler” to identify areas of increased tissue perfu-
sion.54 This may be helpful in localizing inflammation. The
presence of lesions in tendons, such as an inflamed Achilles
or patellar tendon, and the compromise of smaller tendons
by rheumatoid nodules can also be identified by ultrasonog-
raphy. The evaluation of structural abnormalities of joints,
such as osteoarthritis or internal derangement, is more
­difficult.
SYNOVIAL AND BONE BIOPSY
Although rarely needed, synovial biopsy may play a role
in the diagnosis of chronic, unexplained monarticular
arthritis.55 Tuberculous or fungal synovitis is more fre-
quently identified by staining and culture of open biopsy
material than by similar studies of synovial fluid. Closed-
needle biopsy or arthroscopic biopsy of the knee is prefer-
able to open biopsy because of reduced morbidity. In acute
inflammatory arthritis, a surgical biopsy is indicated in the
diagnosis of infection of fibrocartilaginous joints, such as
the sacroiliac and sternoclavicular joints, and probably the
symphysis pubis if an initial attempt at aspiration is not
diagnostic.56 General anesthesia may be required for pain
control. If osteomyelitis is suspected, the physician should
consider obtaining a bone biopsy specimen before initiat-
ing antibiotic therapy.
REFERENCES
  1. Radin EL: Chondromalacia of the patella. Bull Rheum Dis 34:1, 1984.
  2. Lagier R: Synovial reaction caused by adjacent malignant tumors:
Anatomico-pathological study of three cases. J Rheumatol 4:65,
1997.
  3. Feagin JA Jr: The office diagnosis and documentation of common
knee problems. Clin Sports Med 8:453, 1989.
  4. Isenberg DA, Schoenfield Y: The rheumatologic complications of
hematologic disorders. Semin Arthritis Rheum 12:348, 1983.
  5. Hadjipavlou AG, Gaintanis IN, Kontalis GM: Paget’s disease of bone
and its management. J Bone Joint Surg Am 84:160, 2002.
  6. Larsson LG, Baum J: The syndromes of bursitis. Bull Rheum Dis
36:1, 1986.
  7. Viggiano DA, Garrett JC, Clayton ML: Septic arthritis presenting as
olecranon bursitis in patients with rheumatoid arthritis. J Bone Joint
Surg Am 62:1011, 1980.
  8. Ho O Jr, Su EY: Antibiotic therapy of septic bursitis. Arthritis Rheum
24:905, 1981.
  9. Cucurull E, Espinoza LR: Gonococcal arthritis [review]. Rheum
Dis Clin North Am 24:305-322, 1998.
10. Gotuzzo E, Alarcon GS, Bocanegra TS, et al: Articular involvement
in human brucellosis: A retrospective analysis of 304 cases. Semin
Arthritis Rheum 12:245, 1982.
11. Grassi W, Tittarelli E, Blasetti P, et al: Finger tendon involvement
in rheumatoid arthritis: Evaluation with high-frequency sonography.
Arthritis Rheum 38:786, 1995.
12. Dowart BB: Carpal tunnel syndrome: A review. Semin Arthritis
Rheum 14:134, 1984.
13. Putten J: Neurological Differential Diagnosis. New York, Springer-
Verlag, 1980.
14. Gibson RK, Rosenthal SJ, Lukert BP: Pyomyositis. Am J Med 11:421,
1982.
15. Kallen PS, Louie JS, Nies KM, et al: Infectious myositis and related
syndromes. Am J Med 11:421, 1982.
16. Goldenberg DL: Infectious arthritis complicating rheumatoid
arthritis and other chronic rheumatic disorders. Arthritis Rheum
32:496, 1989.
17. Resnick D, Niwayana G: Reiter’s disease. In Diagnosis of Bone and Joint
Disorders, 2nd ed. Philadelphia, WB Saunders, 1988, pp 1218-1251.
 544 GOLBUS  |  Monarticular Arthritis
18. Resnick D, Niwayana G: Psoriatic arthritis. In Diagnosis of Bone
and Joint Disorders, 2nd ed. Philadelphia, WB Saunders, 1988, pp
1218-1251.
19. Cimmino MA: Recognition and management of bacterial arthritis.
Drugs 54:50-60, 1997.
20. Seifert MH, Warin AP, Miller A: Articular and cutaneous manifestations
of gonorrhea: Review of sixteen cases. Ann Rheum Dis 33:140, 1974.
21. Goldenberg DL: Septic arthritis. Lancet 351:197, 1998.
22. Rynes RI, Goldenberg DL, DiGiacomo R, et al: Acquired-
immunodeficiency syndrome-associated arthritis. Am J Med 84:810,
1988.
23. Bomalaski JS, Lluberas G, Schumacher HR Jr: Monosodium urate
­crystals in the knee joint of patients with asymptomatic ­nontophaceous
gout. Arthritis Rheum 29:1480, 1986.
24. Masuda I, Ishikawa K: Clinical features of pseudogout attack:
A review of fifty cases. Clin Orthop Rel Res 229:123, 1988.
25. Gatter RA, Schumacher HR Jr: A Practical Handbook of Synovial
Fluid Analysis. Philadelphia, Lea & Febiger, 1991.
26. Yu T: Diversity of clinical features in gouty arthritis. Semin Arthri-
tis Rheum 13:360, 1984.
27. McCarty DJ: Calcium pyrophosphate dihydrate crystal deposition
­disease—1975. Arthritis Rheum 19:275, 1976.
28. Chylack LT Jr: The ocular manifestations of juvenile rheumatoid
arthritis. Arthritis Rheum 20(Suppl):224, 1976.
29. Weiner SR, Utsinger P: Whipple disease. Semin Arthritis Rheum
15:157, 1986.
30. Zizic TM, Hungerford DS, Stevens MB: Ischemic bone necrosis in
­systemic lupus erythematosus. Medicine (Baltimore) 59:134, 1980.
31. Spilberg I, Siltzbach LE, McEwen C: The arthritis of sarcoidosis.
Arthritis Rheum 12:126, 1969.
32. Cream JJ, Gumpel JM, Peachey RDG: Schönlein-Henoch purpura in
the adult. Q J Med 39:461, 1940.
33. Hall S, Barr W, Lie JT, et al: Takayasu arthritis. Medicine 64:89,
1985.
34. Bennett RM, O’Connell DJ: Mixed connective tissue disease: A clini-
copathologic study of 20 cases. Semin Arthritis Rheum 10:25, 1980.
35. Healey L: Long-term follow-up of polymyalgia rheumatica: Evidence
for synovitis. Semin Arthritis Rheum 23:322, 1984.
36. Ginsberg WW, Cohen MD, Hall SB, et al: Seronegative polyarthritis
in giant cell arthritis. Arthritis Rheum 28:1362, 1985.
37. Meyerhoff J: Familial Mediterranean fever: Report of a large family,
review of the literature, and discussion of the frequency of amyloidosis.
Medicine (Baltimore) 59:66, 1980.
38. Sohar E, Pras M, Gafni J: Familial Mediterranean fever and its ­articular
manifestations. Clin Rheum Dis 1:195, 1975.
39. van de Putte LB, Wouters JM: Adult-onset Still’s disease. Baillieres
Clin Rheumatol 5:263, 1991.
40. Ben-Dov I, Berry E: Acute rheumatic fever in adults over the age
of 45 years: An analysis of 23 patients together with a review of the
­literature. Semin Arthritis Rheum 10:100, 1980.
41. Nathanson L, Cohen W: A statistical and roentgen analysis of two
hundred cases of bone and joint tuberculosis. Radiology 36:550,
1940.
42. Wilson DE, Mann JJ, Bennett JE, Utz P: Clinical features of
­extracutaneous sporotrichosis. Medicine (Baltimore) 63:25, 1984.
43. Docken WP: Pigmented villonodular synovitis. Semin Arthritis
Rheum 9:1, 1979.
44. Wilson PD, Jacobs B, Schecter L: Slipped capital femoral epiphysis.
J Bone Joint Surg Am 14:549, 1967.
45. Ponseti IV, McClintock R: The pathology of slipping of the upper
femoral epiphysis. J Bone Joint Surg Am 38:71, 1956.
46. Pappa AM: The osteochondroses. Pediatr Clin North Am 14:549,
1967.
47. Harcke HT, Mandell GA, Cassell IL: Imaging techniques in child-
hood arthritis. Rheum Dis Clin North Am 23:523, 1997.
48. American College of Rheumatology Ad Hoc Committee on Clinical
Guidelines: Guidelines for the initial evaluation of the adult patient
with acute musculoskeletal symptoms. Arthritis Rheum 39:1,
1996.
49. Imhof H, Breitenseher M, Trattnig S, et al: Imaging of avascular necro-
sis of bone. Eur Radiol 7:160, 1997.
50. Lamer S, Dorgeret S, Khairouni A, et al: Femoral head vascularization
in Legg-Calvé-Perthes disease: Comparison of dynamic gadolinium-
enhanced subtraction MRI with bone scintigraphy. Pediatr Radiol
32:580, 2002.
51. Sadro C: Current concepts in magnetic resonance imaging of the adult
hip and pelvis. Semin Roentgenol 35:231, 2000.
52. Crotty JM, Monu JU, Pope TL: Magnetic resonance imaging of
the musculoskeletal system. Part 4. The knee. Clin Orthop Rel Res
330:288, 1996.
53. Strouse PJ, DiPietro MA, Adler RS: Pediatric hip effusions: ­Evaluation
with power Doppler sonography. Radiology 206:731, 1998.
54. Weintraub JC, Cohen JM, Maravilla KR: Iliopsoas muscles: MR study
of normal anatomy and disease. Radiology 156:435, 1985.
55. Schumacher HR: Joint pathology in infectious arthritis. Clin Rheum
Dis 4:33, 1978.
56. Gordon G, Kabins SA: Pyogenic sacroiliitis. Am J Med 69:50, 1980.
 37
KEY POINTS
Synovial fluid analysis can differentiate inflammatory from
noninflammatory arthritis.
Synovial fluid analysis can establish a diagnosis of gout or
pseudogout.
Osteoarthritis is largely defined by radiographic abnormali-
ties, and another diagnosis must be considered when no
radiographic abnormalities are present.
Pretest probability of a specific diagnosis must be sufficiently
high for laboratory testing to aid in diagnosis.
History and physical examination findings are much more
important than laboratory findings in establishing the
­diagnosis in a patient with inflammatory polyarthritis.
Severe, prolonged morning stiffness, swelling in multiple
joints, ­fatigue, weight loss, or fever suggests a systemic
­inflammatory process.
Extra-articular manifestations often provide definitive
­diagnostic information in a patient with polyarthritis,
­particularly ocular and dermatologic involvement.
Joint pain is extremely common and is almost always referred
to as “arthritis” by the lay public. Because most patients
who believe they have arthritis actually have some other
cause for their pain, the initial challenge for the physician
is to learn to differentiate between true arthritis and other
causes of pain in and around joints.1,2 For patients whose
polyarticular pain does seem to represent polyarthritis, the
challenge is first to use the history, physical examination,
and ancillary tests to develop a differential diagnosis, and
then to establish a probable or, in most cases, a definitive
diagnosis. Because polyarticular pain is the most frequent
complaint bringing patients to rheumatologists, it is imper-
ative that students of the field become proficient in this
area.
HISTORY, PHYSICAL EXAMINATION,
AND LABORATORY TESTS
HISTORY
As Mackenzie3 and others have pointed out, the clini-
cal history is the most important diagnostic tool in the
evaluation of polyarticular disorders. Many clinicians
augment the history with the use of self-report patient
questionnaires, which may be especially beneficial in fol-
lowing patients over time.4 Many items warrant special
attention.
Polyarticular Arthritis
John S. Sergent  • Howard A. Fuchs
Onset
The physician should direct attention early to the nature
of the first symptoms. This is not always easy, especially if
months or years have passed, because most patients prefer
to discuss their current symptoms. It also may be difficult
because many patients tend to discuss all the joint problems
they have experienced in their lifetime, assuming all previ-
ous joint symptoms have been part of the same process, even
if obviously caused by trauma.
Course
The physician must determine what has occurred since the
onset of symptoms and at what rate. Chronic arthritis may be
relentlessly progressive from the onset, or it may be intermit-
tent, with periods of partial or complete remission. As indi-
vidual joints become involved, the process may be migratory or
additive. The term migratory polyarthritis implies that previously
involved joints become asymptomatic as new joints become
inflamed; the disease seems to migrate from joint to joint.
Specific Joint Symptoms
Specific symptoms include locking, localized pain, “giv-
ing way” without warning, palpable or audible crepitation,
warmth, and swelling.
Systemic Symptoms
Patients who seek a physician’s help because of joint pain
often do not perceive, unless asked, relationships between
joint pain and other complaints, such as fever, night sweats,
weight loss, and generalized muscle stiffness. The history
of these symptoms should be sought specifically and, when
possible, quantified.
Rheumatic Disease Systems Review
In addition to systemic symptoms, patients with arthritis
must be asked specifically about conditions associated with
various forms of arthritis, including rash (e.g., photosensitive,
psoriatic, purpuric, or petechial), areas of alopecia, Raynaud’s
phenomenon, sicca syndrome, uveitis, scleritis, oral and gen-
ital ulcers, urethritis or cervicitis, symptoms of inflammatory
bowel disease, and pleuropericardial symptoms.
Past History
Previous diagnoses may be incorrect. A childhood his-
tory of rheumatic fever needs to be explored because many
children will have had Still’s disease or another form of
545
 546 Sergent  |  Polyarticular Arthritis
juvenile chronic polyarthritis instead; the reverse also
may be true. Special emphasis should be given to events
of the immediate weeks or months preceding the onset of
joint disease, including sore throats, febrile illnesses, vene-
real disease, sexual contacts, diarrhea, rashes, and ocular
inflammation.
Family History
In addition to asking about any type of arthritis, the exam-
iner should inquire about a family history of any associated
condition, such as psoriasis, uveitis, or inflammatory bowel
disease. In patients suspected to have a spondyloarthropa-
thy, it is important to obtain the history of any family mem-
bers with chronic back pain and to attempt to determine the
nature of that condition.
PHYSICAL EXAMINATION
A complete physical examination is essential. Special con-
sideration must be given to searching for nonarticular fea-
tures, such as rashes, patches of psoriasis, psoriatic nails,
oral or genital ulcers, ocular abnormalities, murmurs, rubs,
bruits, and abnormal peripheral pulses. A careful neurologic
examination also is warranted.
The details of a complete musculoskeletal examination
are reviewed in Chapter 35. Each joint should be exam-
ined for warmth, synovial thickening, effusions, crepitation,
deformity, and tenderness. The distribution also should
be noted (e.g., distal interphalangeal, proximal interpha-
langeal, metacarpophalangeal, tendon insertions, a “ray”
distribution of a whole digit) because specific patterns of
involvement are seen with different disorders. Active and
passive ranges of motion should be tested. The spinal exami-
nation should include the range of motion of the cervical
and lumbar regions, chest expansion, tenderness of the spi-
nous processes and sacroiliac joints, abnormal curves, and
muscle spasm.
LABORATORY TESTS
Nonspecific tests of inflammation include hematocrit, eryth-
rocyte sedimentation rate, C-reactive protein, and white
blood cell count. Table 37-1 presents tests that are some-
times helpful in specific diseases. They must be ordered
wisely because the background false-positive rate can be
quite high.
If obtainable, synovial fluid should be examined as
described in Chapter 48. The primary benefit of synovial
fluid examination is to differentiate among noninflammatory
arthritis, inflammatory arthritis, and infection. Tests other
than appearance, total white blood cell count and differen-
tial, crystal examination, and culture rarely are indicated.
RADIOGRAPHIC FEATURES
Important radiographic features that may help in patient
evaluation include fracture, focal or diffuse cartilage loss,
erosion, periarticular osteoporosis, osteophytes, subchondral
sclerosis, periostitis, and soft tissue changes. In many cases,
properly chosen radiographs may be virtually diagnostic or
may eliminate certain diseases from further consideration.
Table 37-1  Laboratory Tests for Polyarthritis
Significance
Test Positive Negative
Rheumatoid factor   Helpful in young Prognostic significance
or anti–cyclic   ­individuals, in   only, not helpful in
citrullinated   whom background ­  individual cases
peptide positivity is low
Antinuclear   High titer, suggestive   Virtually rules out  
antibody of a rheumatic   active systemic lu-
disease pus erythematosus
Uric acid Elevated levels,   If repeated levels are  
indicating that   normal, gout
gout is possible unlikely
Antistreptolysin O Recent streptococcal Rheumatic fever
exposure unlikely
HLA-B27 Possibly useful in   No benefit
early-onset spondy­
loarthropathy
Anti-Borrelia Only helpful if pretest Chronic Lyme disease
probability is high unlikely
PRINCIPLES OF DIAGNOSIS
Physicians are expert in pattern recognition, and in no
area is this more important than in rheumatology, where
the specific joints involved, the sequence of events, and
associated features—as opposed to specific diagnostic
tests—define most diseases. Being competent in pattern
recognition sometimes can be a two-edged sword if the
clinician settles too quickly on a diagnosis or is inflex-
ible about changing the presumed diagnosis as new data
become available.
After other causes of joint pain have been eliminated,
and it seems that the patient does have polyarticular arthri-
tis, numerous classification schemes may aid in determining
the correct diagnosis. One might consider the size of the
involved joints (small [digits] versus large), the age of onset,
or other features. We have chosen a system based on the
presence or absence of inflammation and the number and
pattern of joint involvement.
In most situations, the best determinant of inflamma-
tion is the synovial fluid examination (see Chapter 48)
and especially the synovial fluid white blood cell count,
which is less than 1000/mm3 in most noninflammatory
conditions and significantly greater in inflammatory
disease. There also are clinical features that suggest the
arthritis is inflammatory in nature, including prolonged
and severe morning stiffness, fever, night sweats, weight
loss, and spontaneous (not activity-related) joint swell-
ing. Physical examination in inflammatory arthritis often
reveals local warmth, synovial thickening, and large effu-
sions. In addition to inflammatory synovial fluid, other
features suggesting inflammation include anemia, throm-
bocytosis, an increased erythrocyte sedimentation rate,
and increased acute-phase reactants such as C-reactive
protein and ferritin.
Table 37-2 shows a classification of polyarthritis based on
two features: the presence or absence of inflammation and
the number and pattern of joint involvement. Figure 37-1
is an algorithm based on history and examination, which
helps lead to appropriate further investigation.
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS 547

Table 37-2  Classification of Polyarthritis

SYSTEMIC LUPUS ERYTHEMATOSUS
Inflammatory
Peripheral polyarticular Systemic lupus erythematosus (SLE; see Chapter 75) often
Rheumatoid arthritis manifests as chronic polyarthritis and may be confused
Systemic lupus erythematosus with RA in such patients. The arthritis is typically inter-
Viral arthritis
Psoriatic arthritis (occasionally) mittent, may be extremely painful, and is almost never
Peripheral pauciarticular erosive. Mixed connective tissue disease may cause an
Psoriatic arthritis identical arthritis.
Reiter’s syndrome
Rheumatic fever
Polyarticular gout SCLERODERMA
Enteropathic arthritis
Behçet’s disease The systemic sclerosis variant of scleroderma (see Chapter
Bacterial endocarditis 77) typically begins as painful swollen hands, with early
Peripheral with axial involvement contractures as prominent features. The combination of
Ankylosing spondylitis (especially juvenile onset)
Reiter’s syndrome puffy hands and Raynaud’s phenomenon may be referred to
Enteropathic arthritis as undifferentiated connective tissue disease in recognition
Psoriatic arthritis of the fact that the condition of the patient may evolve into
Noninflammatory (osteoarthritis) SLE, scleroderma, mixed connective tissue disease, or RA.
Hereditary Some individuals may remain in the undifferentiated state
Osteoarthritis of the hands for many years. Rarely, patients are seen with characteristic
Primary generalized osteoarthritis
Traumatic osteoarthritis
features of scleroderma and RA.7
Osteoarthritis after local injury
Osteoarthritis of the knees in obese individuals
Chondromalacia after aggressive exercise programs
PSORIATIC ARTHRITIS
Osteoarthritis in the elderly Although typically pauciarticular at onset, psoriatic arthritis
Metabolic diseases (may have an unusual pattern)
Hemochromatosis may evolve into a polyarticular disease that resembles RA
Ochronosis (see Chapter 72). Particular diagnostic problems occur in
Acromegaly children, whose disease may resemble pauciarticular juve-
Idiopathic nile chronic arthritis at onset and may then evolve into a
pattern resembling polyarticular juvenile or adult RA.8

POLYARTICULAR PERIPHERAL ARTHRITIS
RHEUMATOID ARTHRITIS
Rheumatoid arthritis (RA) is the prototype of the polyar-
ticular peripheral arthritides (see Chapter 66). RA accounts
for about one fourth of all patients referred to rheumatolo-
gists,1 and other forms of peripheral joint disease are often
defined partly by how they differ from RA.
Typically beginning in multiple small joints of the
hands and feet in a symmetric fashion, RA has many
variations, including months or years of recurrent mon-
arthritis (palindromic rheumatism)5 before a typical pat-
tern evolves. Disease duration of 12 weeks or more is
strongly predictive of persistent RA.6 The symmetry of
RA is sometimes overemphasized, and it must be appre-
ciated that this is a general, rough symmetry. RA rarely
causes extensive damage to one hand and completely
spares the other.
The arthritis of RA is typically additive, with sequential
involvement of groups of joints. Most joints remain more
or less symptomatic as new joints are involved. The earliest
joints involved are usually small joints of the hands and feet,
but the distal interphalangeal joints are spared until late in
the course.
Although arthritis is usually the presenting feature of
RA, occasional patients have extra-articular features of the
disease at roughly the same time or even earlier. Episcleri-
tis, subcutaneous nodules, and pleural effusions are the most
frequent early extra-articular features of the disease.
GONOCOCCAL ARTHRITIS
In contrast to the monarthritis typical of most septic joint
diseases, gonococcal arthritis usually manifests with fever,
tenosynovitis, and papular or pustular skin lesions. As it
evolves over several days, it may gradually involve one or
more joints in a frankly purulent arthritis (see Chapter 99).
VIRAL ARTHRITIS
Viral arthritis can cause impressive polyarticular pain, often
lasting for weeks or months with pain out of proportion to
physical examination findings (see Chapter 104). Parvovi-
rus B19 is the prototype,9 but similar symptoms have been
reported with rubella,10 mumps,11 hepatitis B,12 and other
viruses. Reactive arthritis, psoriatic arthritis, or an acute
painful polyarthritis may be the presenting manifestation of
human immunodeficiency virus (HIV) infection.13 Another
presentation of HIV infection resembles SLE, with fever,
rash, polyarthritis, proteinuria, and hematologic abnor-
malities.14 Infectious polyarthritis also may complicate HIV
infection.
PAUCIARTICULAR PERIPHERAL ARTHRITIS
In general, the term pauciarticular is used to describe arthritis
that affects four or fewer joints, although certain latitude
may be taken, such as counting the midfoot or wrist as a
single joint. This term usually is limited to the early phase
of the disease because many of these conditions gradually
become polyarticular as the course progresses.
 548 Sergent  |  Polyarticular Arthritis

Polyarticular joint pain

Joint effusion YES Inflammation? NO Noninflammatory joint pain

Crystals YES Gout or pseudogout

NO
Joint inflammation? NO Noninflammatory joint pain
YES
Spinal involvement YES Spondyloarthropathy
NO

Extra/juxtaarticular inflammation
Skin/subcutaneous nodules (See Chapter 47)
Eye (see Chapter 46)
NO
Specific radiographic abnormalities of peripheral joints
Osteoarthritis
Periostiitis (spondyloarthropathy, acne-associate arthritis, HPOA-Chapter 113)
Inflammatory arthritis
NO, OR inflammatory arthritis on radiograph
Order specific laboratory testing only if pretest probability is favorable
RF
ANA
ASO
Viral titers (e.g. Parvovirus B-19, hepatitis B and C)
HLA-B27
Anti-Borrelia Ab
Uric acid
Blood culture

Noninflammatory joint pain

Joint based pain NO Muscular tenderpoints YES Fibromyalgia

in specific distribution?
NO

Insertional tenderness NO Nonarticular

soft tissue pain
YES YES

Tendinitis/bursitis

Bony enlargement on exam or radiographic focal joint space

narrowing, osteophytes, subchondral sclerosis
YES

Evidence for endocrine or metabolic abnormality

or chondrocalcinosis on radiograph YES Specific laboratory testing
NO Growth hormone
Iron studies
OSTEOARTHRITIS Homogentistic acid level
Parathyroid hormone

Figure 37-1  Algorithm for assessment of polyarthritis. ANA, antinuclear antibody; ASO, antistreptolysin O; HPOA, hypertrophic pulmonary
­osteoarthropathy; RF, rheumatoid factor.
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
PSORIATIC ARTHRITIS
Subsets of psoriatic arthritis include rheumatoid-like, spon-
dyloarthropathy with dactylitis and inflammation of tendon
insertions (enthesitis), and a subset with a predilection for
distal interphalangeal joint involvement, particularly in the
presence of psoriatic nail involvement. This condition may
precede skin disease in a few patients and usually begins as
pauciarticular disease. The arthritis is typically asymmetric
(Fig. 37-2), and occasional patients show severe damage
to one group of joints with no involvement of the contra-
lateral side (see Chapter 72). In many cases, entire digits
are involved with arthritis and periostitis, producing the
appearance of a sausage digit.
REACTIVE ARTHRITIS
Previously known as Reiter’s syndrome (arthritis, conjunc-
tivitis, psoriasiform rash, or mucosal lesions following infec-
tions of the gut or genitourinary tract), reactive arthritis
may become manifest in a manner similar to that of psori-
atic arthritis, but with a greater predilection for the lower
extremity. The arthritis is often asymmetric and is frequently
associated with involvement of the heel, other entheses, or
the sacroiliac joints (see Chapter 71).
ADULT RHEUMATIC FEVER
Apparently increasing in frequency of occurrence, adult
rheumatic fever often causes a painful pauciarticular disease,
which is most prominent in the larger joints of the lower
extremities. The typical migratory pattern of childhood
rheumatic fever is uncommon in adults,15 and the response to
aspirin is less dramatic. Although some fever is usually pres-
ent, carditis is uncommon, and other features, such as chorea,
rash, and subcutaneous nodules, are rare. The ­presentation in
adults may be less acute than in children, with a more insidi-
ous arthritis developing over several days or 1 to 2 weeks.
GOUT
Gout often manifests to the rheumatologist as undiagnosed
chronic polyarthritis (see Chapter 87). Particularly confus-
ing can be the syndrome of acute and chronic gouty arthritis
Figure 37-2  Psoriatic arthritis. Note the asymmetry of the distal interphalangeal joint involvement and the associated psoriatic nail disease.
549
superimposed on osteoarthritis of the finger joints.16 Exami-
nation of synovial fluid or material draining from a tophus
yields the diagnosis.
CALCIUM PYROPHOSPHATE DEPOSITION DISEASE
Calcium pyrophosphate deposition disease is most eas-
ily recognized when it manifests as an acute monarthritis
(pseudogout). It has many forms, however, including a
chronic polyarthritis (see Chapter 88). Radiographically,
it may manifest as osteoarthritis in an unusual area (e.g.,
wrists without previous trauma or metacarpophalangeal
rows) or with characteristic chondrocalcinosis that is most
commonly seen in the fibrocartilaginous portions of the
joint.
BEHÇET’S DISEASE
Uncommon in the United States, Behçet’s disease almost
invariably causes chronic polyarthritis limited to a few
joints. It is characterized by painful acute flare-ups associ-
ated with oral, genital, and ophthalmologic manifestations
(see Chapter 86).
ENTEROPATHIC ARTHRITIS
Enteropathic arthritis is associated with inflammatory
bowel disease and characteristically involves large joints
of the lower extremity or the lumbosacral spine (see
Chapter 73).
RELAPSING POLYCHONDRITIS
Relapsing polychondritis usually causes severe inflam­
mation of the ears, nose, and sclerae and is associated
with a pauciarticular arthritis of the knees or wrists (see
Chapter 95).
SARCOIDOSIS
Sarcoidosis causes numerous articular problems. The most
frequent is an oligoarthritis of the knees or ankles in associa-
tion with erythema nodosum and bilateral hilar adenopa-
thy. Joint effusions are rare. Occasionally, in patients with ­
 550 Sergent  |  Polyarticular Arthritis
long-standing sarcoidosis, a chronic, destructive ­polyarthritis
resembling RA may develop with lytic bone lesions on
radiographs (see Chapter 107).
LYME DISEASE
The arthritis of Lyme disease is usually monarticular or oli-
goarticular, but a chronic symmetric polyarthritis also has
been described (see Chapter 100).17
BACTERIAL ENDOCARDITIS
The arthritis of bacterial endocarditis also is typically mon-
articular or oligoarticular, usually in the lower extremities,
although back pain also may be a presenting feature. About
one quarter of patients have rheumatoid factor, which can
add to the diagnostic difficulties.18
AMYLOID ARTHROPATHY
Amyloid arthropathy may include virtually all joints, but
pauciarticular involvement of the upper extremities is most
frequent. Bilateral shoulder disease may cause joint enlarge-
ment known as the “shoulder pad” sign. This finding,
especially in association with carpal tunnel syndrome and
purpura in skin folds, should always prompt the examiner
to suspect primary amyloidosis. The amyloidosis caused by
β2-microglobulin deposition in patients on long-term dialy-
sis may manifest with an impressive destructive arthritis of
large joints, especially the knees, shoulders, and hips (see
Chapter 106).19
INFLAMMATORY POLYARTHRITIS
WITH AXIAL INVOLVEMENT
Early involvement of the axial skeleton can be an extremely
important clue to the correct diagnosis of inflammatory
polyarthritis. For that reason, it may be helpful to ques-
tion the patient carefully about neck and lumbar spine pain
and prolonged stiffness to rule out unusual presentations of
ankylosing spondylitis, reactive arthritis, or other spondylo-
arthropathies (see Chapters 70 and 71).
ANKYLOSING SPONDYLITIS
Ankylosing spondylitis is the prototype of this group of
diseases. Although it typically begins in the patient’s late
teens or early 20s with back pain and stiffness, when it
begins in children or young teenagers, peripheral arthritis
is common and may precede back symptoms by months
or years. The arthritis involves predominantly the lower
extremities and includes knees, ankles, and feet in most
patients.20
reactive arthritis
Although only one fourth to one third of patients with
reactive arthritis have overt back symptoms on presenta-
tion, most of these patients have unilateral or bilateral
sacroiliitis and spine disease during the course of the
disease.21
ENTEROPATHIC ARTHRITIS
The arthritis of inflammatory bowel disease, enteropathic
arthritis may manifest as axial or peripheral arthritis,
or the two may coexist.22 The axial arthritis is identi-
cal to ankylosing spondylitis and is strongly associated
with HLA-B27. When present, the spondylitis runs a
progressive course regardless of the activity of the bowel
disease.23 Spondylitis typically develops at around the
same time as the bowel disease, but may precede it by
years.
PSORIATIC ARTHRITIS
Psoriatic arthritis may cause an axial and a peripheral
arthritis, but the axial disease is not a typical presenting
feature.
WHIPPLE’S DISEASE
Most patients with Whipple’s disease may present with
a chronic nondeforming migratory polyarthritis usually
involving only a few joints at a time. The arthritis may pre-
cede other symptoms, such as weight loss, diarrhea, hyper-
pigmentation, or central nervous system involvement, by
years.24
NONINFLAMMATORY POLYARTHRITIS
Many of the features that support the diagnosis of nonin-
flammatory polyarthritis (i.e., osteoarthritis) are simply the
absence of features that suggest inflammation. Many specific
findings are helpful, however, in establishing a diagnosis of
osteoarthritis (see Chapter 90).
The patient usually describes a history of pain primarily
during and after use of the affected joints, with minimal pain
at rest and minimal morning stiffness. Physical examination
often reveals coarse crepitation, and loose bodies and other
debris may be palpable while the joint is moved through its
range of motion. Bony osteophytes may be palpable, espe-
cially in the fingers.
Results of routine tests of inflammation are normal, unless
another disease is present. Because osteoarthritis and rheu-
matoid factor positivity increase in frequency with advanc-
ing age, positive test results for rheumatoid factor become
less valuable in older individuals. In a French study, nearly
half of the patients seeking care for osteoarthritis had poly-
articular arthritis.25
The ultimate diagnostic test for osteoarthritis is the
radiograph. Numerous important biochemical changes in
cartilage precede radiographic changes, but for all practi-
cal purposes, symptomatic osteoarthritis is nearly always
accompanied by radiographic changes (see Chapter 53) and
manifests as specific syndromes.
OSTEOARTHRITIS OF THE HANDS
A hereditary disease much more prevalent in women,
osteoarthritis of the hands typically develops within a
few years of menopause and is often associated with mild
inflammation for the first 1 or 2 years that a particular joint
is involved. The joints may be intermittently warm and
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
tender. After 1 or 2 years, the evidence of inflammation,
if any, gradually subsides, and the typical osteophytes of
Heberden’s and Bouchard’s nodes develop in the distal and
proximal interphalangeal joints. The disease is strikingly
symmetric, although the degree of involvement may vary
(Fig. 37-3).26
PRIMARY GENERALIZED OSTEOARTHRITIS
A rare, hereditary disease with osteoarthritis of multiple
joints, primary generalized osteoarthritis usually begins in
middle age and involves “typical” joints, such as the hips,
knees, and hands.27
OSTEOARTHRITIS SECONDARY TO METABOLIC
DISEASES
Osteoarthritis secondary to metabolic diseases is increasingly
recognized as particular patterns of osteoarthritis have been
described in association with several disorders discussed next.
Calcium Pyrophosphate Deposition Disease
Calcium pyrophosphate deposition disease (CPPD) may be
associated with generalized osteoarthritis, including severe
involvement of specific joints (pseudo-Charcot joints) and
involvement of joints not generally involved with osteo-
arthritis (wrists, ankles, elbows, metacarpophalangeal
joints). The frequency of CPPD increases with age, and
some elderly patients with osteoarthritis of the knee have
chondrocalcinosis on radiographic examination or CPPD
crystals on synovial fluid examination. More often than
not, tricompartmental osteoarthritis with effusion indi-
cates the presence of CPPD, especially with severe patel-
lofemoral joint involvement (see Chapter 88). Underlying
causes for which screening should be performed include
hemochromatosis, hyperparathyroidism, and hypothyroid-
ism. In patients with hand involvement, radiographs are
sensitive for establishing the diagnosis early in the disease
course.28
Figure 37-3  Osteoarthritis. Although the left third proximal interpha-
langeal joint is involved most severely, the right also is involved, and
there are several early Heberden’s nodes. The patient is a 60-year-old sec-
retary who has moderate soreness in these joints at the end of the day.
551
Hemochromatosis
Hemochromatosis, an underdiagnosed but common disor-
der,29 may manifest with osteoarthritis that is associated with
CPPD. Osteoarthritis involving the second and third meta-
carpophalangeal joints is particularly common and may be
confused clinically with RA, but it has distinct radiographic
differences. Early-onset osteoarthritis of weight-bearing and
large non–weight-bearing joints also may be seen in hemo-
chromatosis (see Chapter 108).
Hypothyroidism
Hypothyroidism is associated with symmetric, noninflam-
matory, highly viscous effusions, frequently in joints with
preexisting osteoarthritis (see Chapter 111).
Acromegaly
Acromegaly causes polyarthritis as a result of cartilage over-
growth early in disease and the subsequent development
of significantly debilitating osteoarthritis, with the most
severe involvement occurring in the hips and spine (see
Chapter 111).30
CHONDROMALACIA
Chondromalacia of the patella has multiple causes, but, as
a clinical presentation, it is usually seen in physically active
young and middle-aged women. Physical findings include
small effusions, laxity of the patellar ligaments, and patel-
lofemoral crepitation (see Chapters 42 and 90).
OTHER CONDITIONS
Obesity causes numerous joint problems, the most common
of which is bilateral osteoarthritis of the knees, especially in
women.31 Various developmental defects, such as congeni-
tally shallow acetabula, are often asymptomatic until osteo-
arthritis develops later in life.
DIFFERENTIAL DIAGNOSIS
ARTHRITIS VERSUS ARTHRALGIA
Table 37-3 lists the major nonarticular considerations in
patients with chronic polyarticular pain. The first challenge
to the physician is to differentiate among arthritis, arthral-
gia, and periarticular disorders.
TENDINITIS AND RELATED DISORDERS
Tendinitis and related disorders include painful shoulder
syndromes, tennis elbow (lateral epicondylitis), golfer’s
elbow (medial epicondylitis), trochanteric bursitis, iliotibial
band syndrome, anserine bursitis, prepatellar bursitis, Achil-
les tendinitis, and tendinitis along the radial aspect of the
wrist (de Quervain’s tenosynovitis). In these syndromes,
pain is often maximal at the beginning of an activity and
starts to subside as the activity is continued. A thorough
history, including a description of job and sports activities,
is essential. In many cases, physical examination reveals
 552 Sergent  |  Polyarticular Arthritis

Table 37-3  Differential Diagnosis of Polyarticular  

Pain
Polyarthritis
Tendinitis
Muscle disorders
Polymyalgia rheumatica
Vasculitis
Vaso-occlusive disease
Neuropathies
Diseases of the spine
Primary bone diseases
Periostitis
Fibromyalgia
Malingering

areas of local tenderness near, but distinct from, the joint.

Pain may be exacerbated by movement of the affected struc-
tures against resistance. In tendinitis, swelling, if present, is
usually minimal and limited to tendon sheaths and bursae,
rather than joints. Thickening of tendons and subcutaneous
tissues in patients with diabetes can cause a variety of clini-
cal features, including Dupuytren’s contractures and, espe-
cially in patients with juvenile-onset diabetes, the diabetic
stiff hand syndrome (Fig. 37.4) (see Chapter 111).32
MUSCLE DISORDERS
Although weakness predominates in most of these condi-
tions, and arthritis is not a serious consideration, occasional
patients present with periarticular pain as a primary com-
plaint (see Chapter 78).
POLYMYALGIA RHEUMATICA
Because many patients with polymyalgia rheumatica (see
Chapter 81) have preexisting polyarticular disorders, such as
osteoarthritis and tendinitis, it may require skill and experi-
ence to differentiate among these problems. Several inves-
tigators33-35 have pointed out the difficulty of distinguishing
polymyalgia rheumatica from older-onset RA and have stressed
that in the early stages it may be impossible to tell them apart.
VASCULITIS
Frank arthritis is an uncommon manifestation of systemic
vasculitis. Joint pain may be deep, aching, and constant.
VASO-OCCLUSIVE DISEASES
Vaso-occlusive diseases include atherosclerosis; cholesterol
emboli; and emboli from cardiac sources such as a myxoma,
diabetes, Raynaud’s disease, Buerger’s disease, and the
antiphospholipid syndrome. These conditions rarely cause
polyarticular pain without other manifestations of disease.
Patients with cholesterol emboli may present with polyar-
thralgias, myalgias, renal disease, elevated erythrocyte sedi-
mentation rates, eosinophilia, and positive test results for
rheumatoid factor and antinuclear antibody.36
Figure 37-4  “Prayer sign” in the diabetic stiff hand syndrome. As a re-
sult of progressive thickening of tendons, joint capsules, and subcuta-
neous tissues, progressive stiffness and flexion contractures develop in
these patients.
NEUROLOGIC DISEASES
Neurologic diseases include peripheral neuropathies, com-
pression neuropathies such as carpal tunnel syndrome, and
infiltrative diseases such as amyloidosis and Waldenström’s
macroglobulinemia. Pain usually is associated with pares-
thesias and usually is worse at night. Although patients may
have pain localized to joints, it is more typically diffuse, and
examination reveals no objective joint abnormalities.
DISEASES OF THE SPINE
A variety of diseases of the neck and spine, including spi-
nal stenosis (congenital or acquired), spondylolisthesis, and
tumors of the lower cord and cauda equina, may manifest
in a similar fashion. The pain is usually primarily in the
buttocks and is worse with certain postures or activities. In
spinal stenosis, pain is typically that of neurogenic claudica-
tion, with aching in the buttocks and thighs brought on by
certain activities and that is often worse going down hills or
steps (see Chapter 41).
PRIMARY BONE DISEASES AND MALIGNANCY
Metastatic tumors and myeloproliferative disorders may
masquerade as polyarticular arthritis. Children with sickle
cell crises and leukemia are especially likely to present with
widespread joint pain, and any large series of children with
the initial diagnosis of juvenile RA contains a small percent-
age who ultimately are found to have leukemia.37 In adults,
myeloma and a variety of widely metastatic cancers manifest
as bone and joint pain. In childhood leukemia, joint effu-
sions, warmth, and tenderness may be present (see Chapter
112). These objective findings are rare in adults with leuke-
mia or other malignancies.
Other widespread bone diseases, especially osteonecrosis
(see Chapter 94), occasionally may resemble polyarticular
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
arthritis. When osteonecrosis involves peripheral joints,
such as the knee and ankle, swelling and tenderness may be
impressive.38 It is common to see stress fractures in runners
or weekend athletes mimicking polyarthritis in the feet and
ankles.
PERIOSTITIS
Periostitis, especially as part of the syndrome of hypertrophic
pulmonary osteoarthropathy, may cause severe widespread
joint pain, tenderness, and warmth. Careful examination
reveals tenderness around joint structures and along the
shafts of the involved long bones (see Chapter 112) typi-
cally associated with clubbing of the fingernail beds.
FIBROMYALGIA AND PSYCHOGENIC
PAIN SYNDROMES
Objective examination of patients with the chronic pain
syndrome of fibromyalgia reveals generalized tenderness
that is not joint based, but distributed preferentially in typi-
cal trigger points (see Chapter 38).
SUMMARY
Polyarticular arthritis, the most common indication for
rheumatologic consultation, represents a challenge to the
skills and experience of the clinician. By careful history
taking and physical examination, along with appropriate
tests, the physician almost always can establish the correct
diagnosis, however, and institute appropriate therapy. In
an era of increasing demand that medicine show that it is
cost-effective, the bedside skills required to evaluate these
patients prove particularly valuable.
REFERENCES
  1. Hooker RS, Brown JB: Rheumatology referral patterns. HMO Practice
4:61, 1990.
  2. Pauker SG, Kassirer JP: Medical progress: Decision analysis. N Engl J
Med 316:250, 1987.
  3. Mackenzie AH: Differential diagnosis of rheumatoid arthritis. Am
J Med 85(Suppl 4):2, 1985.
  4. Wolfe F, Pincus T: Listening to the patient: A practical guide to self-
report questionnaires in clinical care. Arthritis Rheum 42:1797,
1999.
  5. Hannonen P, Mottonen T, Oka M: Palindromic rheumatism: A clini-
cal survey of sixty patients. Scand J Rheumatol 16:413, 1987.
  6. Green M, Marzo-Ortega H, McGonagle D, et al: Persistence of mild,
early inflammatory arthritis: The importance of disease duration,
rheumatoid factor, and the shared epitope. Arthritis Rheum 42:2184,
1999.
  7. Zimmerman C, Steiner G, Skriner K, et al: The concurrence of rheuma-
toid arthritis and limited systemic sclerosis: Clinical and serologic char-
acteristics of an overlap syndrome. Arthritis Rheum 41:1938, 1998.
  8. Southwood TR, Petty RE, Malleson PN, et al: Psoriatic arthritis in
children. Arthritis Rheum 32:1007, 1989.
  9. Moore TL: Parvovirus-associated arthritis. Curr Opinion Rheum
12:289, 2000.
10. Chambers RJ, Bywaters EG: Rubella synovitis. Ann Rheum Dis
22:263, 1963.
553
11. Gordon SC, Lauter CB: Mumps arthritis: A review of the literature.
Rev Infect Dis 6:338, 1984.
12. Sergent JS: Extrahepatic manifestations of hepatitis B infection. Bull
Rheum Dis 33:1, 1983.
13. Winchester R: AIDS and the rheumatic diseases. Bull Rheum Dis
39:1, 1990.
14. Kopelman RH, Zolla-Pazner S: Association of human immunode-
ficiency virus infection and autoimmune phenomenon. Am J Med
84:82, 1988.
15. Wallace MR, Garst PD, Papadimos TJ, et al: The return of acute
rheumatic fever in young adults. JAMA 262:2557, 1989.
16. Lally EV, Zimmerman B, Ho G Jr, et al: Urate-mediated inflammation
in nodal osteoarthritis: Clinical and roentgenographic correlations.
Arthritis Rheum 32:86, 1989.
17. Steere AC, Schoen RT, Taylor E: The clinical evolution of Lyme
arthritis. Ann Intern Med 107:725, 1987.
18. Churchill MA, Geraci JE, Hunder GG: Musculoskeletal manifesta-
tions of bacterial endocarditis. Ann Intern Med 87:754, 1977.
19. Alfrey AC: Beta2-microglobulin amyloidosis. Nephrol Lett 6:27,
1989.
20. Khan MA: Editorial comment. J Rheumatol 16:634, 1989.
21. Lionarons RJ, van Zoeren M, Verhagen JN, et al: HLA-B27-­associated
reactive spondyloarthropathies in a Dutch military hospital. Ann
Rheum Dis 45:141, 1986.
22. Gravallese EM, Kantrowitz FG: Arthritic manifestations of inflam-
matory bowel disease. Am J Gastroenterol 83:703, 1987.
23. Burgos-Vargas R, Clark P: Axial involvement in the seronegative
enteropathy and arthropathy syndrome and its progression to ankylos-
ing spondylitis. J Rheumatol 16:192, 1989.
24. Durand DV, Leomte C, Cathebras P, et al: Whipple disease: Clinical
review of 52 cases. The SNFMI Research group on Whipple Disease.
Medicine 76:170-184, 1997.
25. Fautrel B, Hilliquiin P, Allaert F-A, et al: Who are the patients
who consult for osteoarthritis? Arthritis Rheum 44:5237, 2001
(abstract).
26. Cushnaghan J, Dieppe P: Study of 500 patients with limb joint
osteoarthritis, I: Analysis by age, sex, and distribution of symptom-
atic joint sites. Ann Rheum Dis 50:8, 1991.
27. Ala-Kokko L, Baldwin CT, Moskowitz RW, et al: Single base muta-
tion in the type II procollagen gene (COL2A1) as a cause of primary
osteoarthritis associated with a mild chondrodysplasia. Proc Natl Acad
Sci U S A 87:6565, 1990.
28. Devauchelle-Pensec V, Berthelot JM, Jousse S, et al: Performance
of hand radiographs in predicting the diagnosis in patients with early
arthritis. J Rheumatol 33:1511-1515, 2006.
29. Edwards CQ, Griffen LM, Goldgar D, et al: Prevalence of hemo-
chromatosis among 11,065 presumably healthy blood donors.
N Engl J Med 318:1355, 1988.
30. Biermasz NR, Pereira AM, Smit JW, et al: Morbidity after long-term
remission for acromegaly: Persisting joint-related complaints cause
reduced quality of life. J Clin Enodcrinol Metab 90:2731-2739, 2005.
31. Felson DT, Anderson JJ, Naimark A, et al: Obesity and knee osteo-
arthritis: The Framingham Study. Ann Intern Med 109:18, 1988.
32. Kapoor A, Sibbitt WL Jr: Contractures in diabetes mellitus: The
syndrome of limited joint mobility. Semin Arthritis Rheum 18:168,
1989.
33. Healey LA: Late-onset rheumatoid arthritis vs. polymyalgia rheumat-
ica: Making the diagnosis. Geriatrics 43:65, 1988.
34. Deal CL, Meenan RF, Goldenberg DL, et al: The clinical features of
elderly-onset rheumatoid arthritis. Arthritis Rheum 28:987, 1985.
35. Healy LA: Polymyalgia rheumatica and seronegative RA may be the
same entity. J Rheumatol 19:270, 1991.
36. Cappiello RA, Espinoza LR, Adelman H, et al: Cholesterol embolism:
A pseudovasculitic syndrome. Semin Arthritis Rheum 18:240, 1989.
37. Kunnamo I, Kallio P, Pelkonen P, et al: Clinical signs and laboratory
tests in the differential diagnosis of arthritis in children. Am J Dis
Child 141:34, 1987.
38. Lotke PA, Steinberg ME: Osteonecrosis of the hip and knee. Bull
Rheum Dis 35:1, 1985.
 38
KEY POINTS
Fibromyalgia is a commonly recognized syndrome characterized
by pain, sleep disturbance, and fatigue combined with a general
increase in medical symptoms, including problems of memory or
thinking, and often psychological distress.
A formal diagnosis of fibromyalgia is unnecessary as long as
fibromyalgia symptoms are recognized. There is no evidence
that diagnosing fibromyalgia improves its outcome.
Fibromyalgia has a quality of inexplicability and unexpected-
ness. The clinician is surprised by the extent and severity of
symptoms and surprised at unexpected emotional distress.
The prevalence of fibromyalgia is 2% to 4% worldwide.
Treatment has a modest short-term effect. There is no
­evidence that treatments alter the long-term outcome.
There is substantial evidence against the existence of fibro-
myalgia as a distinct clinical or epidemiologic entity.1-5
However, right or wrong, fibromyalgia is a commonly diag-
nosed syndrome, with its own ICD code and criteria.6 The
term fibromyalgia is used here to indicate an individual who
satisfies the American College of Rheumatology (ACR)
fibromyalgia criteria.6 In taking the schizoid position of writ-
ing about a condition that may not exist, we ask the reader
to remember that “… all models are wrong; the practical
question is how wrong do they have to be to not be useful,”7
a judgment we leave to the reader.
Fibromyalgia is a bitterly controversial disorder. What
is it about fibromyalgia, asked White,8 that provokes such
venom and ire? Rarely, if ever, written about, but often spo-
ken about within small groups of physicians and medical
workers, fibromyalgia patients are often identified and char-
acterized by unusual behaviors and psychological character-
istics. Barsky and Borus9 characterize fibromyalgia as one of
a group of “functional somatic syndromes.” Individuals with
such syndromes:
… share similar phenomenologies, high rates of co-
occurrence, similar epidemiologic characteristics, and
higher-than-expected prevalences of psychiatric comorbid-
ity …. Suffering … is exacerbated by a self-perpetuating,
self-validating cycle in which common, endemic, somatic
symptoms are incorrectly attributed to serious abnormality
…. The climate surrounding functional somatic syndromes
includes sensationalized media coverage, profound suspicion
of medical expertise and physicians, the mobilization of par-
ties with a vested self-interest in the status of functional
somatic syndromes, litigation, and a clinical approach that
Fibromyalgia
Frederick Wolfe  •  Johannes J. Rasker
overemphasizes the biomedical and ignores psychosocial
factors …. These influences exacerbate and perpetuate the
somatic distress of patients …, heighten their fears and pes-
simistic expectations, prolong their disability, and reinforce
their sick role.9
To a large extent, the “venomous”8 opposition to fibro-
myalgia comes from opposition to the idea of characterizing
this particular functional somatic syndrome as a “disease.”
HISTORICAL DEVELOPMENT
Attempts to characterize and diagnose fibromyalgia have
gone through several changes in conceptualization. The
earliest roots of fibromyalgia can be found in the 19th cen-
tury perception of abnormal connective tissue and muscles.
In various forms, this concept held sway until the late 1970s,
when a new emphasis on sleep disturbance and tender points
led to proposed clinical criteria that included sleep distur-
bance and tenderness to palpation at 12 of 14 selected sites.10
In the early1980s, most of the other fibromyalgia-associated
symptoms were identified, and criteria were proposed that
combined these symptoms with tenderness. With the pub-
lication of the ACR criteria in 1990,6 opposition to fibro-
myalgia increased and has continued until the present.11-17
Among proponents, fibromyalgia was characterized first as
an “affective symptom disorder”18,19 and later as the result of
“… aberrant central pain transmission … [in which] purely
behavioral or psychological factors are not primarily respon-
sible for the pain and tenderness ….”20 This view is held
by proponents in rheumatology and pain specialties; how-
ever, different views predominate in epidemiology, physical
medicine, and psychiatry.
FIBROMYALGIA DEFINITIONS
AND PROBLEMS
In 1990, the ACR criteria for fibromyalgia were published
(Table 38-1 and Fig. 38-1).6 They defined fibromyalgia as being
present in patients who had “widespread pain” and tenderness
at 11 of 18 tender point sites. The criteria rapidly came under
attack. For individuals who did not believe in fibromyalgia
as an entity, the criteria represented the false codifications of
symptoms and the establishment of a nondisease.11-16 A second
set of criticism addressed the circularity of the ACR criteria
study, arguing that because the authors approached the study
from the point of view of tender points, they could not fail to
find that tender points were important.17 Later, epidemiologic
and clinical studies showed that the fibromyalgia criteria iden-
tified a cut-point along a pain-distress continuum, rather than
identifying a discrete entity.1-5 Despite these objections, the
criteria and the underlying definition gained wide support.
555
 556 wolfe  |  Fibromyalgia

Table 38-1  1990 AMERICAN COLLEGE  

OF ­RHEUMATOLOGY CRITERIA FOR  
THE CLASSIFICATION OF FIBROMYALGIA*
1. History of widespread pain
Definition: Pain is considered widespread when all of the following
are present: pain in the left side of the body, pain in the right side
of the body, pain above the waist, and pain below the waist. In
addition, axial skeletal pain (cervical spine or anterior chest or
thoracic spine or low back) must be present. In this definition,
shoulder and buttock pain is considered as pain for each involved
side. Low back pain is considered lower segment pain.
2. Pain in 11 of 18 tender point sites on digital palpation
Definition: Pain, on digital palpation, must be present in at least 11 of
the following 18 tender point sites:
  Occiput: bilateral, at suboccipital muscle insertions
  Low cervical: bilateral, at anterior aspects of intertransverse spaces
at C5-C7
  Trapezius: bilateral, at midpoint of upper border
  Supraspinatus: bilateral, at origins, above the scapula spine near
the medial border
  Second rib: bilateral, at second costochondral junctions, just lateral
to junctions on upper surfaces
  Lateral epicondyle: bilateral, 2 cm distal to epicondyles
  Gluteal: bilateral, in upper outer quadrants of buttocks in anterior
fold of muscle
  Greater trochanter: bilateral, posterior to trochanteric prominence
  Knees: bilateral, at medial fat pad proximal to joint line
Digital palpation should be performed with an approximate force
of 4 kg
For a tender point to be considered “positive” the subject must state
that the palpation was painful; “tender” is not to be considered
painful
*For classification purposes, patients are said to have fibromyalgia if criteria
1 and 2 are satisfied. Widespread pain must have been present for at least
3 months. The presence of a second clinical disorder does not exclude the
diagnosis of fibromyalgia.6
From Wolfe F, Smythe HA, Yunus MB, et al: The American College of Rheu-
matology 1990 Criteria for the Classification of Fibromyalgia: Report of the
Multicenter Criteria Committee. Arthritis Rheum 33:160-172, 1990.
Figure 38-1  Tender point sites of the 1990 American College of Rheu-
matology criteria for fibromyalgia. (From Wolfe F, Smythe HA, Yunus MB,
et al: The American College of Rheumatology 1990 Criteria for the Classifica-
tion of Fibromyalgia: Report of the Multicenter Criteria Committee. Arthritis
Rheum 33:160-172, 1990.)
Although the ACR criteria are classification criteria that
are designed for use in research studies, they do not include
many of the central symptoms of the syndrome. The domi-
nant symptom of fibromyalgia in the ACR criteria study, but
one not included in the criteria,6 was identified by the ques- of ­ diagnostic criteria is inconsistent with other rheumatic
tion, “Do you hurt all over?” In addition, some individuals disease criteria and is not workable clinically. In clinical
who do not satisfy the ACR criteria seem to have fibromyal- practice, however, when an individual is diagnosed with
gia, which can be identified by clinical criteria in individuals fibromyalgia, the diagnosis devolves to permanency. Clini-
with less than 11 tender points in the presence of character- cians who use the term fibromyalgia generally mean that the
istic fibromyalgia symptoms21; there is evidence that clini- individual has fibromyalgia traits—pain and fatigue, which
cians endorse this broader definition in their practices. In the may vary in intensity from time to time and in response
ACR criteria study, various candidate definitions of criteria to stressors—and that fibromyalgia behaves as if it were a
performed almost as well as the official criteria.6 One might “trait,” not a state, a type of response to known or unknown
conclude that the ACR criteria represent a manageable defi- stressors. From the point of view of clinical diagnosis and
nition of fibromyalgia, perhaps for research studies, but only care, an individual who satisfies the criteria at one time gen-
an incomplete and approximate definition for clinical care. erally can be considered to have fibromyalgia even when
There are other problems with diagnosis and definition not subsequently satisfying the criteria.
by ACR criteria. Using the continuum of tender points as
the central diagnostic variable, not only does an individ- OBJECTIONS TO THE CONCEPT
ual with 10 tender points not satisfy the diagnostic crite-
ria, but also an individual who has 11 tender points during
OF FIBROMYALGIA
one examination and 10 tender points at the next exami- There are a number of interrelated objections to ­fibromyalgia.
nation has fibromyalgia at the first examination and does The first is that what is called fibromyalgia is a nondisease,
not have fibromyalgia at the latter examination. Such a use merely the end of a pain-distress continuum. The ­contrary
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
view, expressed directly or acquiesced to, sees fibromyalgia
as a disease, rather than as a collection of symptoms. This
­fundamental disagreement is called into view when patients
ask, “Is it my fibromyalgia that causes me to be so tired (or to
have any one of many symptoms)?” Clinicians who act as if
fibromyalgia is a disease would answer, “Yes”; the objection-
ists, “No.” Stated slightly differently, “Do I have pain because
I have fibromyalgia or do I have fibromyalgia because I have
pain?” Individuals who object to the fibromyalgia concept
go further. They see the term fibromyalgia as part of the prob-
lem. Use of the term de facto, even if not intended, results in
fibromyalgia being seen or treated as a disease.12,13
There are consequences to treating a nondisease as a dis-
ease according to fibromyalgia opponents12,13: Fibromyalgia
symptoms are seen as manifestations of a disease and subject
to compensation for disability and injury; various traumas
can “cause” this disease; individuals whose predominant
problem is psychosocial distress can have their symptoms
designated as a disease; insurance costs increase; court deci-
sions institutionalize fibromyalgia; support groups multiply
in support of this nondisease; medical practitioners spring
up to treat nonillness, and pharmaceutical companies mar-
ket drugs for its treatment. These problems are exactly those
that Barsky and Borus9 identified as associated generally
with “functional somatic syndromes.”
Clinically, fibromyalgia seems to represent a cut-point
along a continuum of pain, fatigue, and other symptoms
in which “patients” with high-intensity symptoms (Fig.
38-2) and increased symptom prevalence can be found
(Fig. 38-3). Observations similar to these are found in epide-
miologic studies and may be summed up in this observation:
“The evidence that fatigue is dimensionally distributed in
the community, and that no cut-off exists to separate nor-
mal from abnormal fatigue, is overwhelming.”5 In addition,
fibromyalgia overlaps with chronic fatigue and “… ­virtually
Figure 38-2  A fibromyalgia pain diagram as completed in the clinic.
557
every other medically unexplained syndrome, including
tension headache, chemical sensitivity, irritable bowel
syndrome, atypical chest pain, gynaecological syndromes,
­temporomandibular disorders, and mitral valve prolapse.”5
EPIDEMIOLOGY
Fibromyalgia is diagnosed more frequently in women
(9:1 ratio). Using ACR criteria, the prevalence of fibromyal-
gia in the adult general population is generally similar across
the world. The prevalence of fibromyalgia in Wichita, Kan-
sas, was 3.4% (95% confidence interval [CI] 2.3, 4.6) among
women, 0.5% (95% CI 0.0, 1.0) among men, and 2% (95%
CI 1.4, 2.7) overall22; among women in New York City,
it was 3.7% (95% CI 3.2, 4.4).23 In Ontario, Canada, the
estimated prevalence was 4.9% (95% CI 4.7, 5.1) among
women, 1.6% (95% CI 1.3%, 1.9%) among men,24 and
3.3% (95% CI 3.2, 2.4) overall. The prevalence of fibromy-
algia in these studies increased with age until about age 70,
after which it decreased slightly. Outside of North America,
reports indicate the prevalence in Bangladesh was 5.3% to
7.5% in women and 0.2% to 1.4% in men25; in North Paki-
stan, it was 2.1% overall26; in Italy, it was 2.2% (95% CI
1.4-3.2)27; in Turkey, it was 3.6% (95% CI 2.8-4.4) for ages
20 to 6428; in Brazil, it was 2.5% (95% CI 1.97-3.12)29; and
in Southwest Sweden, it was 1.3% (95% CI 0.8-1.7).30
The prevalence of fibromyalgia in children in three stud-
ies was 1.2%,31 1.4%,32 and 6.2%.33 At a follow-up time of 1
year, approximately 25% of individuals meeting ACR crite-
ria initially still satisfied the criteria.32,34 These data should
not be interpreted as evidence of prognosis because some
individuals not meeting criteria initially meet them at the
1-year follow-up. Instead, the data suggest that the concept
of fibromyalgia in children may be dubious, particularly
when dependent on tender point assessment.
 558 wolfe  |  Fibromyalgia

1
.8 .8

sleep disturbance
Probability of
parasthesias

.6 .6

Probability of
.4 .4
.2 .2
0 0

0 2 4 6 8 10 0 2 4 6 8 10
Symptom intensity scale Symptom intensity scale

.8 .8

memory problem
Probability of
.6 .6
Probability of
depression

.4 .4
.2 .2
0 0

0 2 4 6 8 10 0 2 4 6 8 10
Symptom intensity scale Symptom intensity scale
Figure 38-3  Graphs of characteristic fibromyalgia-type symptoms as a function of symptom intensity (SI) scale values, adjusted for age and sex. Pre-
dicted values and their 95% confidence intervals are generated by running line smooths of the symptom variable on SI scale, adjusted for age and sex.
The vertical lines define the 5% and 95% confidence intervals for having and not having fibromyalgia by survey fibromyalgia criteria. The SI scale is com-
bined measure of the extent of regional pain and the intensity of fatigue. (Modified from Wolfe F, Rasker JJ: The symptom intensity (SI) scale, fibromyalgia,
and the meaning of fibromyalgia-like symptoms. J Rheumatol 2006; 33:2291-2299.)

The prevalence of fibromyalgia is generally greater in clin-
ical settings than in epidemiologic studies. It was noted to be
5.7% in general medical clinics35 and 2.1% in family practice
settings.36 In rheumatology clinics, fibromyalgia prevalence
was expectedly higher: 12%37 to 20%38 of new patients.
ETIOLOGY AND PATHOPHYSIOLOGY
Many theories have been proposed for fibromyalgia, but
neither etiology nor the pathophysiologic mechanisms are
known, and no model explains more than a little of the
available data. In addition, because fibromyalgia symptoms
are distributed as a continuum, pathophysiologic investiga-
tions that consider fibromyalgia as a discrete entity to be
compared with “normal” controls distort the importance
and understanding of observed mechanisms.
Theories have shifted over time from peripheral pathol-
ogy (muscles and insertions) to central dysfunction (pain
processing), and from unicausal to multicausal hypoth-
eses. It is likely that many mechanisms play overlapping
roles in the pathophysiology of fibromyalgia symptoms.
Central mechanisms, suggested by the presence of sleep
disturbance, blunted stress response, hyperalgesia, and the
diffuse nature of fibromyalgia pain, have been reviewed in
detail.39-44
MUSCLES AND MICROTRAUMA
Originally thought to be important in pathogenesis, muscle
and tendon disorders have fallen out of favor because they do
not explain adequately the systemic symptoms of fibromyal-
gia. In addition, changes found in muscle biopsy ­specimens
are nonspecific, consistent with many types of muscle dam-
age, ranging from ischemia to simple deconditioning, and
are not different from changes found in individuals without
fibromyalgia.
PSYCHOSOCIAL FACTORS
Psychosocial factors, which include reduced education,
nonmarried status, lower household income, smoking, and
obesity, have been identified in many studies.
GENETIC AND FAMILIAL FACTORS
Fibromyalgia aggregates in families.45,46 Genetic factors may
predispose individuals to fibromyalgia. A possible genetic
linkage in the HLA region has been reported, based on sib-
ship analysis.47 Patients with chronic widespread pain and
fibromyalgia have been found to have low gene expression
for the proinflammatory cytokines interleukin-4 and inter-
leukin-10 and reduced levels of serum concentrations com-
pared with controls. These findings might indicate a role
for cytokines in the pathophysiology of fibromyalgia or as a
sequela of chronic pain and its treatment.48
SLEEP DISTURBANCE
Fibromyalgia patients often report unrefreshing and nonrestor-
ative sleep.49 Electroencephalographic abnormalities initially
were thought to play a major role in the pathogenesis of fibro-
myalgia, but it is now clear that such abnormalities are non-
specific findings. Sleep electroencephalographic studies show
abnormalities of delta wave or stage 4 sleep by repeated alpha
wave intrusion. Similar abnormalities are found in healthy
individuals, and in individuals with emotional stress, fever,
osteoarthritis, rheumatoid arthritis, and Sjögren’s syndrome.
STRESS-RELATED NEUROENDOCRINE
DYSFUNCTION
Stress responses and endocrine axes are disturbed in fibro-
myalgia, but many of these changes are commonly seen in
patients who have known external sources of ­chronic pain.
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
It is unclear whether these endocrine disturbances in fibro-
myalgia are primary to the disorder or are secondary to the
pain or distress associated with fibromyalgia. Patients with
fibromyalgia report more past stressful life events and more
daily stressful hassles than patients with rheumatoid arthri-
tis or pain-free healthy controls. Similarly, fibromyalgia is
associated with increased reports of virus infections and
traumas preceding fibromyalgia and a higher frequency of
sexual abuse in childhood. Work-related psychological fac-
tors such as work demands and factors such as job control,
social support, and psychological distress are associated with
reporting of musculoskeletal pain, particularly when pain is
reported at multiple sites.50
PRIMARY NEUROENDOCRINE DYSREGULATION
Primary neuroendocrine dysregulation found in fibromyalgia
can be divided into changes in the two major stress systems,
the hypothalamic-pituitary-adrenal axis and the autonomous
nervous system. In fibromyalgia, almost all hormonal feedback
mechanisms controlled by the hypothalamus are disrupted.
After stimulation of the hypothalamic-pituitary-adrenal
axis with exogenous corticotrophin-releasing hormone or
by insulin-induced hypoglycemia, an exaggerated pituitary
adrenocorticotropic hormone release has been observed
with relative adrenal hyporesponsiveness.51 Serum thyroid
hormone levels are normal, but after intravenous injection
of thyrotropin-releasing hormone, patients with primary
fibromyalgia responded with a significantly reduced secretion
of thyrotropin and thyroid hormones.52 Growth hormone
is secreted during stage 4 sleep and is important for muscle
repair and strength. Low levels might explain extended peri-
ods of muscle pain after exertion in fibromyalgia patients.
Serum growth hormone levels and levels of somatomedin C
(insulin-like growth factor-I) have often been reported to be
low, but results are inconsistent.53 It is possible that physical
deconditioning, related to avoidance of physical activities
because of pain, could lead to more fatigue, stiffness, and, via
altered growth hormone metabolism, sleep disturbance.
AUTONOMOUS NERVOUS SYSTEM
Sympathetic function in fibromyalgia patients has been
reported as low, normal, or functionally high. There is a
derangement of sympathetic tone and reaction in some
patients, being high or low, depending on the situation. One
explanation for this finding may be that most studies did not
control for physical activity levels of participants.54 It also
has been suggested that fibromyalgia is a generalized form of
reflex sympathetic dystrophy.55
ABNORMAL PAIN PROCESSING
There are major differences between the sexes with respect
to analgesic responses, across all animal species. This may
explain the decreased pain tolerance in women with fibro-
myalgia compared with men. Patients with fibromyalgia
have reduced pain tolerance to stimuli that are normally
not painful, such as pressure, heat, and electric pulse, at the
classic tender points and control points (allodynia). They
also perceive pain as being more intense and extending for a
longer time (hyperalgesia).
559
This abnormal sensory pain processing could be explained
by increased pain facilitation and reduced pain-inhibiting
mechanisms on the spinal and cerebral levels. Fibromyalgia
patients also displayed abnormal temporal summation of pain
after a series of thermal stimulations, called “wind-up.”56
The concentration of substance P, a neuromodulator
of pain, in the cerebrospinal fluid was threefold greater in
fibromyalgia patients than in controls. Substance P may
play a role in spreading of muscle pain. This elevation of
substance P is not specific to fibromyalgia, however, and
has been shown in patients with pain due to other causes.
Measures of pain intensity in fibromyalgia patients are cor-
related with levels of metabolites of the excitatory amino
acid neurotransmitters glutamate and aspartate. Sensitiza-
tion of nociceptive neurons in the spinal dorsal horn by
hyperexcitable receptors, such as the glutamate receptor
N-methyl-d-aspartate, could be one of the mechanisms
responsible for pain in fibromyalgia.44
DECREASED PAIN INHIBITION
Pain inhibitory pathways, descending from the cortex, limbic
system, hypothalamus, thalamus, and brainstem, modulate
the activity of spinal nociceptive neurons. In fibromyalgia
patients, regional blood flow seems to be reduced in the most
important pain processing areas in the brain, the thalamus
and caudatum, compared with controls.57
Serotonin is a neurotransmitter in the descending inhibi-
tory pathways that inhibits release of substance P and excit-
atory amino acids from the terminals of primary afferent
neurons. Serotonin also regulates NREM sleep. Low levels
of serotonin metabolites have been reported in the cerebro-
spinal fluid and serum of patients with fibromyalgia and low
back pain.44 Serotonin antibodies are found in fibromyalgia
patients four times as frequently as in controls, but have no
diagnostic relevance.58 The role of serotonin in the patho-
physiology of fibromyalgia is unclear. Drugs that affect sero-
tonin metabolism or action do not have a dramatic effect.
PSYCHOLOGICAL ABNORMALITIES
There has been disagreement as to whether psychiat-
ric abnormalities represent reactions to chronic pain, or
whether the symptoms of fibromyalgia are a reflection of
psychiatric disturbance. Psychiatric disorders may interact
with the neuroendocrine system as part of a stress reac-
tion.59 The most common psychiatric conditions observed
in patients with fibromyalgia include depression, dysthymia,
panic disorder, and simple phobia.60 In the National Data
Bank for Rheumatic Diseases,61 64% of patients report prior
depression, and 8% report mental illness. Fibromyalgia also
occurs in patients without significant psychiatric problems,
however. Some individuals with fibromyalgia satisfy the
American Psychiatric Association criteria for somatoform
disorders (DSM 307.80 and 307.89).62
CLINICAL FEATURES
Fibromyalgia is characterized by high levels of pain, sleep
disturbance, and fatigue combined with a general increase
in medical symptoms (Table 38-2), including problems of
memory or thinking, and often ­ psychological distress.63
 560 wolfe  |  Fibromyalgia

Table 38-2  Prevalence of Specific Symptoms   1

among 2784 Patients with Fibromyalgia in   Fibromyalgia probability
the National Data Bank for Rheumatic Diseases
Symptom % .8
Sleep problems 89.1

Probability of Fibromyalgia
Fatigue or tiredness 88.6
Muscle pain 85.2 .6
Muscle weakness 70.2
Paresthesias 67.6
Cognitive problems 66.3 .4

Headache 64.7
Dry mouth 53.3
.2 Symptom count
Insomnia 51.8
Easy bruising 49.1
Dry eyes 47.5
0
Depression 47.5
0 10 20 30 40
Blurred vision 47
Symptom count (0-37)
Irritable bowel syndrome 46.3
Figure 38-4  The relationship between fibromyalgia diagnosis and
Heartburn 44.4 the count of somatic symptoms in 2613 fibromyalgia patients and 3525
Itching 44.3 ­patients with other noninflammatory disorders. (From the National Data
Bank for Rheumatic Diseases, 2006.)
Dizziness 42.1
Constipation 41.9
Pain/cramps in abdomen 41.5
Ringing in ears 41.4 Upper and lower back pain is the most common pain
Pain in upper abdomen 40.3 problem (>80%). Many patients, at the clinical inter-
Nervousness 39.7 view, emphasize only a few areas of pain. Questions
Nausea 37.7
specifically directed to other areas may elicit reports of pain
that were not stated spontaneously. Patients with fibromy-
Diarrhea 33.6
algia may complain of greater pain in an osteoarthritic joint
Shortness of breath 32.3 than patients without fibromyalgia. Although musculoskel-
Hearing difficulties 29.8 etal pain is central to fibromyalgia, patients may be more
Hair loss 23.6 concerned about fatigue or memory problems.
Oral ulcers 22.4 Fibromyalgia patients perform more poorly in formal
cognitive testing than age-matched controls.64 In the
Wheezing 21.4
National Data Bank for Rheumatic Diseases in 2006, 66%
Loss of appetite 21.1 of 2784 fibromyalgia patients complained of memory or
Raynaud’s phenomenon 20.1 thinking problems compared with 31% of 24,479 patients
Chest pain 19.2 with other rheumatic conditions. The most common symp-
Rash 17.1 toms, found in more than two thirds of patients, are sleep
Sun sensitivity 16.7
problems, fatigue, muscle pain, paresthesias, and cognitive
problems (see Table 38-2). In addition, the prevalence of
Loss/change in taste 14.4
other important symptoms is as follows: headache 65%,
Fever 13.4 depression 48%, and irritable bowel syndrome 46% (see
Hives/welts 9.3 Table 38-2). A high count of symptoms is characteristic of
Vomiting 9.1 fibromyalgia and is frequently a key to diagnosis (Fig. 38-4).
Seizures 1.7 Fibromyalgia also is associated with increased reporting of
comorbid conditions.65 The typical picture of fibromyalgia
emphasizes certain symptoms (pain, fatigue, sleep distur-
I­ ndividuals with this syndrome are unusually sensitive to bance, cognitive problems) and an abundance of symptoms
digital pressure (tender points) in certain body areas. Clini- and comorbidities.
cally, fibromyalgia is often identified or suspected by the Given the high levels of symptom variables and mem-
inexplicability and severity of symptoms and by their num- bership at the tail of the pain-distress continuum, it is
ber. The most common defining symptom is that of general- not surprising that evidence of psychosocial disruption is
ized pain (“pain all over”).6 The clinician may be surprised found. Considered as a whole, patients with fibromyalgia
by the extent and severity of symptoms (see Table 38-2 and have less education and household income than individu-
Fig. 38-2) and surprised at unexpected emotional distress als without fibromyalgia. They are less likely to be mar-
(see Fig. 38-2). Fibromyalgia has a quality of inexplicability ried, and they have greater rates of lifetime psychiatric
and unexpectedness. illness.66
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
Fibromyalgia occurs frequently in other rheumatic dis-
orders, including rheumatoid arthritis, osteoarthritis, and
systemic lupus erythematosus, in which the prevalence
of fibromyalgia exceeds 20%. The clues to identifying
fibromyalgia in the presence of other painful disorders
are location of pain (nonarticular), continued pain and
distress despite objective improvement, and unusual
fatigue.
ASSESSMENT OF A PATIENT
WITH FIBROMYALGIA
LABORATORY AND IMAGING TESTS
No laboratory or imaging tests are routinely helpful or
­indicated in the diagnosis or management of fibromyalgia.
SELF-REPORT MEASURES
Symptom severity, physical function, and work status are the
key status and outcome variables in fibromyalgia, as in other
rheumatic disorders. Assessments that can be useful routinely
to clinicians include measurements of pain, fatigue, physical
function, sleep quality, anxiety, depression, and work status.
As a minimum, assessments should include visual analog
scales (VAS) for pain and fatigue and a measure of func-
tional status. Function can be assessed by one of the family
of health assessment questionnaires, including the Health
Assessment Questionnaire (HAQ),67 the Health Assess-
ment Questionnaire–II (HAQ-II),68 and the Multi-Dimen-
sional Health Assessment Questionnaire (MDHAQ).69 The
HAQ is a 33-item questionnaire; the function scale of the
HAQ-II and MDHAQ is a 10-item questionnaire. Scales for
the assessment of anxiety, depression, and sleep disturbance
also can be added.70,71 For simplicity and ease of administra-
tion, however, we recommend VAS assessments of pain and
fatigue and either the HAQ-II or MDHAQ.
The Fibromyalgia Impact Questionnaire (FIQ) is a widely
used 20-item research assessment scale that addresses all of
the key fibromyalgia variables and can be used in clinical
care.72,73 The limitation of the FIQ is that it is suitable only
for use in fibromyalgia patients, whereas the above-men-
tioned health assessment questionnaires are useful and have
been used across the entire range of rheumatic disorders. In
addition, the FIQ total scale has no simple interpretation.
Functional questionnaire results have reduced validity
among fibromyalgia patients. Compared with patients with
rheumatoid arthritis and ankylosing spondylitis, there was
striking discordance between observed and questionnaire-
reported activities in patients with fibromyalgia.74 This
discordance limits slightly the usefulness of functional ques-
tionnaires and alters their interpretation: Results may repre-
sent perceived rather than actual functional difficulties.
Research Questionnaires
Almost any generic research scale is suitable for use in fibro-
myalgia, including commonly used scales such as the Medical
Outcomes Scale Short-Form 36 (SF-36).75,76 Two fibromyal-
gia-related scales may be of special interest. The Regional
Pain Scale is a questionnaire assessment of the number of
painful nonarticular body regions.77 The Symptom Intensity
561
Scale combines the Regional Pain Scale and a VAS fatigue
scale to quantitate fibromyalgia symptom intensity.2
PHYSICAL MEASURES
With the exception of the performance of the tender point
examination, the physical examination of a patient suspected
to have fibromyalgia does not differ from the examination of
any other rheumatic disease patient or pain patient. Measure-
ment of pain threshold by the tender point examination is the
only routinely useful physical measurement. Although helpful
for diagnosis (see Table 38-1 and Fig. 38-1), the tender point
count is poorly correlated with other fibromyalgia symptoms
and with change in symptom severity among fibromyalgia
patients.78 Patients may improve or worsen substantially with-
out important differences in the tender point count.
How to Perform the Tender Point Examination
Fibromyalgia patients have a lower threshold for pain than
do subjects without fibromyalgia.79 In the clinic, two meth-
ods exist by which tenderness can be elicited and mea-
sured21—digital palpation and dolorimetry.80 Tender point
sites represent specific areas of muscle, tendon, and fat pads
that are much more tender to palpation than surrounding
sites. Sites selected as part of ACR criteria6 represent tender
point sites that best discriminate between patients with and
without fibromyalgia. To test for pain with digital palpation,
the ACR criteria indicate that the examiner should press the
tender point site with an approximate force of 4 kg. Usually
the second and third fingers or the thumb is used for palpa-
tion, and a rolling motion is helpful in eliciting tenderness.
The amount of force that the examiner uses is important
because a large force would elicit pain in a subject with-
out fibromyalgia, whereas a small force may miss tenderness.
Because the 4-kg force requirement is a theoretic goal, and
difficult to implement, clinicians need other guidelines for
determining the amount of pressure to use. A good method
for determining this is to palpate “normal” individuals of
the same build and stature. The amount of force that does
not elicit tenderness in an individual without fibromyalgia
(just below the pressure pain threshold) is the correct force
to use. In practice, less force is required in smaller, thinner,
less muscled individuals. More force is needed in heavier
and more muscular individuals.
The pressure used by the examiner and the examiner’s
interpretation of the patient’s response can influence results
of palpation. The best and most appropriate way to perform
the tender point count is to ask the patient if the palpation
is painful,6 accepting only a “yes” as a positive reply, regard-
less of facial expression or body movement. Specifically, the
frequently heard comment of patients to the digital exam-
iner’s question regarding pain, “It’s tender,” is a negative
rather than a positive response, and best should be followed
by another question, such as, “Yes, but is it painful?”
Limitations to the Tender Point Examination
Although the tender point examination can provide clinically
useful information when properly performed, it can be influ-
enced by external factors. Physicians who believe the patient
does or does not have fibromyalgia can influence the results by
 562 wolfe  |  Fibromyalgia
the amount of pressure applied. The meaning and use of the
examination are widely known among physicians, patients,
and patient support groups; in some circumstances where a
positive or negative examination would seem to be desir-
able (e.g., in a disability or medicolegal examination), results
might differ from those obtained during a routine examina-
tion. In addition, the tender point examination is inherently
inaccurate around the “diagnostic” tender point count of 11.
DIAGNOSIS
The approach to fibromyalgia diagnosis should differ accord-
ing to the setting and the physician’s underlying beliefs
about fibromyalgia acceptability. Because of the limitations
of the ACR criteria with respect to sensitivity and accuracy,
clinical criteria may be the best method for diagnosis in the
clinical setting because they consider all aspects of fibromy-
algia, rather than just the number of tender points.21 Clinical
criteria require 2 of 3 tender points of widespread pain, � 11
tender points, and characteristic fibromyalgia symptoms. The
ACR criteria are a reasonable but not required alternative in
the clinic.6 A diagnosis of chronic pain syndrome or the rec-
ognition of the presence of a chronic pain syndrome in the
context of another illness also is sufficient as a diagnosis.
Clinical trials require use of the ACR criteria.6 It is usu-
ally the case that patients with fibromyalgia have to satisfy
the ACR criteria at the time of entry into the study whether
or not they satisfied the criteria previously. In that respect,
the ACR criteria represent not only a diagnostic method,
but also a measure of severity because of the requirement
for current high levels of tender points. You cannot improve
and still have fibromyalgia according to the ACR criteria.
The ACR criteria also present a problem for epidemio-
logic research because they require physical examination of
any individual satisfying the widespread pain criterion. Such
studies are complex and expensive to perform. In addition,
they have the potential of bias that many “healthy” people
would not voluntarily undergo a physical examination. The
extent or importance of this bias is unknown. ACR crite-
ria cannot be used in studies where physical examination
is impossible, such as survey-based observational studies.
Survey fibromyalgia criteria have been described and seem
to work as well as ACR criteria.81 These criteria require
high levels of fatigue and widespread pain, using a continu-
ous scale for each measure. Survey criteria have an added
advantage in the research setting, in that a clinical diagnosis
is not required for their use; patients can be studied without
labeling the subjects with a diagnosis.
Diagnostic criteria also cause problems for physicians
who recognize fibromyalgia symptoms, but disagree with the
legitimacy or consequences of a diagnosis of fibromyalgia.
Because there are no data to suggest that “correctly” diag-
nosing fibromyalgia improves treatment or outcome, formal
diagnosis of fibromyalgia is unnecessary as long as fibromyal-
gia symptoms are recognized.
PRIMARY, SECONDARY, AND SECONDARY-
CONCOMITANT FIBROMYALGIA
Fibromyalgia is sometimes divided into primary, secondary,
and secondary-concomitant fibromyalgia. The term primary
fibromyalgia is most often used when there is not another
condition present whose symptoms could explain fibromyal-
gia symptoms. This division between primary and secondary
fibromyalgia is artificial, however. Back pain in older indi-
viduals when age-related radiographic changes are present
might be considered secondary fibromyalgia, whereas the
same symptoms in younger individuals might be consid-
ered primary fibromyalgia. The ACR criteria study showed
no difference between primary and secondary fibromyalgia
with regard to symptoms and diagnosis.6 The usefulness of
primary fibromyalgia occurs in clinical trials, in which it is
desirable to be sure that symptoms are not coming from
another well-established illness. A fibromyalgia diagnosis
implies understanding of issues such as pain, fatigue, sleep,
and cognitive and emotional problems. When fibromyalgia
is considered only in patients without other musculoskel-
etal conditions, the “benefit” of fibromyalgia diagnosis—its
consideration of such issues—is lost. If fibromyalgia is to
be diagnosed or considered, such consideration should be
applied to all patients.
DIFFERENTIAL DIAGNOSIS
Fibromyalgia is more frequently present in individuals with
identifiable medical disorders than in individuals without
such disorders. It is virtually never the case that there is a dif-
ferential diagnosis problem between fibromyalgia and another
medical disorder. When diagnosis is problematic, it is because
the other medical condition is difficult to diagnose or has not
been evaluated properly. It is not a case of fibromyalgia or
lupus, but rather fibromyalgia and lupus. Disorders sometimes
thought to be difficult to separate from fibromyalgia include
early rheumatoid arthritis, systemic lupus erythematosus,
polymyositis, hypermobility syndromes, and endocrine dis-
orders. In each of these cases, the clue to understanding the
patient’s illness is thoroughly evaluating the patient.
MANAGEMENT OF
FIBROMYALGIA—RESEARCH
STUDIES AND RECOMMENDATIONS
Most long-term observational studies do not show improve-
ment in fibromyalgia symptoms and outcomes, even when
patients are followed in centers with special interest and
knowledge of fibromyalgia.82,83 Service utilization (a mea-
sure of symptom activity) does not lessen after diagnosis.84
Benefit of treatment is generally not sustained in long-term
randomized clinical trials.85 These data should be kept in
mind when evaluating the results of clinical trials. The null
hypothesis for a chronic, painful disorder should not be no
short-term treatment effect, but instead no long-term treat-
ment effect. Short-term studies should be regarded with sus-
picion, and most fibromyalgia studies are short-term.
Compliance with treatment prescription is an important
problem in fibromyalgia, and in fibromyalgia clinical tri-
als, the dropout rate is high. Even when intention-to-treat
analyses are performed, the effectiveness of treatment is
overestimated. Patients who follow exercise recommenda-
tions have better outcomes than patients who do not; how-
ever, most patients in clinical practice do not or will not
perform aerobic exercises. It is fair to conclude that ­exercise
prescription is often an ineffective ­recommendation, rather
than concluding that it is an effective treatment.
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
Treatments without a true, contemporaneous control
group cannot provide meaningful estimates of efficacy
because they often exaggerate efficacy. In evaluating study
results, the degree of improvement must be examined, and
the degree of improvement must be clinically meaning-
ful. Even when improvement is clinically meaningful, the
baseline and final outcome values, such as values of pain
and fatigue, must be considered. If the patients are selected
for trials in relative (temporary) flare conditions, they may
improve, but still have very high levels of the outcome vari-
ables at the conclusion of the trial.
Numerous useful reviews of treatment in fibromyalgia
are available.86,87 Most such reviews rely on the concept of
efficacy and rank evidence as a function of study quality.
One review indicates that “Evidence for treatment efficacy
was ranked as strong (positive results from a meta-analysis
or consistently positive results from more than 1 randomized
controlled trial [RCT]), moderate (positive results from 1
RCT or largely positive results from multiple RCTs or con-
sistently positive results from multiple non-RCT studies),
and weak (positive results from descriptive and case stud-
ies, inconsistent results from RCTs, or both).”86 As noted by
these authors, studies are needed “… to determine whether
the improvement is maintained over months or years.” To
which we would add, not only maintenance of improve-
ment, but also maintenance of meaningful improvement
and nondiscontinuation of treatment.
Still another problem with the interpretation of fibro-
myalgia studies relates to study scales. Because patients
diagnosed as having fibromyalgia have problems with pain,
fatigue, cognition, and anxiety and depression, to name some
issues in fibromyalgia, different studies may select different
scales and outcomes according to the interests of the inves-
tigators. This leads to problems in comparing study results.
In addition, when multiple outcomes and study instruments
are selected, frequently studies can show positive results for
one outcome and negative results for another. Even when
an outcome such as pain is being measured, if there is more
than one pain scale, positive results may be found with one
pain scale and not with another. Complex scales also are
difficult to interpret, as is the case with the commonly used
FIQ total scale. This composite summary scale has no simple
interpretation: A reader may note an improvement, but not
have a clear idea of what such improvement means.
From 6750 fibromyalgia patients screened in the National
Data Bank for rheumatic diseases, the mean (standard devi-
ation) VAS pain and fatigue scores were 6.3 (2.5) and 7.0
(2.5). As an aid in interpreting effect sizes, the following data
are presented; assuming a baseline score of 7.0, the following
are the effect size, change score, post-treatment score, and
percent improvement: 0.3, 0.75, 6.25, 10.7%; 0.4, 1.25, 6.0,
14.3%; 0.5, 1.25, 5.75, 17.9%; 0.6, 1.5, 5.5, 21.4%.
DIAGNOSIS
Diagnosis may be an important aspect of treatment. Diag-
nosing fibromyalgia in individuals with short-term stress-
related illnesses is harmful and leads to prolonged illness
and medicalization. There is no valid evidence to support
the assertion that diagnosis of fibromyalgia in patients with
long-term symptoms has a salutary effect. A study of pri-
mary care patients in the United Kingdom reported that
563
“… patients who had been diagnosed as having [fibromyal-
gia] reported higher rates of illness and health care resource
use for at least 10 years prior to their diagnosis, which sug-
gests that illness behavior may play a role …. Diagnosis has
a limited impact on health care resource use in the longer
term, possibly because there is little effective treatment.”84
There is no evidence at the patient level that diagnosis is
harmful. Using the diagnostic term in the presence of severe
symptoms often makes it easier for physicians and patients
to discuss the condition; when fibromyalgia is not diagnosed,
patients sometimes ask directly, “Do I have fibromyalgia?” In
considering making the diagnosis of fibromyalgia, the physi-
cian should consider the following comment by Barsky and
Borus:9 “The hyperbole, litigation, compensation, and self-
interested advocacy surrounding the functional somatic syn-
dromes can exacerbate and perpetuate symptoms, heighten
fears and concerns, prolong disability, and reinforce the
sick role. Excessive medical testing and treatment expose
patients to iatrogenic harm and amplify symptoms.” If fibro-
myalgia is “diagnosed,” however, it is important to be clear
to the patient that fibromyalgia is a name given to the symp-
toms, not a cause of the symptoms.
EDUCATION
Education in some reports may have a modest effect on fibro-
myalgia symptoms, such as fatigue, anxiety, and depression,
but has limited to no effect on pain.88,89 What is called edu-
cation is actually composed of two components—­education
and rapport or engagement—and it is impossible to dis-
tinguish the two components. Most education studies are
derived from formal university-based treatment programs;
only one study was applicable to clinical practice,88 and the
sample size was too small to evaluate the effect of the inter-
vention in fibromyalgia. All studies had deficiencies in the
validity of the control groups; there are no long-term data
on the effect of education. Although it is sensible that edu-
cation should always be part of any treatment program, and
is part of establishing rapport, its content should depend on
the patient, the duration of illness, and the diagnostic label
already present. The goal of education is to help the patient
understand and manage his or her symptoms optimally,
reduce dependence on the medical system, and work effec-
tively within that system when necessary. There are no data,
however, as to whether, within the clinical setting, exten-
sive education is more or less effective than limited educa-
tion. In a group of 100 consecutive enrollees in a 1.5-day
multidisciplinary group outpatient fibromyalgia treatment
program, after 30 days a 12.8% improvement was noted in
the 78 who completed the study.90
EXERCISE
Aerobic exercise increases cardiovascular fitness and reduces
pain and other fibromyalgia symptoms. In a short-term
RCT, exercise improved aerobic performance by 16% and
pain by 13%.91 A carefully done, well-powered RCT of a
12-week community-based exercise program compared with
relaxation controls showed a 4% difference in FIQ scores
at 1 year, but nonsignificant changes in McGill pain scores
and SF-36 scores.92 At the 12-month follow-up, 38% of sub-
jects in the exercise arm and 22% in the control arm rated
 564 wolfe  |  Fibromyalgia
t­ hemselves much better or very much better. Only 53% of
patients attended more than half of the intervention sessions.
A follow-up report at 12 months on patients who participated
in a 23-week, three-times-per-week exercise program indi-
cated general improvement compared with baseline values.93
The degree of improvement as measured by the FIQ was 5%.
A Cochrane collaboration meta-analysis of “four high
quality aerobic training studies” reported significantly greater
improvements in the exercise groups versus control groups in
aerobic performance (17.1% increase in aerobic performance
with exercise versus 0.5% increase in the control groups),
tender point pain pressure threshold (28.1% increase versus
7% decrease), and improvements in pain (11.4% decrease in
pain versus 1.6% increase).91 A more recent noncontrolled
study comparing water-based exercise with land-based exer-
cise showed an average 36% reduction in pain.94 Exclusions
in this study included 67 for work schedule incompatibility
and 32 for nonspecified refusals; 60 patients were randomly
assigned, and 52 completed the study.
Practically, the problem with exercise prescription is that
it is difficult to get fibromyalgia patients to participate. Exer-
cise may produce “short-term increases in pain and fatigue
that should abate within the first few weeks of exercising,”95
but this may be unacceptable to patients in ordinary clinical
settings. Even in formal programs, adherence to exercise is
poor.96,97 In a 4.5-year follow-up of a randomized trial of exer-
cise, only 20% of patients maintained an adequate physical
activity level.98 In the National Data Bank for rheumatic
diseases in 2006, 24% of 2784 fibromyalgia patients reported
performing some aerobic exercise weekly, but only 9% per-
formed at levels substantial enough to result in increasing or
maintaining aerobic fitness.
COGNITIVE BEHAVIORAL THERAPY
Cognitive behavioral therapy is a form of short-term, goal-
oriented psychotherapy. It has been the subject of some
positive reports,99-102 some less positive reports,85,103 and some
completely negative studies.104,105
PHARMACOTHERAPY
Analgesics and Nonsteroidal
Anti-inflammatory Drugs
Many drugs frequently used by patients diagnosed as having
fibromyalgia have not been formally evaluated for efficacy
or effectiveness.86 With respect to analgesics and nonsteroi-
dal anti-inflammatory drugs (NSAIDs), a 1998 multicenter
study of 538 fibromyalgia patients noted the following usage
in a 6-month period: aspirin 20.6%, NSAIDs 55.9%, acet-
aminophen 27.6%, opioid analgesics 6.4%, and nonopioid
analgesics 21.5%.106 Tramadol use was 15%. These data are
important because it is often suggested that NSAIDs are
ineffective.86
A few analgesic and NSAID treatments have been for­­­
mally evaluated. Naproxen, 500 mg twice daily (n = approxi­­
mately 15), which is the only NSAID that has been studied,
was indistinguishable from placebo (n = approximately 15) in
a controlled clinical trial of relatively young subjects (age 48
years).107 The combination of tramadol and acetaminophen
reduced pain 18.5% more than did the use of placebo.108 In
this trial, 48% in the active treatment group and 62% of
placebo users were noncompleters in this 3-month trial.
Psychotropic Agents
Many drugs that have antidepressant and other psychotro-
pic attributes have been used in fibromyalgia treatment.
Such drugs reduce pain centrally, even in the absence of
depression, and often are employed at doses that are insuffi-
cient to treat depression. Among drugs that have shown effi-
cacy, effect sizes are in the range of 0.35 to 0.55, with pain
improvement of 11% to 18% noted in short-term trials.
A careful meta-analysis of the effect of amitriptyline and
a review of other antidepressants was reported by Arnold and
colleagues.109 The meta-analysis showed that amitriptyline had
an overall effect size for all studied outcomes of approximately
0.44 and effect sizes for pain of 0.57 and for fatigue of 0.521.
Of the nine amitriptyline trials studied, one lasted 26 weeks,
and others lasted 3 to 12 weeks. A series of N-of-1 studies sug-
gested that one third of fibromyalgia patients are benefited110;
Arnold and colleagues109 indicated that significant clinical
response to tricyclic agents was observed in 25% to 37% of
patients with fibromyalgia, and that the overall degree of effi-
cacy was modest in most studies. Similar, although slightly
weaker, results are noted with cyclobenzaprine.
Among newer agents, milnacipran showed efficacy in a
3-month trial.111 Duloxetine, a serotonin and norepineph-
rine reuptake inhibitor, generally improved symptoms and
pain threshold in a 12-week RCT. Improvements included
a 10% change in total FIQ score, improvement in the Brief
Pain Inventory, but nonsignificant changes in FIQ pain,
fatigue, and morning tiredness. Forty percent of patients did
not complete the study. There is some evidence for efficacy
of fluoxetine, sertraline, and venlafaxine.86
Compared with clinical trial results, results in longitu-
dinal studies and clinical practice show marginal effective-
ness of tricyclic antidepressants and similar treatments.
A high-quality RCT found no difference in the response to
amitriptyline and cyclobenzaprine.
Other Pharmacologic Treatments
An 8-week trial of pregabalin resulted in a 13% reduction
in pain compared with placebo.112 A 50% improvement
was noted by 29% of the pregabalin group compared with
13% of the placebo-treated patients. The most common
adverse effects were dizziness (49.9%) and somnolence
(28%). Pregabalin has been approved by the U.S. Food
and Drug Administration. The recommended dose is 300
to 450 mg/day. Dosing should begin at 150 mg two times
per day, but may be increased after 1 week, as required.
Based on the clinical trial criterion for efficacy, there is no
evidence for efficacy of NSAIDs, corticosteroids, benzodiaze-
pine and nonbenzodiazepene hypnotics, guaifenesin, melatonin,
calcitonin, opioids, thyroid hormone, dehydroepiandrosterone
and magnesium, or anti–tumor necrosis factor therapy.86
NONPHARMACOLOGIC TREATMENTS
There is some evidence for efficacy of numerous nonmain-
stream treatments, including strength training91,113 and hyp-
nosis.114 There is weak evidence for chiropractic, manual,
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
and massage therapy, and no evidence of efficacy for tender
or trigger point injections or flexibility exercise. Evidence
for acupuncture is contradictory,115,116 as is evidence for the
efficacy of biofeedback117-119 and balneotherapy.120-122
PRACTICAL RECOMMENDATIONS IN THE
APPROACH TO A PATIENT WITH FIBROMYALGIA
The goal of fibromyalgia treatment is to improve the physi-
cal and mental health of patients and their quality of life.
This goal implies helping patients manage distressing symp-
toms, but with decreased dependence on the medical care
system. There are no studies as to how often the simple
recommendations of education, exercise, and limited phar-
macologic treatment provide results at an acceptable level
of symptoms and functional ability. Data from the National
Data Bank for Rheumatic Diseases show, however, that 62%
of 2772 fibromyalgia patients were somewhat or very dis-
satisfied with their health compared with 34% of 20,909
patients with rheumatoid arthritis. These data indicate that
contemporary treatment of fibromyalgia is generally unsat-
isfactory.
This high level of dissatisfaction is reflected in physician
and patient interactions. An unknown but probably small
proportion of rheumatology experts refuse to accept refer-
ral of fibromyalgia patients. A larger proportion is unhappy
seeing such patients or is uncomfortable providing care.
Patients, sensing this attitude, are equally unhappy with
physicians: Patient support groups provide specific advice on
finding positive, sympathetic physicians, including identify-
ing them by name. Physician behavior results from a general
uncomfortableness with illnesses that are often unrespon-
sive to treatment and have strong psychological and psy-
chosocial components. There is no simple resolution to this
problem. Physicians who are unable to provide helpful care
to patients with fibromyalgia should make that known to
the patients.
In considering fibromyalgia treatment, physicians should
determine what resources are available in the commu-
nity, and whether the resources are effective and helpful.
The educational, exercise, and cognitive behavioral ther-
apy programs described in the research studies earlier are
almost never available to community physicians. Avail-
able programs may or may not be competent, appropriate,
or helpful. Pain management programs sometimes mean
little more than spinal blocks and “trigger point” injections,
and physical therapy referral often results in treatments
that are ineffective for fibromyalgia. The referring physi-
cians should investigate the quality and outcomes of referral
resources.
Although the common recommendations of education,
exercise, and pharmacotherapy are often appropriate, par-
ticularly in newly diagnosed cases, patients with established
fibromyalgia have often experienced these recommenda-
tions and treatments. Whether such treatments have strong
evidence for effectiveness or not, as measured by clinical
trials, they are often not clinically effective enough, and
patients return to the physician for additional suggestions
and care. In circumstances such as these, Goldenberg and
associates86 recommend, “Trials with selective serotonin
reuptake inhibitor[s], serotonin and norepinephrine reup-
take inhibitor[s], or tramadol …. Consider combination
565
medication trial[s] or anticonvulsant[s].” Local injections in
muscular areas of pain also are commonly employed by rheu-
matologists. The authors surveyed rheumatologists regarding
the use of injections and found them to be used frequently,
in agreement with others.86 Rheumatologists reported that
patients “like injections,” but also that the rheumatologists
did not know what else to do.
The question arises as to how to approach a resistant
patient with fibromyalgia, given the knowledge that after
failure with several standard treatments, success with other
medications is unlikely. Should the physician simply go from
one (dubious) treatment to another? Should the physician
use treatments of dubious or uncertain value? The adverse
effects of inappropriate or unnecessary treatments are not
inconsequential and include dependence, medicalization
of common symptoms, overuse of medical care, increased
costs, and side effects.
The physician must be friendly and interested—a resource
the patient can rely on. Testing should be limited and reserved
for times when it is truly necessary to investigate comorbid
conditions. Comorbid conditions, such as arthritis and obe-
sity, should be treated because they can contribute to increas-
ing physical and mental symptoms. The worst problem should
be identified. Sometimes identifying where the pain problem
began can offer clues to appropriate treatment of the coex-
isting condition. If many fibromyalgia treatments have been
tried and have been unsuccessful, it is generally not a good
idea to try even more similar, and soon to be unsuccessful,
therapies. We often ask patients, “Which treatment has been
most helpful,” and suggest (assuming treatment is needed and
helped at all) that they return to that treatment.
There is no blanket rule on the use of opioids. Experience
has shown that they often do not truly help and very often
cause problems. Opioids are generally not recommended.
There are exceptions to this recommendation, however,
and physicians should exercise clinical judgment and use
opioids when they think such therapy is necessary, provided
that appropriate guidelines are followed.123 “Tender points”
never need injection therapy. Painful areas in muscle may
respond to local injections of local anesthetics; corticoste-
roids are never indicated. If injections relieve pain for more
than short periods of time, they may represent a reasonable
therapy. In illnesses with strong psychosocial components,
medically ineffective therapies can result in overall benefit
to patients. The circumstances where dubious therapies
might be used are limited; and the physician should under-
stand clearly why he or she is administering such therapies,
and what results are anticipated.
Physical therapy is not recommended because the
aerobic exercise required in fibromyalgia does not usually
require formal physical therapy and increases medicaliza-
tion. Because medical therapy is unsatisfactory, patients find
their way to alternative therapies. Some of these therapies
may be helpful to individual patients, such as massage, water
therapy, spa treatment, and acupuncture. These therapies
tend to have high cost-effectiveness ratios, and the decision
to use such therapies is often best left to the patients and the
reimbursement authority. That is not say that such treat-
ments do not help—everything helps—but they do not help
often enough and importantly enough, and some decision
point is required. One important goal of therapy is to reduce
medicalization and increase independence.
 566 wolfe  |  Fibromyalgia
A frustrated physician may not know where to turn next
in a nonresponsive patient. Should the patient be referred
to a pain clinic? Sometimes such a referral is inevitable.
The quality of pain clinics varies, however, and the results
in fibromyalgia are often not good. The decision to refer
should depend on the experience with the available clinics
and the results that they have produced. In some countries,
reimbursement authorities limit referrals, providing a cost-
­effectiveness analysis that may be alien to the physician-
patient relationship.
Treatment options sort themselves out over time. Deci-
sions that are difficult resolve. In the end, the physician who
provides support and interest is a strong resource and a guide
for patients with fibromyalgia, even when medical therapies
are limited.
MEDICOLEGAL ISSUES
AND FIBROMYALGIA
Frequently, fibromyalgia becomes a medicolegal issue when
an individual with fibromyalgia asserts that he or she is
unable to work because of fibromyalgia. Because fibromyal-
gia symptoms are felt only by the patient, there are no objec-
tive medical findings to help in the disability assessment.
Gaining a disability award is complex, depending on the
source of payment (e.g., government versus private insur-
ance), the physician’s belief and documentation, the avail-
ability of legal services, and the impact of the illness on the
patient. Various guidelines have been suggested for evaluat-
ing disability as they apply to fibromyalgia. Determination
of disability does not depend on proving the existence of
fibromyalgia.
The second medicolegal issue arises when an individual
claims that trauma caused him or her to develop or exac-
erbate fibromyalgia, and that the fibromyalgia is disabling.
Although it is proposed that trauma can alter the central
nervous system (“neural plasticity”) and cause fibromyalgia,
the relationship between the severity of trauma and the
report of fibromyalgia is very weak. There is no way to deter-
mine scientifically if trauma causes or caused fibromyalgia.
In addition, it is often difficult to establish the severity of the
fibromyalgia symptoms. In reality, the relationship between
trauma and disability does not require a diagnosis of fibro-
myalgia because symptom severity and work impairment are
important, not the presence of absence of fibromyalgia.
OUTCOME OF FIBROMYALGIA
The outcome of fibromyalgia can be studied in the context
of change and level of symptoms, use of services, and work
disability. Interpretation of results is complex, however.
Although some studies show improvement over time, these
results might simply represent a shifting within the distri-
bution of fibromyalgia symptoms, allied to random change,
which in part can be thought of as representing regression
to the mean. To illustrate this point, we examined 20,248
patients in the National Data Bank for Rheumatic Diseases
on two consecutive occasions 6 months apart. At the start,
19.9% of patients satisfied survey fibromyalgia criteria. At
follow-up, only 61.4% of patients who met criteria now sat-
isfied the criteria. In addition, their pain scores decreased
from 6.8 to 6.3. Some patients who did not meet the ­criteria
at the first observation now did meet criteria, however,
with the result that the 19.9% fibromyalgia prevalence was
reduced only to 19.5%, and the overall pain scale changed
from 4.1 to 4.0. Because criteria reflect symptom change
rather than “disease,” improvement does not really mea-
sure improvement unless worsening or the development of
new cases is considered. When applied to tender points, the
inherent inaccuracy of the measure always leads to shifting
of the tender point count in individuals with 10 to 13 tender
points.
Many studies have addressed the issue of outcome.
Some have suggested that “… knowledge of the poten-
tial reversibility of the syndrome [is] resulting in improved
outcomes”124 and that “… outcome is good with minimal
intervention.”125 In a prospective study of fibromyalgia of
patients referred to a specialty clinic, 70 of 82 were reas-
sessed after 3 years. The returnees were generally improved
(pain reduced from 6.8 to 5.4 and fatigue reduced from 6.8 to
5.7). The authors concluded that, “The overall outcome …
was favorable.”126 In 33 of 51 patients seen 6 to 8 years after
initial participation in a fibromyalgia treatment study, pain
was reduced from 6.7 to 5.3, and fatigue was reduced from
7.5 to 6.5. The authors concluded that the results of these
returnees “… [suggest] a benign long-term outcome in these
patients with [fibromyalgia].”127 A six-center, 7-year study
of 538 patients noted that, “Although functional disability
worsened slightly and health satisfaction improved slightly,
measures of pain, global severity, fatigue, sleep disturbance,
anxiety, depression, and health status were markedly abnor-
mal at study initiation and were essentially unchanged over
the study period. Half the patients are dissatisfied with their
health, and 59% rate their health as fair or poor.”83 In one
report of 45 of 70 patients who had participated in a 3-week
trial 6 years earlier, symptoms of fibromyalgia persisted over
6 years.82 A study of prediagnosis and postdiagnosis use of
services found that no changes in the high use rates were
seen over time.84
Based on available data, it seems that among patients
who return for follow-up, improvement of 15% to 20%
may occur, although patients continue to have substan-
tial symptom severity. Patients who did not return may be
worse, however. Use of services remains high, and in a large
multicenter study no change in symptom status occurred
over 7 years.83 Finally, improvement may partly represent an
artifact in diagnosis classification methodology, such that it
would be almost impossible not to improve.
REFERENCES
  1. Wolfe F: The relation between tender points and fibromyalgia symp-
tom variables: Evidence that fibromyalgia is not a discrete disorder in
the clinic. Ann Rheum Dis 56:268-271, 1997.
  2. Wolfe F, Rasker JJ: The symptom intensity (SI) scale, fibromyal-
gia, and the meaning of fibromyalgia-like symptoms. J Rheumatol
2006; 33:2291-2299.
  3. Macfarlane GJ: Generalized pain, fibromyalgia and regional pain:
An epidemiological view. Baillieres Best Pract Res Clin Rheumatol
13:403-414, 1999.
  4. Makela MO: Is fibromyalgia a distinct clinical entity? The epidemi-
ologist’s evidence. Baillieres Best Pract Res Clin Rheumatol 13:415-
419, 1999.
  5. Wessely S, Hotopf M: Is fibromyalgia a distinct clinical entity?
Historical and epidemiological evidence. Baillieres Best Pract Res
Clin Rheumatol 13:427-436, 1999.
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
  6. Wolfe F, Smythe HA, Yunus MB, et al: The American College
of Rheumatology 1990 Criteria for the Classification of Fibro-
myalgia: Report of the Multicenter Criteria Committee. Arthritis
Rheum 33:160-172, 1990.
  7. Box GEP, Draper NR: Empirical Model-Building and Response Sur-
faces. New York, Wiley, 1987.
  8. White KP: Fibromyalgia: The answer is blowin’ in the wind. J Rheu-
matol 31:636-639, 2004.
  9. Barsky AJ, Borus JF: Functional somatic syndromes. Ann Intern Med
130:910-921, 1999.
10. Smythe HA, Moldofsky H: Two contributions to understanding of
the “fibrositis” syndrome. Bull Rheum Dis 28:928-931, 1977.
11. Ehrlich GE: Pain is real; fibromyalgia isn’t. J Rheumatol 30:1666-
1667, 2003.
12. Hadler NM, Greenhalgh S: Labeling woefulness: The social con-
struction of fibromyalgia. Spine 30:1-4, 2005.
13. Hadler NM: “Fibromyalgia” and the medicalization of misery. J Rheu-
matol 30:1668-1670, 2003.
14. Hazemeijer I, Rasker JJ: Fibromyalgia and the therapeutic domain: A
philosophical study on the origins of fibromyalgia in a specific social
setting. Rheumatology (Oxf) 42:507-515, 2003.
15. Block SR: Fibromyalgia and the rheumatisms—common sense and
sensibility. Rheum Dis Clin North Am 19:61-78, 1993.
16. Quintner JL, Cohen ML: Fibromyalgia falls foul of a fallacy. Lancet
353:1092-1094, 1999.
17. Cohen ML, Quintner JL: Fibromyalgia syndrome, a problem of tau-
tology. Lancet 342:906-909, 1993.
18. Hudson JI, Mangweth B, Pope HG Jr, et al: Family study of affective
spectrum disorder. Arch Gen Psychiatry 60:170-177, 2003.
19. Hudson JI, Hudson MS, Pliner LF, et al: Fibromyalgia and major
affective disorder: A controlled phenomenology and family history
study. Am J Psychiatry 142:441-446, 1985.
20. Crofford LJ, Clauw DJ: Fibromyalgia: Where are we a decade after
the American College of Rheumatology classification criteria were
developed? Arthritis Rheum 46:1136-1138, 2002.
21. Wolfe F: When to diagnose fibromyalgia. Rheum Dis Clin North Am
20:485-501, 1994.
22. Wolfe F, Ross K, Anderson J, et al: The prevalence and character-
istics of fibromyalgia in the general population. Arthritis Rheum
38:19-28, 1995.
23. Raphael KG, Janal MN, Nayak S, et al: Psychiatric comorbidities
in a community sample of women with fibromyalgia. Pain 124(1-2):
117-125, 2006.
24. White KP, Speechley M, Harth M, et al: The London Fibromyalgia
Epidemiology Study: The prevalence of fibromyalgia syndrome in
London, Ontario. J Rheumatol 26:1570-1576, 1999.
25. Haq SA, Darmawan J, Islam MN, et al: Prevalence of rheumatic
diseases and associated outcomes in rural and urban communities in
Bangladesh: A COPCORD study. J Rheumatol 32:348-353, 2005.
26. Farooqi A, Gibson T: Prevalence of the major rheumatic disorders in
the adult population of north Pakistan. Br J Rheumatol 37:491-495,
1998.
27. Salaffi F, De AR, Grassi W: Prevalence of musculoskeletal condi-
tions in an Italian population sample: Results of a regional commu-
nity-based study, I: The MAPPING study. Clin Exp Rheumatol 23:
819-828, 2005.
28. Topbas M, Cakirbay H, Gulec H, et al: The prevalence of fibromyal-
gia in women aged 20-64 in Turkey. Scand J Rheumatol 34:140-144,
2005.
29. Senna ER, De Barros AL, Silva EO, et al: Prevalence of rheumatic
diseases in Brazil: A study using the COPCORD approach. J Rheu-
matol 31:594-597, 2004.
30. Lindell L, Bergman S, Petersson IF, et al: Prevalence of fibromyal-
gia and chronic widespread pain. Scand J Primary Health Care 18:
149-153, 2000.
31. Clark P, Burgos Vargas R, Medina Palma C, et al: Prevalence of fibro-
myalgia in children: A clinical study of Mexican children. J Rheuma-
tol 25:2009-2014, 1998.
32. Mikkelsson M: One year outcome of preadolescents with fibromyal-
gia. J Rheumatol 26:674-682, 1999.
33. Buskila D, Press J, Gedalia A, et al: Assessment of nonarticular ten-
derness and prevalence of fibromyalgia in children. J Rheumatol 20:
368-370, 1993.
34. Buskila D, Neumann L, Hershman E, et al: Fibromyalgia syndrome in
children—an outcome study. J Rheumatol 22:525-528, 1995.
567
35. Campbell SM, Clark S, Tindall EA, et al: Clinical characteristics of
fibrositis, I: A “blinded,” controlled study of symptoms and tender
points. Arthritis Rheum 26:817-824, 1983.
36. Hartz A, Kirchdoerfer E: Undetected fibrositis in primary care prac-
tice. J Fam Pract 25:365-369, 1987.
37. Wolfe F, Cathey MA: Prevalence of primary and secondary fibrositis.
J Rheumatol 10:965-968, 1983.
38. Yunus MB, Masi AT, Calabro JJ, et al: Primary fibromyalgia (fibro-
sitis): Clinical study of 50 patients with matched normal controls.
Semin Arthritis Rheum 11:151-171, 1981.
39. Rowbotham MC: Is fibromyalgia a neuropathic pain syndrome?
J Rheumatol Suppl 75:38-40, 2005.
40. Dworkin RH, Fields HL: Fibromyalgia from the perspective of neuro-
pathic pain. J Rheumatol Suppl 75:1-5, 2005.
41. Levine JD, Reichling DB: Fibromyalgia: The nerve of that disease.
J Rheumatol Suppl 75:29-37, 2005.
42. Price DD, Staud R: Neurobiology of fibromyalgia syndrome. J Rheu-
matol Suppl 75:22-28, 2005.
43. Crofford LJ: The relationship of fibromyalgia to neuropathic pain
syndromes. J Rheumatol Suppl 75:41-45, 2005.
44. Mease P: Fibromyalgia syndrome: Review of clinical presentation,
pathogenesis, outcome measures, and treatment. J Rheumatol Suppl
75:6-21, 2005.
45. Arnold LM, Hudson JI, Hess EV, et al: Family study of fibromyalgia.
Arthritis Rheum 50:944-952, 2004.
46. Raphael KG, Janal MN, Nayak S, et al: Familial aggregation of
depression in fibromyalgia: A community-based test of alternate
hypotheses. Pain 110(1-2):449-460, 2004.
47. Yunus MB, Khan MA, Rawlings KK, et al: Genetic linkage analy-
sis of multicase families with fibromyalgia syndrome. J Rheumatol
26:408-412, 1999.
48. Uceyler N, Valenza R, Stock M, et al: Reduced levels of antiinflam-
matory cytokines in patients with chronic widespread pain. Arthritis
Rheum 54:2656-2664, 2006.
49. Roizenblatt S, Moldofsky H, Benedito-Silva AA, et al: Alpha sleep
characteristics in fibromyalgia. Arthritis Rheum 44:222-230, 2001.
50. Bergman S, Herrstrom P, Hogstrom K, et al: Chronic musculosk-
eletal pain, prevalence rates, and sociodemographic associations
in a Swedish population study 1338. J Rheumatol 28:1369-1377,
2001.
51. Griep EN, Boersma JW, Dekloet ER: Altered reactivity of the hypo-
thalamic-pituitary-adrenal axis in the primary fibromyalgia syndrome.
J Rheumatol 20:469-474, 1993.
52. Neeck G, Riedel W: Thyroid function in patients with fibromyalgia
syndrome. J Rheumatol 19:1120-1122, 1992.
53. Dinser R, Halama T, Hoffmann A: Stringent endocrinological testing
reveals subnormal growth hormone secretion in some patients with
fibromyalgia syndrome but rarely severe growth hormone deficiency.
J Rheumatol 27:2482-2488, 2000.
54. Petzke F, Clauw DJ: Sympathetic nervous system function in fibro-
myalgia. Curr Rheumatol Rep 2:116-123, 2000.
55. Martinez-Lavin M: Is fibromyalgia a generalized reflex sympathetic
dystrophy? Clin Exp Rheumatol 19:1-3, 2001.
56. Staud R, Vierck CJ, Cannon RL, et al: Abnormal sensitization and
temporal summation of second pain (wind-up) in patients with fibro-
myalgia syndrome. Pain 91(1-2):165-175, 2001.
57. Kwiatek R, Barnden L, Tedman R, et al: Regional cerebral blood flow
in fibromyalgia: Single-photon-emission computed tomography evi-
dence of reduction in the pontine tegmentum and thalami. Arthritis
Rheum 43:2823-2833, 2000.
58. Werle E, Fischer HP, Muller A, et al: Antibodies against serotonin
have no diagnostic relevance in patients with fibromyalgia syndrome.
J Rheumatol 28:595-600, 2001.
59. McBeth J, Silman AJ: The role of psychiatric disorders in fibromyal-
gia. Curr Rheumatol Rep 3:157-164, 2001.
60. Epstein SA, Kay G, Clauw D, et al: Psychiatric disorders in patients
with fibromyalgia—a multicenter investigation. Psychosomatics
40:57-63, 1999.
61. Wolfe F, Michaud K: A brief introduction to the National Data Bank
for Rheumatic Diseases. Clin Exp Rheumatol 23:S168-S171, 2005.
62. American Psychiatric Association: Diagnostic and Statistical Man-
ual of Mental Disorders (DSM-IV), 4th ed. Washington, American
­Psychiatric Association, 1994.
63. Wolfe F: Fibromyalgia: The clinical syndrome. Rheum Dis Clin
North Am 15:1-18, 1989.
 568 wolfe  |  Fibromyalgia
64. Park DC, Glass JM, Minear M, et al: Cognitive function in fibromy-
algia patients. Arthritis Rheum 44:2125-2133, 2001.
65. Wolfe F, Hawley DJ: Evidence of disordered symptom appraisal in
fibromyalgia: Increased rates of reported comorbidity and comorbid-
ity severity. Clin Exp Rheumatol 17:297-303, 1999.
66. McBeth J, Silman AJ: The role of psychiatric disorders in fibromyal-
gia. Curr Rheumatol Rep 3:157-164, 2001.
67. Fries JF, Spitz PW, Kraines RG, et al: Measurement of patient out-
come in arthritis. Arthritis Rheum 23:137-145, 1980.
68. Wolfe F, Michaud K, Pincus T: Development and validation of the
health assessment questionnaire, II: A revised version of the health
assessment questionnaire. Arthritis Rheum 50:3296-3305, 2004.
69. Pincus T, Swearingen C, Wolfe F: Toward a multidimensional Health
Assessment Questionnaire (MDHAQ)—assessment of advanced
activities of daily living and psychological status in the patient-
friendly health assessment questionnaire format. Arthritis Rheum
42:2220-2230, 1999.
70. Wolfe F, Skevington SM: Measuring the epidemiology of distress:
The rheumatology distress index. J Rheumatol 27:2000-2009, 2000.
71. Neumann L, Press J, Glibitzki M, et al: CLINHAQ scale—validation
of a Hebrew version in patients with fibromyalgia. Clin Rheumatol
19:265-269, 2000.
72. Burckhardt CS, Clark SR, Bennett RM: The fibromyalgia impact
questionnaire: Development and validation. J Rheumatol 18:728-
733, 1991.
73. Bennett R: The Fibromyalgia Impact Questionnaire (FIQ): A review
of its development, current version, operating characteristics and
uses. Clin Exp Rheumatol 23(5 Suppl 39):S154-S162, 2005.
74. Hidding A, Vansanten M, Deklerk E, et al: Comparison between
self-report measures and clinical observations of functional disabil-
ity in ankylosing spondylitis, rheumatoid arthritis and fibromyalgia. J
Rheumatol 21:818-823, 1994.
75. Ware JE, Sherbourne CD: The MOS 36-Item Short-Form Health
Survey (SF-36), 1: conceptual framework and item selection. Med
Care 30:473-483, 1992.
76. Picavet HS, Hoeymans N: Health related quality of life in multiple
musculoskeletal diseases: SF-36 and EQ-5D in the DMC3 study. Ann
Rheum Dis 63:723-729, 2004.
77. Wolfe F: Pain extent and diagnosis: Development and validation of
the regional pain scale in 12,799 patients with rheumatic disease.
J Rheumatol 30:369-378, 2003.
78. Jacobs JW, Rasker JJ, Van der Heide A, et al: Lack of correlation
between the mean tender point score and self-reported pain in fibro-
myalgia. Arthritis Care Res 9:105-111, 1996.
79. Simms RW, Goldenberg DL, Felson DT, et al: Tenderness in 75
anatomic sites: Distinguishing fibromyalgia patients from controls.
Arthritis Rheum 31:182-187, 1988.
80. Fischer AA: Pressure algometry (dolorimetry) in the differential
diagnosis of muscle pain. In Rachlin ES (ed): Myofascial Pain and
Fibromyalgia: Trigger Point Management, St Louis, Mosby, 1994, pp
121-140.
81. Katz RS, Wolfe F, Michaud K: Fibromyalgia diagnosis: A comparison
of clinical, survey, and American College of Rheumatology criteria.
Arthritis Rheum 54:169-176, 2006.
82. Baumgartner E, Finckh A, Cedraschi C, et al: A six year prospec-
tive study of a cohort of patients with fibromyalgia. Ann Rheum Dis
61:644-645, 2002.
83. Wolfe F, Anderson J, Harkness D, et al: Health status and disease
severity in fibromyalgia: results of a six-center longitudinal study.
Arthritis Rheum 40:1571-1579, 1997.
84. Hughes G, Martinez C, Myon E, et al: The impact of a diagnosis of
fibromyalgia on health care resource use by primary care patients in
the UK: An observational study based on clinical practice. Arthritis
Rheum 54:177-183, 2006.
85. Redondo JR, Justo CM, Moraleda FV, et al: Long-term efficacy of
therapy in patients with fibromyalgia: A physical exercise-based pro-
gram and a cognitive-behavioral approach. Arthritis Rheum 51:184-
192, 2004.
86. Goldenberg DL, Burckhardt C, Crofford L: Management of fibro-
myalgia syndrome. JAMA 292:2388-2395, 2004.
87. Littlejohn GO: Balanced treatments for fibromyalgia. Arthritis
Rheum 50:2725-2729, 2004.
88. Alamo MM, Moral RR, Perula de Torres LA: Evaluation of a patient-
centered approach in generalized musculoskeletal chronic pain/fibro-
myalgia patients in primary care. Patient Educ Couns 48:23-31, 2002.
89. Cedraschi C, Desmeules J, Rapiti E, et al: Fibromyalgia: A ran-
domised, controlled trial of a treatment programme based on self
management. Ann Rheum Dis 63:290-296, 2004.
90. Pfeiffer A, Thompson JM, Nelson A, et al: Effects of a 1.5-day mul-
tidisciplinary outpatient treatment program for fibromyalgia: A pilot
study. Am J Phys Med Rehabil 82:186-191, 2003.
91. Busch A, Schachter CL, Peloso PM, et al: Exercise for treating fibro-
myalgia syndrome. Cochrane Database Syst Rev 3:CD003786, 2002.
92. Richards SC, Scott DL: Prescribed exercise in people with fibromyal-
gia: Parallel group randomised controlled trial. BMJ 325:185, 2002.
93. Gowans SE, deHueck A, Voss S, et al: Six-month and one-year fol-
lowup of 23 weeks of aerobic exercise for individuals with fibromyal-
gia. Arthritis Rheum 51:890-898, 2004.
94. Assis MR, Silva LE, Alves AM, et al: A randomized controlled trial
of deep water running: Clinical effectiveness of aquatic exercise to
treat fibromyalgia. Arthritis Rheum 55:57-65, 2006.
95. Gowans SE, deHueck A: Effectiveness of exercise in management of
fibromyalgia. Curr Opin Rheumatol 16:138-142, 2004.
96. Dobkin PL, Da CD, Abrahamowicz M, et al: Adherence during an
individualized home based 12-week exercise program in women with
fibromyalgia. J Rheumatol 33:333-341, 2006.
97. Dobkin PL, Abrahamowicz M, Fitzcharles MA, et al: Maintenance of
exercise in women with fibromyalgia. Arthritis Rheum 53:724-731,
2005.
98. Wigers SH: Fibromyalgia outcome: The predictive values of symptom
duration, physical activity, disability pension, and critical life events—a
4.5 year prospective study. J Psychosom Res 41:235-243, 1996.
99. Bennett R, Nelson D: Cognitive behavioral therapy for fibromyalgia.
Nat Clin Pract Rheumatol 2:416-424, 2006.
100. Thieme K, Gromnica-Ihle E, Flor H: Operant behavioral treatment
of fibromyalgia: A controlled study. Arthritis Rheum 49:314-320,
2003.
101. Singh BB, Berman BM, Hadhazy VA, et al: A pilot study of cognitive
behavioral therapy in fibromyalgia. Altern Ther Health Med 4:67-
70, 1998.
102. White KP, Nielson WR: Cognitive behavioral treatment of fibro-
myalgia syndrome: A followup assessment. J Rheumatol 22:717-721,
1995.
103. Hadhazy VA, Ezzo J, Creamer P, et al: Mind-body therapies for the
treatment of fibromyalgia: A systematic review. J Rheumatol 27:2911-
2918, 2000.
104. Goossens ME, Rutten-van Molken MP, Leidl RM, et al: Cognitive-
educational treatment of fibromyalgia: A randomized clinical trial, II:
Economic evaluation. J Rheumatol 23:1246-1254, 1996.
105. Vlaeyen JW, Teeken-Gruben NJ, Goossens ME, et al: Cognitive-
educational treatment of fibromyalgia: A randomized clinical trial, I:
Clinical effects. J Rheumatol 23:1237-1245, 1996.
106. Wolfe F, Anderson J, Harkness D, et al: A prospective, longitudi-
nal, multicenter study of service utilization and costs in fibromyal-
gia. Arthritis Rheum 40:1560-1570, 1997.
107. Goldenberg DL, Felson DT, Dinerman H: A randomized, controlled
trial of amitriptyline and naproxen in the treatment of patients with
fibromyalgia. Arthritis Rheum 29:1371-1377, 1986.
108. Bennett RM, Kamin M, Karim R, et al: Tramadol and acetamino-
phen combination tablets in the treatment of fibromyalgia pain:
A double-blind, randomized, placebo-controlled study. Am J Med
114:537-545, 2003.
109. Arnold LM, Keck PE, Welge JA: Antidepressant treatment of
fibromyalgia: A meta-analysis and review. Psychosomatics 41:
104-113, 2000.
110. Jaeschke R, Adachi JD, Guyatt G, et al: Clinical usefulness of ami-
triptyline in fibromyalgia: The results of 23 N-of-1 randomized con-
trolled trials. J Rheumatol 18:447-451, 1991.
111. Gendreau RM, Thorn MD, Gendreau JF, et al: Efficacy of milnaci­
pran in patients with fibromyalgia. J Rheumatol 32:1975-1985, 2005.
112. Crofford LJ, Rowbotham MC, Mease PJ, et al: Pregabalin for the
treatment of fibromyalgia syndrome: Results of a randomized, dou-
ble-blind, placebo-controlled trial. Arthritis Rheum 52:1264-1273,
2005.
113. Wigers SH, Stiles TC, Vogel PA: Effects of aerobic exercise versus
stress management treatment in fibromyalgia: A 4.5 year prospective
study. Scand J Rheumatol 25:77-86, 1996.
114. Haanen HCM, Hoenderdos HTW, Van Romunde LKJ, et al: Con-
trolled trial of hypnotherapy in the treatment of refractory fibromyal-
gia. J Rheumatol 18:72-75, 1991.
 PART 5  |  EVALUATION OF GENERALIZED AND LOCALIZED SYMPTOMS
115. Deluze C, Vischer TL: Electroacupuncture in fibromyalgia—reply.
BMJ 306:393, 1993.
116. Assefi NP, Sherman KJ, Jacobsen C, et al: A randomized clinical trial
of acupuncture compared with sham acupuncture in fibromyalgia.
Ann Intern Med 143:10-19, 2005.
117. Drexler AR, Mur EJ, Gunther VC: Efficacy of an EMG-biofeedback
therapy in fibromyalgia patients: A comparative study of patients
with and without abnormality in (MMPI) psychological scales. Clin
Exp Rheumatol 20:677-682, 2002.
118. van Santen M, Bolwijn P, Verstappen F, et al: A randomized clini-
cal trial comparing fitness and biofeedback training versus basic
treatment in patients with fibromyalgia. J Rheumatol 29:575-581,
2002.
119. Ferraccioli GF, Ghirelli L, Scita F, et al: EMG-biofeedback training
in fibromyalgia syndrome. J Rheumatol 14:820-825, 1987.
120. Altan L, Bingol U, Aykac M, et al: Investigation of the effects of pool-
based exercise on fibromyalgia syndrome. Rheumatol Int 24:272-277,
2004.
569
121. Neumann L, Sukenik S, Bolotin A, et al: The effect of balneotherapy
at the Dead Sea on the quality of life of patients with fibromyalgia
syndrome. Clin Rheumatol 20:15-19, 2001.
122. Zijlstra TR, van de Laar MA, Bernelot Moens HJ, et al: Spa treat-
ment for primary fibromyalgia syndrome: A combination of thalas-
sotherapy, exercise and patient education improves symptoms and
quality of life. Rheumatology (Oxf) 44:539-546, 2005.
123. Ballantyne JC, Mao J: Opioid therapy for chronic pain. N Engl J Med
349:1943-1953, 2003.
124. Littlejohn GO, Walker J: A realistic approach to managing patients
with fibromyalgia. Curr Rheumatol Rep 4:286-292, 2002.
125. Littlejohn G: The fibromyalgia syndrome: Outcome is good with
minimal intervention [letter]. BMJ 310:1406, 1995.
126. Fitzcharles MA, Costa DD, Poyhia R: A study of standard care in
fibromyalgia syndrome: A favorable outcome. J Rheumatol 30:154-
159, 2003.
127. Mengshoel AM, Haugen M: Health status in fibromyalgia—a fol-
lowup study. J Rheumatol 28:2085-2089, 2001.
 PART DIFFERENTIAL DIAGNOSIS OF
6 REGIONAL MUSCULOSKELETAL PAIN
39
KEY POINTS
Neck pain is a ubiquitous condition associated with enormous
medical and legal costs in the United States.
Physicians need to differentiate causes of neck pain that can
be managed conservatively from causes that require more
aggressive treatments.
Knowledge of the anatomy helps diagnosis and the
­differentiation of symptoms from a musculoskeletal,
­neurogenic, or vascular etiology.
The history and clinical examination help focus the
­differential diagnosis and help to identify the origin of the
neck pain based on the anatomy and physiology.
Indicated imaging studies, neurophysiologic procedures, and
laboratory studies aid in diagnosis and determining a treat-
ment plan for the patient’s symptoms.
In the absence of spinal instability, neurologic deficit, infec-
tious process, or neoplastic process, the patient may benefit
from conservative treatment with expectant recovery in time.
EPIDEMIOLOGY
Pain is an evolutionary protective mechanism to prevent
further tissue damage, with neck pain being a nearly ubiqui-
tous condition with a lifetime prevalence of 67% to 71%.1
The point prevalence of neck pain ranges from 10% to 15%
with the annual total costs for neck and low back pain cor-
responding to 1% of the gross national product in Sweden,
with direct health care costs representing only a small frac-
tion of this percentage.2-4 The medical and legal expenses
associated with neck pain can be enormous, such as in whip-
lash injuries, which costs an estimated $29 billion annually
in the United States.5
Neck pain may originate from various anatomic struc-
tures, including paraspinal soft tissues, intervertebral joints
and disks, compression of the spinal cord or nerves, and
referred visceral pain (Fig. 39-1). The etiology of neck pain
has a wide differential diagnosis, which can include trauma,
degenerative changes, infection, and autoimmune disorders,
such as rheumatoid arthritis and ankylosing spondylitis.
Neck Pain
Clinton Devin  •  Karl Sillay  • 
Joseph s. Cheng
The perception and resultant reporting of neck pain var-
ies significantly based on cultural and social circumstances.
Honeyman and Jacobs6 noted that Australian aborigines
significantly underreport pain and are rarely disabled by
pain. Social circumstances also play an important role in an
individual’s ability to cope with and overcome neck pain.
Studies have shown worse outcomes after diskectomy for
patients with a workers’ compensation claim or litigation
surrounding their condition.7 These studies indicate nonor-
ganic contributions to neck pain for secondary gain. Most
episodes of acute neck pain resolve with patient education
and the passage of time.
Physicians need to be able to differentiate causes of neck
pain that can be managed with a conservative approach
from causes that require more aggressive treatments. An
understanding of the anatomy and physiology and their
association with the pathogenesis of neck pain provides the
basis for obtaining a thorough history, physical examina-
tion, and ancillary data with the ultimate goal of effective
treatment.
ANATOMY
The cervical spine consists of seven vertebrae denoted as C1
through C7. The bony anatomy of the atlas (C1) and axis
(C2) are unique, whereas C3 through C7 have fairly con-
sistent anatomy (Fig. 39-2). The atlas is a ring, consisting
of anterior and posterior arches with two lateral masses and
no vertebral body. The superior aspects of the lateral masses
articulate with the skull through the occipital condyles and
form the atlanto-occipital joints, which are supported fur-
ther by the anterior and posterior occipital membranes.8
The atlanto-occipital joint is responsible for approximately
50% of total flexion and extension in the neck, with clear
functional implications when this motion is lost. The axis
consists of two lamina, a spinous process, two lateral masses,
two pedicles, a vertebral body, and the dens or odontoid
peg, which projects upward and anterior to articulate with
the posterior aspect of the anterior arch of the atlas. The
principal stabilizer of the odontoid to the anterior arch of
the atlas is the transverse ligament, with the alar and api-
cal ligaments acting as secondary stabilizers. This is a true
571
 572 DEVIN  |  Neck Pain

Neck pain

History
clinical exam

Suspected
Axial neck pain Radiculopathy Myelopathy neoplasm or Trauma
infection

No
Conservative care improvement Radiological studies Rigid collar
soft collar, X-ray, CT, MRI, spine
medications, PT bone scan precautions

Continue
conservative Mild findings
treatments Negative neural compression Severe findings
findings bone lesion neural compression
deformity bone lesion deformity

Neurodiagnostics Request spine

Conservative treatments with EMG, NCS, SSEP surgeon consult
Evidence of
negative results nerve or cord
compression
Figure 39-1  Neck pain algorithm.

Foramen for Lambdoid

vertebral suture
artery
Atlas

Synovial External Superior

joint occipital nuchal
Axis
protuberance line
3
Intervertebral
disk
4 Foramen
True
transverse magnum
5
element of
transverse Transverse
6
process Odontoid peg process
Costo- (dens) and
7
transverse bar Tubercle on
posterior arch
Costal of atlas
element
of transverse
process
First rib
A B
Figure 39-2  Cervical spine anatomy. A, Cervical spine. Anterior view of articulated cervical vertebrae. B, Posterior view of the skull, seven cervical
vertebrae, and first thoracic vertebra. (From Nakano KK: Neck pain. In Ruddy S, Harris ED Jr, Sledge CB [eds]: Kelley’s Textbook of Rheumatology, 6th ed.
Philadelphia, Saunders, 2001, p 458.)
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
synovial joint and susceptible to inflammatory processes,
such as seen with rheumatoid arthritis. There is no interver-
tebral disk between the atlanto-occipital joint and atlanto-
axial joint, and without the stability conferred by a disk, the
area is often involved by destructive inflammatory arthri-
tides, which may result in instability.9 The axis articulates
with the vertebra above and below through the superior and
inferior facets, also termed the zygapophyseal joints. Posteri-
orly, the axis has a large spinous process, which can be easily
palpated just below the occiput. The atlantoaxial articula-
tion also provides approximately 50% of rotatory motion of
the cervical motion.
The subaxial cervical spine consists of the C3 through
C7 vertebrae, all with fairly similar anatomy. Each vertebra
consists of a body, two interconnecting pedicles, two lateral
masses, two transverse processes, two laminae, and a spinous
process. The transverse and spinous processes project out-
ward, providing attachment for ligaments and muscles and
creating a moment arm to facilitate motion. The spinous
processes of C3 through C6 are bifid, whereas the C7 spi-
nous process usually is not. The C7 spinous process is large,
however, and the next most prominent and easily palpable
spinous process below C2.
There are five articulations between each vertebra
from C2 through C7, including the intervertebral disk,
two uncovertebral joints, and two facet or zygapophyseal
joints. The facet joints are true apophyseal joints with hya-
line cartilage articulations, intervening menisci, synovial
lining, and a joint capsule. This composition makes them
susceptible to degenerative changes and systemic arthriti-
des. The cartilage and synovial lining are not innervated,
whereas the joint capsule is highly innervated by the dorsal
primary ramus. The facet joints are angled approximately
45 degrees from the transverse plane, articulating with
the vertebrae above and below in a pattern that has been
described as shingles on a roof. The facet joints in con-
cert with the uncovertebral joints and ligaments help limit
excess movement of the vertebral column, which helps
protect the spinal cord.
The intervertebral disks increase in size from C2 down-
ward, giving the cervical spine its characteristic lordotic
shape. Each disk consists of an outer anulus fibrosus and
an inner nucleus pulposus and a cephalad and caudad end
plate. The anulus fibrosus consists of type I collagen, help-
ing to give form to the disk and provide tensile strength.
The anulus fibrosus is innervated by the sinuvertebral
nerve, formed by branches of the ventral nerve root and
the sympathetic plexus.10 The nucleus pulposus consists
of type II collagen and proteoglycans, which interact with
water to resist compressive stress. The pressure within
the disk is highest with flexion, which may explain why
individuals with a disk herniation find this position most
uncomfortable.11 Disk degeneration with aging includes
loss of water content with resultant loss of height, annu-
lar tears, and myxomatous changes, increasing the risk of
disk herniation. Herniation typically occurs in the pos-
terolateral aspect of the disk, where the posterior longi-
tudinal ligament is not present and the anulus fibrosus is
at its weakest. The disk is bordered on both sides by the
uncovertebral joints, which are formed by an up-curving
of the superior end plate to articulate with the vertebra
above.
573
Spheno-occipital
synchondrosis
Tectorial membrane
PONS
te
Pala
Apical
ligament
Atlas
Remnant of disk uniting
body and dens of axis
Margins of
foramen
magnum
Dura
Spinal cord
Posterior
longitudinal
ligament
Anterior
longitudinal
ligament
Figure 39-3  Cervical spine anatomy. Sagittal view of the lower head
and neck to show the relationship of the spinal cord and brainstem to
the bones, ligaments, and joints between the bodies of the cervical ver-
tebrae. The cervical lordosis and the relationship of the anterior and pos-
terior longitudinal ligaments to intervertebral disks and the ligaments at
the craniovertebral junction can be seen. (From Nakano KK: Neck pain. In
Ruddy S, Harris ED Jr, Sledge CB [eds]: Kelley’s Textbook of Rheumatology,
6th ed. Philadelphia, Saunders, 2001, p 459.)
The spinal column is supported by an interplay of liga-
ments and muscles (Fig. 39-3). The anterior longitudinal lig-
ament and posterior longitudinal ligament originate on the
occiput and extend downward to the sacrum, coursing along
the anterior and posterior aspects of the vertebral bodies.
The anterior longitudinal ligament resists hyperextension,
and the posterior longitudinal ligament resists hyperflexion.
The posterior longitudinal ligament can become ossified,
limiting motion and contributing to the degenerative pro-
cess. The ligamentum flavum is a short ligament that joins
the laminae of adjacent vertebrae. It can become thick-
ened over time creating stenosis in the spinal canal and
compressing the spinal cord and exiting nerve roots. In a
similar manner, the intraspinous ligament joins the spinous
processes of adjacent vertebrae. The supraspinous ligament
originates as the nuchal ligament at the occiput and extends
caudally as an aponeurosis until it attaches to the tip of the
spinous processes of C7 and then continues to the lumbar
region. There are 14 paired anterior, lateral, and posterior
muscles that help orchestrate the complex movements of
the neck.
A brief review of the spinal cord and nerve roots is ben-
eficial for assisting with a thorough evaluation. There is
significantly more room for the spinal cord in the upper
cervical spine than the lower cervical spine, making the
upper cervical spine a less likely site of spinal cord com-
pression. Grossly, the spinal cord is divided into the poste-
rior column, the lateral columns, and the anterior column.
 574 DEVIN  |  Neck Pain

Posterior longitudinal ligament

Sympathetic ganglion
Rami communicantes

Anterior ramus
Sinuvertebral nerves

Posterior ramus
Figure 39-4 Cervical spine neural elements. ­ Localized ­ cervical pain
is mediated primarily through the posterior primary ramus and the
­recurrent meningeal (sinuvertebral) nerves, which supply structures
within the spinal canal. The recurrent meningeal nerves arise from rami
­communicantes and enter the spinal canal via intervertebral foramina;
branches ascend and descend one or more levels, interconnecting with
the recurrent ­meningeal nerves from other levels and innervating, among
other structures, the anterior and posterior longitudinal ligaments, the Dorsal root
anterior and posterior portion of the dura, and blood vessels. (From Levin ganglion
KH [ed]: Neck and Back Pain. Continuum 7 (no. 1), Philadelphia, Lippincott Ligamentum
Williams & Wilkins, 2002, p 9.) flavum

The posterior column consists of the fasciculus cuneatus
laterally and the fasciculus gracilis medially, which mediate
proprioceptive, vibratory, and tactile sensation. The lateral
column consists of the lateral corticospinal tract, which
provides the conduit for motor fibers, and the spinotha-
lamic tract, which provides pain and temperature sensation
from the contralateral side of the body. The anterior col-
umn conveys crude touch sensation. There are eight total
cervical nerve roots on each side as the dorsal and ventral
roots converge to form the spinal nerve within the verte-
bral foramen. Cervical nerve roots enter the intervertebral
foramina by passing over top of the corresponding pedicle
except C8, which lies between C7 and T1. A C5-C6 pos-
terolateral disk herniation affects the C6 nerve root. The
nerve root occupies approximately one third of the fora-
men (Fig. 39-4). The space available for the nerve root is
decreased with neck extension and degenerative changes
and increased with neck flexion.
The spinal cord is supplied by the anterior spinal artery
and two posterior spinal arteries. The anterior spinal artery
arises from the vertebral arteries and supplies most of the
spinal cord, excluding the posterior columns. The ­posterior
columns receive their blood supply from the two posterior
spinal arteries, which originate from either the inferior
­cerebellar artery or the vertebral arteries. Blood supply
to the spinal cord may be impaired in diseases such as
­arteriosclerosis, diabetes, and syphilis, with exacerbation of
symptoms with various head positions, usually extension.
The vertebral arteries arise from the subclavian arteries and
course through the C6 transverse foramen cephalad, passing
anterior to the emerging cervical nerve root at each level.
They pass behind the lateral mass of C1 and enter the fora-
men magnum, where they join to form the basilar artery.
Diseases such as vertebral artery dissection can be associ-
ated with severe neck pain and impairment of blood flow
through the vertebral artery and can result in posterior cir-
culation signs, including nystagmus, vertigo, drop attacks,
Table 39-1  Age and Normal Cervical Movement
Flexion-­Extension Lateral Rotation Lateral Flexion
Age (yr) (degrees) (degrees) (degrees)
<30 90 90 45
31-50 70 90 45
>50 60 90 30
From Nakano KK: Neck pain. In Kelley WN, Harris ED Jr, Ruddy S, et al (eds):
Kelley’s Textbook of Rheumatology, 5th ed. Philadelphia, WB Saunders, 1997,
p 396.
dysarthria, and weakness. These symptoms are often associ-
ated with head position, and if a critical reduction of blood
flow occurs, it can result in a cerebellar infarction.
The cervical spine is the most mobile segment of the
spine with approximately a 90-degree arc of motion in flex-
ion and extension, with three fourths of this due to extension
(Table 39-1). The maximal range of motion in the sagit-
tal plane within the subaxial spine is at the C5-C6 level,
making it a common site of disk degeneration. Rotation
encompasses approximately 80 to 90 degrees of motion with
50% of this occurring at the atlantoaxial joint. Similar to
extension, rotation also reduces the cross-sectional area of
the spinal canal. The cervical spine has 30 degrees of lateral
mobility in each direction, and this typically occurs with
some degree of rotation secondary to the orientation of the
facet joints.
AXIAL NECK PAIN
Axial neck pain describes a pattern of pain that is local-
ized to the occiput and neck region. It may originate from
any tissue that receives innervation, including the facet
joints, cervical disks, vertebral periosteum, posterior neck
muscles, cervical dura mater, occipito-atlantoaxial joints,
and vertebral artery. The etiology may include degenerative,
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
traumatic, malignant, infectious, or systemic inflammatory
processes. The facet joints and cervical disks have the most
direct supporting data linking these sites as the origin of
axial neck pain. Provocative injections into the facet joint
in asymptomatic volunteers invoke a reproducible pattern
of occipital or axial neck pain.12 This pattern of pain can be
accurately diagnosed and treated, for at least a short time,
with anesthetic injections targeted at the joint capsule itself
or by blocking its respective dorsal primary ramus.13-15 Degen-
erative arthritis within the upper cervical spine can manifest
as suboccipital headache and localized pain. This is termed
cervicogenic headache and is thought to result from irritation
of the greater occipital nerve. Typically, arthritis within the
atlanto-occipital joints is worsened with provocative neck
flexion and extension, whereas atlantoaxial arthritis is wors-
ened with rotation. A study in which asymptomatic vol-
unteers underwent injections at the atlanto-occipital and
atlantoaxial joints reproduced this pattern of pain.16 Relief
of suboccipital pain may be obtained by fluoroscopically
guided injection of corticosteroid into the diseased joint or
by fusion of these joints in recalcitrant cases.17
The cervical disk is a more controversial source of axial
neck pain with pain secondary to injury to the highly inner-
vated anulus fibrosus. This idea is based on provocative dis-
cography, whereby a diseased disk is fluoroscopically injected
to a given pressure with reproduction of pain in reliable pat-
terns. To be a true-positive study or a “concordant study,”
an adjacent normal disk should not produce pain when
injected. Using this method, several studies have implicated
cervical disks as a source of axial neck pain.18-20 Even with
careful technique, a false-positive study can occur, and it
is not unusual to have a “nonconcordant study” with mul-
tiple disks eliciting a pain response despite being normal.18
Operative interventions directed at treating the disk for iso-
lated axial neck pain have often been unsuccessful despite
the fact that the cervical disk likely contributed to the neck
pain. In addition, degeneration of the disk affects the space
available for the nerve root with resultant radiculopathy,
which is discussed in more detail later.
Myofascial pain secondary to irritation of the muscles
around the neck can contribute to axial neck pain. Patients
with chronic myofascial pain have been shown to have a
lower level of high-energy phosphates in the involved mus-
cle tissue.21 It can be the original source of neck pain or,
more commonly, a manifestation of postural adaptations
and compensatory overuse of normal tissue that remains
after the injured structure heals. A more generalized form
is fibromyalgia, which is a widespread disorder, not causing
isolated neck pain. By definition, fibromyalgia is a diffuse
pain affecting all four quadrants of the body with at least
11 of 18 pressure points being positive. Patients have asso-
ciated symptoms of fatigue; cognitive difficulties; irritable
bowel syndrome; and a nondermatomal pattern of dysesthe-
sias, weakness, and paresthesias.22
Systemic inflammatory arthropathies rarely cause isolated
neck pain because these illnesses typically show the clas-
sic pattern of morning stiffness, polyarticular involvement,
rigidity, and often cutaneous manifestations. Rheumatoid
arthritis often involves the cervical spine, initially causing
stiffness and later causing pain. This involvement can often
affect the occipito-atlantoaxial joint causing instability and
the potential for neurologic compromise. After the hands
575
and feet, the cervical spine is the most common site of
disease involvement in rheumatoid arthritis.23 Seronegative
spondyloarthropathies that can manifest with neck pain
include ankylosing spondylitis, psoriatic arthritis, and reac-
tive arthritis. Psoriatic arthritis manifests with skin lesions
before the development of arthritis in 70% of patients, and
reactive arthritis rarely involves the cervical spine.
Ankylosing spondylitis often affects the entire axial
skeleton with early limitation of lumbar motion and chest
expansion and later involvement of the cervical spine. In
progressive patterns, the cervical spine takes on a kyphotic
deformity, and as the spine fuses, it biomechanically becomes
similar to a long bone. This condition has implications when
ankylosing spondylitis patients are involved in even minor
trauma, and neck pain should be taken very seriously in these
patients. Even in the face of negative plain radiographs, these
patients should be worked up extensively with strict spine
precautions, with neutral alignment varying with baseline
spinal curvature, and with frequent ­ neurologic evaluations
to assess for development of an epidural hematoma.
Infections and neoplasms can cause axial neck pain
through bone destruction with irritation of vertebral body
periosteal nerves and altered biomechanics of the facet
joints and cervical disks. The onus is on the clinician to
identify these patients at the initial visit because a delay in
diagnosis can have catastrophic consequences. Red flags for
axial neck pain that require further workup at the initial
presentation include advanced age, history of malignancy,
immunocompromise, fevers, chills, unexplained weight loss,
fatigue, nighttime awakening, recent antecedent bactere-
mia, and severe nonmechanical neck pain.22
RADICULOPATHY AND MYELOPATHY
The clinician must determine if there is evidence of nerve
root compression, termed radiculopathy, versus spinal cord
compression, termed myelopathy. Cervical radiculopathy
resulting from nerve root compression is most often caused
by degenerative changes, such as spondylosis. Initially, there
are changes within the disk causing a loss of height with pos-
terior bulging of the disk into the spinal canal and foramen.
As the disk collapses, the posterior soft tissue structures,
including the ligamentum flavum and facet joint capsule,
fold inward, further compromising the spinal canal and neu-
ral foramen. Pressure that was once dispersed throughout
the disk is transferred to the facet joints and uncinate pro-
cesses, resulting in the development of bone overgrowth or
osteophytes leading to extrinsic pressure on the nerve root
or spinal cord.
In radiculopathy, there is mechanical distortion of the
nerve leading to increased vascular permeability, resulting in
chronic edema and eventually fibrosis. This situation leads
to hypersensitivity of the nerve root with an inflammatory
response mediated by substances released from the cell bod-
ies of sensory neurons and cervical disks.24 Compression of
the dorsal root ganglion is believed to be especially impor-
tant in producing radicular pain.25 Clinically, this compres-
sion manifests with pain in a dermatomal distribution. The
dermatomes for the higher cervical nerve roots, including
C3 and C4, are along the posterior scapula and should not
be confused with isolated axial neck pain.26 When it is estab-
lished that a patient has radiculopathy, it must be determined
 576 DEVIN  |  Neck Pain
if a neurologic deficit is present. Persistent compression on
a nerve root can lead to sensory loss and weakness. If these
deficits are minor and tolerable, it is reasonable to treat with
conservative care with close follow-up to ensure that the
deficit is not progressive. Disabling deficits should be treated
operatively because prolonged nerve compression can result
in irreversible changes. In patients without a neurologic
deficit, it is reasonable to expect a good outcome with con-
servative care.27
Myelopathy is the clinical presentation of long tract signs
resulting from compression of the spinal cord. Myelopa-
thy can be due to mass effect from a tumor or infection or
instability owing to systemic arthritides or connective tissue
disorders, but it is often a result of advanced degenerative
changes within the cervical spine. Factors that contribute
to the development of myelopathy include a congenitally
narrow spinal canal, dynamic cord compression, dynamic
thickening of the spinal cord, and vascular changes. The
anteroposterior diameter in the subaxial spine for a normal
adult measures 17 to 18 mm, and the cord measures 10 mm.
Diameters of less than 13 mm are considered to be congeni-
tally stenotic. The shape of the spinal cord deformity has
a high association with the development of myelopathy;
patients with a banana-shaped cord on axial views had evi-
dence of myelopathy 98% of the time.28 Ono and associates29
described a ratio whereby the anteroposterior diameter of
the spinal cord is divided by the transverse diameter of the
cord. Patients with a ratio of less than 0.40 tended to have
severe neurologic deficits. Some patients may have dynamic
cord compression with signs and symptoms of myelopathy
only during neck flexion and extension. The space available
for the cord is decreased during neck extension as a result of
infolding of the ligamentum flavum and overlapping of the
lamina. In addition, the spinal cord shortens during neck
extension, effectively increasing the diameter and making
it more prone to compression by the posterior structures. In
flexion, the cord lengthens and drapes over anterior degen-
erated disks and osteophytes.30
Myelopathy can be exacerbated by altered biomechan-
ics from degenerated segments because when a given
level stiffens, the level above can become hypermobile.31
A subset of patients can develop myelopathy in the absence
of mechanical compression, which has been attributed to
ischemic insult.32 It also has been shown in a canine model
that in the setting of spinal cord compression, additive
ischemia results in a significantly worse outcome because
over time the spinal cord shows permanent irreversible
changes.33 Caudad to the level of compression, the central
gray matter and lateral columns may show cystic cavita-
tion, demyelination, and gliosis. Cephalad to the level of
compression, the posterior columns may undergo wallerian
degeneration. Patients with mild cases of myelopathy that
does not affect activities of daily living can be followed
closely.34 Patients with more severe deficits tend to deterio-
rate over time with conservative care, and these patients
should undergo surgery to decompress the spinal cord.35
CLINICAL FEATURES
In terms of functional anatomic pathways, neck pain is
mediated via somatic or autonomic pathways.36 Somatic
pain is the most common, being perceived in dermatomes,
myotomes, or sclerotomes. Pain originating in the auto-
nomic pathway, or sympathetic nervous system, may fall
into somatic segmental distributions, vascular supply distri-
butions, peripheral nerve distributions, or nonconforming
patterns. Pain mediated by autonomic and somatic pathways
has significant overlap, potentially confounding localiza-
tion. Additional clinical information regarding characteris-
tics of the neck pain and diagnostic studies complement the
determination of the origin of the pain when localization
based on functional anatomy is insufficient.
PATIENT HISTORY
Neck pain is the most common symptom of cervical spine
pathology, and correctly characterizing it helps to identify
conditions requiring immediate treatment versus condi-
tions for which conservative treatments are indicated.
Important characteristics include onset, distribution, fre-
quency, duration, quality, aggravating factors, and the pres-
ence of neurologic symptoms other than pain. In general,
pain present only intermittently may indicate instability or
motion-related pain, whereas constant and increasing pain
is concerning for a mass effect. Generalized neck pain of
new onset with relatively short duration is likely related to
benign pathology, including muscle strain, whereas a ­longer
duration of symptoms indicates significant or progressive
pathology. Well-localized pain indicates specific nerve root
irritation, whereas poorly defined pain may derive from irri-
tation of deep connective tissue structures, such as muscle,
joint, bone, or disk. Aggravating and relieving factors may
help elucidate biomechanical changes in the cervical spine
that are contributing to the symptoms.
Localized axial neck pain is commonly reported as
originating posteriorly with extension into the shoulder or
occiput. Localized pain of myofascial etiology may worsen
with neck flexion, whereas discogenic neck pain wors-
ens with neck extension or rotation. Referred pain to the
occiput usually indicates pathologic changes in the upper
cervical spine and may radiate down the neck and to the ear.
Shoulder girdle pain develops secondary to postural adapta-
tions from initial neck pain symptoms. Pain commonly is
referred from the shoulder, heart, lungs, viscera, or temporo-
mandibular joint to the neck region owing to overlapping
nerve distribution. Symptoms may arise secondary to irri-
tation or activation of receptors directly as with articular
pain; pseudoarticular pain; vascular headache; cervicogenic
headache; occipital headache; pseudo–angina pectoris; and
eye, ear, or throat conditions.
Articular symptoms arise from the facet and uncoverte-
bral joints causing local pain and stiffness. Patients often
state that their symptoms worsen with inactivity and
describe feelings of clicking, grating, or “sand” in the neck.
Pseudoarticular pain may be felt in the shoulder and elbow,
with the true pathology originating from the neck. Vascular
symptoms result from compression of the vertebral artery by
osteophytes or a protruding disk. Symptoms may intensify
with neck movement or certain postures. Tenosynovitis and
tendinitis may involve the rotator cuff and tendons around
the elbow, wrist, or hand. Stenosis or fibrosis of tendon
sheaths or palmar fascia may be present along with trigger
points over the affected joints, giving a false impression of
local pathology.37
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
Table 39-2  Cervical Nerve Root Segments and Corresponding Clinical Signs and Symptoms
Nerve Root Symptom
C3 Suboccipital pain with extension to back of ear
C4 Pain from caudad aspect of neck to superior aspect of shoulder
C5 Numbness over shoulder and down lateral aspect of arm to
­midportion. Deltoid muscle may be weak and biceps reflex,
which is innervated by C5-C6, may be affected
C6 Radiating pain and numbness down lateral aspect of arm and
forearm to thumb and index finger (“six shooter”). Weakness
in wrist extension, elbow flexion, and supination. Diminished
­brachioradialis and biceps reflex
C7 Numbness and pain down posterior aspect of arm and forearm
to long finger. Weakness in triceps, wrist flexion, and finger
­extensors
C8 Numbness into ulnar two digits. Weakness in FDP to IF and LF and
FPL
T1 Numbness into ulnar aspect of forearm and weakness in hand
intrinsics
FDP, flexor digitorum profundus; FPL, flexor pollicis longus; IF, index finger; LF, long flexor.
LOCALIZATION OF PAIN GENERATORS
When localizing symptoms of pain and paresthesia, one
must consider the classssification and localization of pain
generators. Pain may be somatic or autonomic and is not
always felt in precise anatomic zones. Overlapping sensory
supplies and radiation in spinal segments by recruitment
within the spinal column may be present causing difficulty
in localization. Somatic pain is caused by cervical nerve
root irritation. It is the most common type of pain, and
diabetics are especially susceptible to this nerve root irrita-
tion. Neurologic deficits correspond to the offending disk
level in 80% of patients.38
Neuralgic and myalgic pain describes symptoms second-
ary to compression of different areas of the nerve root. Neu-
ralgic pain originates from irritation of the dorsal sensory
root and has a “lightning” or “electric” sensation, which
tends to be dermatomal and associated with numbness and
paresthesias. The pain tends to manifest more proximally
with paresthesias distally. Myalgic pain occurs with irrita-
tion of the ventral motor root. This pain is described as a
deep, boring, unpleasant sensation, which tends to be poorly
localized because of its referral to sclerotomal areas. These
sensations conform to the areas of muscles present that are
innervated by the compressed nerve root. Autonomic medi-
ated symptoms result in dizziness, blurring of vision, tinni-
tus, retro-ocular pain, facial pain, and jaw pain.
The various origins of pain generators in the cervical
spine include mechanical, vascular, and nerve root sources
and spinal cord compression. These pain generators may
manifest with radicular, sclerotomal, myalgic, or autonomic
symptoms, making the history an important component
to identify key features specific to the origin. An informed
hypothesis as to the origin of the pain can be formed, which
can be tested by physical examination and invasive and
noninvasive diagnostic studies so that a treatment plan can
be established.
It is important to determine if the axial neck pain is
isolated, or if there is associated radiating pain, weakness,
change in sensation, or alteration in proprioception. Com-
pression of a nerve root often can be localized by identifying
577
Correlate
If C3, C4, C5 all are involved, may cause paradoxical breathing
Sensory component can mimic carpal tunnel syndrome
Most frequent. Entrapment of posterior interosseous nerve
can mimic motor component, but no sensory deficits are
present
Anterior interosseous nerve entrapment can mimic a radicu-
lopathy of C8 or T1, but sensory changes and involvement
of thenar muscles are not present. Ulnar nerve entrapment
spares short thenar muscles with exception of adductor
pollicis.
the distribution of pain, paresthesias, or weakness as they
follow the segmental distribution of the respective nerve
root (Table 39-2). Sensory loss may be described by the
patient in precise terms with a description of numbness, or
alternatively there may be vague symptoms of the sensation
of swelling or bogginess to the skin. Complete numbness
indicates a more serious nerve or root lesion. If the face,
head, or tongue is involved, the upper three nerve roots of
the cervical plexus may be affected. Numbness of the neck,
shoulder, arm, forearm, or fingers indicates involvement
of C5-T1. Weakness, as with sensory changes, occurs in a
graded fashion depending on the amount of compression
on the nerve root. This weakness manifests clinically with
an obvious functional deficit or more subtle findings, made
obvious only after repetitive testing. The clinician must be
attuned to these subtle complaints of weakness, which may
be described by the patient as a feeling of heaviness of the
limbs, early fatigue, or insufficient power grip. If obvious
atrophy is present in a muscle, more than one nerve root
is affected as a result of the evolutionary benefit of multiple
levels of innervation for a given muscle. Alterations in pro-
prioception are due to compression of the dorsal column
of the spinal cord; this is described by the patient as symp-
toms of clumsiness, with complaints of tripping or dropping
objects. This is concerning for the more ominous condition
of myelopathy secondary to spinal cord compression.
Cervical spinal disease classically causes isolated axial
neck pain or radicular pain that radiates to the shoulder or
down the upper extremity (Table 39-3). Less commonly, it
can be the cause of headache, pseudo–angina pectoris, and
otolaryngologic sensations. The cause of cervicogenic occip-
ital headache is multifactorial, and headache is most often
seen with degenerative changes in apophyseal joints of the
upper cervical spine. These degenerative changes can be
compounded by adaptive changes in the posterior occipital
muscles. Cervicogenic occipital headaches often spread to
the eye region and manifest as a dull, rather than pulsating,
pain. These headaches are unique in that they are aggra-
vated by neck movements. They typically have migraine-
like symptoms, including phonophobia or photophobia.
 578 DEVIN  |  Neck Pain
Table 39-3  Cervical Pain Referral Pathways
Location of Pain Source
Upper posterolateral cervical region C0-1, C1-2, C2-3
Occipital region C2-3, C3
Upper posterior cervical region C2-3, C3-4, C3
Middle posterior cervical region C3-4, C4-5, C4
Lower posterior cervical region C4-5, C5-6, C4, C5
Suprascapular region C4-5, C5-6, C4
Superior angle of scapula C6-7, C6, C7
Midscapular region C7-T1, C7
From Nakano KK: Neck pain. In Kelley WN, Harris ED Jr, Ruddy S, et al (eds):
Textbook of Rheumatology, 5th ed. Philadelphia, WB Saunders, 1997, p 394.
Pseudo–angina pectoris has been reported to be due to
cervical spinal disease, being confused with angina pecto-
ris or breast pain in women (Fig. 39-5). In the presence of
a C6-C7 lesion, neuralgic or myalgic pain may be present
along with tenderness in the precordium or scapular region.
A pressure sensation is felt in the chest, which increases
with exercise, radiates down the arm, is aggravated by neck
movement, and may be associated with torticollis or muscle
spasm in the neck. Differentiation of heart disease from
symptoms associated with C6-C7 dysfunction is made on
the basis of muscle weakness, fasciculations, and sensory or
reflex changes. Differentiation of these two pathologies may
be difficult when true angina and pseudoangina coexist in
the same patient.39
Cervical spinal disease may manifest in the form of eye,
ear, and throat symptoms (see Fig. 39-4). Eye and ear symp-
toms may arise from irritation of the plexus surrounding the
vertebral and internal carotid arteries. Eye symptoms can
manifest with blurring of vision relieved by changing neck
position, increased tearing, orbital and retro-orbital pain,
and descriptions of eyes being “pulled backward” or “pushed
forward.” Altered equilibrium with associated gait distur-
bances may result from irritation of the surrounding sym-
pathetic plexus or from vertebral insufficiency. Hearing can
be affected with tinnitus and altered auditory acuity. Throat
symptoms, including dysphagia, may be related to ante-
rior vertebral osteophytes causing direct compression and
cranial nerve and sympathetic nerve communications.
Symptoms of dyspnea, cardiac arrhythmia, and drop
attacks may have a cervical spinal origin. Dyspnea can be
related to a deficit in the C3-C5 innervation of the dia-
phragm. Palpitations and tachycardia secondary to cervical
spine pathology can be differentiated from other causes by
the fact that these symptoms are associated with unusual
positions or hyperextension of the neck. This hyperex-
tension is caused by irritation of C4 innervation of the
diaphragm and pericardium or by irritation of the cardiac
sympathetic nerve supply. Drop attacks suggest posterior cir-
culation insufficiency.
Patients with myelopathy, or spinal cord compression,
initially present with subtle complaints of hand clumsi-
ness or difficulty with balance. Patients report worsening
handwriting or difficulty buttoning buttons. Patients also
may have nausea and emesis caused by equilibrium dysfunc-
tion. Paresthesias and dysesthesias may be present, often
involving bilateral upper extremities and not following a
dermatomal distribution. This is often mistaken for periph-
eral neuropathy or carpal tunnel syndrome, but should be
considered when bilateral extremity symptoms are present.
As the disease progresses over time, more advanced mani-
festations include wasting of hand intrinsics and bowel and
bladder dysfunction.
Neck pain may manifest concomitant with systemic
disease with varied symptoms requiring further investiga-
tion. Examples include inflammatory arthritides, infection,
tumor, multiple sclerosis, subacute combined degeneration,
or syrinx. Inflammatory arthritides often manifest with
morning stiffness, polyarticular involvement, rigidity, or
cutaneous manifestations. Fever, weight loss, or night pain
points to an infectious or neoplastic etiology. Pancoast’s
tumor is a neoplastic process of the apical portion of the
lung that can cause a mass effect on the caudad cervical
nerve roots. Pancoast’s tumor always should be considered
in an individual with radicular symptoms and a history of
smoking, with workup including a chest x-ray. Idiopathic
brachial plexus neuritis, formerly known as Parsonage-
Turner syndrome, is caused by viral infection of the brachial
plexus manifesting with severe arm pain involving multiple
nerve roots. When the acute phase resolves, patients are
left with variable deficits. Subacute combined degeneration
from vitamin B12 deficiency is a consideration when there is
greater sensory deficit in the lower extremities.
CLINICAL EXAMINATION
A careful clinical examination provides additional focus to
the differential diagnosis. The clinical examination helps
to identify the origin of neck pain based on the anatomy,
physiology, nature, and distribution of pain. The clinical
examination begins with broadly observing the patient’s
gait and head and neck posture. Further palpation, range of
motion testing, and neurologic signs, including motor signs,
reflexes, sensory signs, autonomic signs, and articular signs,
are assessed (Table 39-4).
Careful palpation with knowledge of key anatomic bony
and soft tissue landmarks in the cervical spine may localize
the lesion to a particular cervical level and location. Ante-
riorly or anterolaterally, the transverse process of C1 is pal-
pated between the angle of the jaw and the styloid process.
C3 is identified by palpation of the hyoid bone. C4-C5 is
at the level of the thyroid cartilage, and C6 is at the level
of the cricoid ring and the carotid tubercle. In the process
of examining the neck, the clinician also notes the sagittal
balance with retention or loss of normal cervical lordosis.
Posteriorly and posterolaterally, the occiput, inion, superior
nuchal line, mastoid processes, and spinous processes of C2
and C7-T1 are palpable.
Soft tissues around the anterior and posterior triangles of
the neck, occipital region, and posterior paraspinal muscles
are examined. Borders of the anterior triangle are the ster-
nocleidomastoid muscle, mandible, and suprasternal notch.
The sternocleidomastoid muscle is involved with whiplash
injuries causing abrupt hyperextension of the neck. The
muscle may be tender to palpation, or the patient may be
splinting the neck with the head turned away from the
injured muscle. This posturing of the neck is termed torticollis,
and the clinician should remember that the head is turned
away from the side of the involved sternocleidomastoid. The
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN 579

Temporo-
mandibular
joint
Hiatal hernia Temporo-
with complications mandibular joint
Chondro- Cystic duct stone
sternal Gallbladder;
Pancreatic disease common duct
joint
Esophageal stone
Cardiac hernia Lesions of
disease Gastric ulcer mediastinum
Gallbladder and lung
disease Perforated
Hiatal hernia peptic ulcer
Costo- Duodenal ulcer involving
chondral with or without pancreas
junction perforation Duodenal ulcer
separation Gastric ulcer
Head of pancreas
Tail of pancreas Gallbladder
Gallbladder Pancreas
A B

Ophthalmic
V C2 V
C2 V
Maxillary V
V V
C2 C3
Mandibular C2,C3
V C2,3 C4 C3 C4
T1
C5 C5
C3 C6 C3 C6
C4 T1
C6 T2 T2 T2 T2
T3 T3 T3 T3 C5
T4 T4 T4 T4
T5 T5 T5 T2
C5 T5 C5
T2
C6
T1 T1
T1
C7 C7 C7

C6 C6 C6
C8 C8
C8
C7

C
Figure 39-5  Patterns of reflex-referred pain from visceral and somatic structures. A, Anterior distribution. B, Posterior distribution. C, Dermatome
distribution of nerve fibers from C1 through T5, carrying senses of pain, heat, cold, vibration, and touch to the head, neck, arm, hand, and thoracic area.
The sclerotomes and myotomes are similar, but with some overlap. Pain arising from structures deep to the deep fascia (myotome and sclerotome) do
not precisely follow the dermatome distribution. (A and B from Nakano KK: Neck pain. In Ruddy S, Harris ED Jr, Sledge CB [eds]: Kelley’s Textbook of Rheuma-
tology, 6th ed. Philadelphia, Saunders, 2001, p 463; C from Nakano KK: Neck pain. In Ruddy S, Harris ED Jr, Sledge CB [eds]: Kelley’s Textbook of Rheumatology,
6th ed. Philadelphia, Saunders, 2001, p 461.)

posterior triangle borders include the sternocleidomastoid
muscle, the trapezius muscle (inion to T12), and the clavicle.
Flexion injuries may traumatize the trapezius muscle. The occip-
ital region and paraspinal muscles from the inion to C7-T1
should be palpated carefully. Midline cervical tenderness is
more concerning for ligament injury, whereas paraspinal mus-
cle tenderness is typically a more benign process.40 The greater
occipital nerves are located lateral to the inion and may be
involved in traumatic inflammation associated with flexion
or extension injuries resulting in suboccipital headaches. Skin
markings or visible trauma should be noted at this time.
Range of motion examination may reveal pain or limi-
tations in flexion-extension, lateral bending, and rotation.
Flexion limitation may be assessed by the examiner placing
 580 DEVIN  |  Neck Pain

Table 39-4  Nerves and Tests of Principal Muscles

Nerve Nerve Roots Muscle Test
Accessory Spinal Trapezius Elevation of shoulders
Abduction of scapula
Spinal Stemocleidomastoid Tilting of head to same side with rotation to opposite
side
Brachial plexus Pectoralis major
C5, C6 Clavicular part Adduction of arm
C7, C8, T1 Sternocostal part Adduction, forward depression of arm
C5, C6, C7 Serratus anterior Fixation of scapula during forward thrusting of the arm
C4, C5 Rhomboid Elevation and fixation of scapula
C4, C5, C6 Supraspinatus Initiate abduction of arm
(C4), C5, C6 Infraspinatus External rotation of arm
C6, C7, C8 Latissimus dorsi Adduction of horizontal, externally rotated arm,
­coughing
Axillary C5, C6 Deltoid Lateral and forward elevation of arm to horizontal
Musculocutaneous C5, C6 Biceps Flexion of supinated forearm
Brachialis
Radial C6, C7, C8 Triceps Extension of forearm
C5, C6 Brachioradialis Flexion of semiprone forearm
C6, C7 Extensor carpi radialis longus Extension of wrist to radial side
Posterior interosseous C5, C6 Supinator Supination of extended forearm
C7, C8 Extensor digitorum Extension of proximal phalanges
C7, C8 Extensor carpi ulnaris Extension of wrist to ulnar side
C7, C8 Extensor indicis Extension of proximal phalanx of index finger
C7, C8 Abductor pollicis longus Abduction of first metacarpal in plane at right angle
to palm
C7, C8 Extensor pollicis longus Extension of first interphalangeal joint
C7, C8 Extensor pollicis brevis Extension of first metacarpophalangeal joint
Median C6, C7 Pronator teres Pronation of extended forearm
C6, C7 Flexor carpi radialis Flexion of wrist to radial side
C7, C8, T1 Flexor digitorum superficialis Flexion of middle phalanges
C8, T1 Flexor digitorum profundus Flexion of terminal phalanges, index and middle fingers
(lateral part)
C8, T1 Flexor pollicis longus Flexion of distal phalanx, thumb
(anterior interosseous nerve)
C8, T1 Abductor pollicis brevis Abduction of first metacarpal in plane at right angle
to palm
C8, T1 Flexor pollicis brevis Flexion of proximal phalanx, thumb
C8, T1 Opponens pollicis Opposition of thumb against fifth finger
C8, T1 First and second lumbricals Extension of middle phalanges while proximal phalan-
ges are fixed in extension
Ulnar C7, C8 Flexor carpi ulnaris Observation of tendons while testing abductor digiti
minimi
C8, T1 Flexor digitorum profundus Flexion of distal phalanges of ring and little fingers
(medial part)
C8, T1 Hypothenar muscles Abduction and opposition of little finger
C8, T1 Third and fourth lumbricals Extension of middle phalanges while proximal phalan-
ges are fixed in extension
C8, T1 Adductor pollicis Adduction of thumb against palmar surface of index
finger
C8, T1 Flexor pollicis brevis Flexion of proximal phalanx, thumb
C8, T1 Interossei Abduction and adduction of fingers
From Nakano KK: Neck pain. In Kelly WN, Harris ED Jr, Ruddy S, et al (eds): Textbook of Rheumatology, 5th ed. Philadelphia, WB Saunders, 1997, p 399.

fingers between the patient’s chin and sternum at maximal
flexion with 50% of the motion occurring at the occiput-C1
joint and the remaining 50% distributed over C2-C7. If the
patient is unable to place the chin on the chest, the inter-
val should be measured. One finger width shows a limita-
tion of 10 degrees; three finger widths indicate a 30-degree
limitation in flexion. On extension, the distance between
the base of the occiput and spinous process of T1 should be
measured. Lateral flexion should allow the ear to touch the
shoulder with motion being shared across all cervical verte-
brae. On rotation, the chin should touch the shoulder with
50% of rotation occurring at C1-C2 and the remaining 50%
distributed in the subaxial spine between C3-C7. There
is a natural decrease in range of motion with age, even in
healthy individuals.41
Range of motion testing is dangerous if the patient is
unable to protect the cervical spine because of sedation,
analgesics, or distracting injuries. After ensuring the patient
is able to comply safely with range of motion testing, active
range of motion, passive range of motion, and motion
against resistance are performed. Range of motion tests the
ligaments, capsules, and fascia, and this range of motion is
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
reduced in the presence of cervical spinal muscular spasm
or pain. Patients with degenerative changes of the cervical
spine have pain with decreased range of motion of the cervi-
cal spine. The most common findings secondary to changes
in the cervical spine articulations are (in order): restriction
of movement with or without pain, pain on movement, and
local tenderness. Lateral flexion is the earliest and most
impaired movement in degenerative diseases with rotation
first impaired in rheumatoid arthritis owing to involvement
of the odontoid peg. A uniformly stiff neck may be caused by
diffuse idiopathic skeletal hyperostosis, which is present in a
quarter of elderly patients, but also may be due to ankylosing
spondylitis or recent trauma to the neck.42 If articular signs
are found, the examiner must evaluate the entire vertebral
column and peripheral joints for evidence of further ­arthritis
and search for extra-articular manifestations.
Motion against resistance testing is performed after active
and passive range of motion is established. Firm resistance to
movement is provided by the examiner to assess the origin
and insertion of tendons and ligaments and motor strength.
Muscle groups tested include the flexors and extensors of
the neck. In testing flexor muscles, a hand is placed between
the forehead and chest. The primary flexor is the sterno-
cleidomastoid muscle with secondary flexors being the three
scalene muscles and small prevertebral muscles. Extensors
are tested by placing a hand on the shoulder and head for
resistance. Primary extensors include the paravertebral
extensor mass, splenius, semispinalis capitis, and trapezius.
Secondary flexors include the small intrinsic muscles of
the neck. Rotators are examined by placing a hand on the
shoulder and chin for resistance. The sternocleidomastoid
muscle and the intrinsic muscles of the neck provide rota-
tional force. Motion against resistance testing should include
active maximal effort strength testing to the extremes of
flexion, extension, and rotation to assess muscle strength.
Causes of decreased range of motion of the cervical spine
include joint locking and bony ankylosis from degenerative
changes or arthritides, fibrous contractures, muscle spasm,
splinting over painful joints, and nerve root or spinal cord
­compression or irritation. Decreased range of motion in
the presence of pain or weakness warrants further inves­
tigation.
Sensation testing for light touch, pin prick, temperature,
and proprioception should be performed. These tests are
subjective, and both upper extremities should be compared
to assess differences in sensation. Comparing an unaffected
area, such as the face, with the area of decreased sensa-
tion also can be helpful. Pin prick can be performed using
a sterile needle and temperature using an alcohol pad to
assess function of the spinothalamic tract that traverses the
anterolateral aspect of the spinal cord. Light touch and pro-
prioception assess function of the posterior spinal column.
Dermatomally, C1 and C2 innervate the occiput region;
C3 and C4, the nape of the neck; C5, the deltoid region;
C6, the radial aspect of the forearm; C7, the long finger; C8,
the ulnar border of the hand; and T1, the medial border of
the arm. T2-T12 provide innervation to the chest and abdo-
men, with T4 being at the nipple line, T10 at the umbilicus,
and T12 at the inguinal ligament (see Fig. 39-5). The lower
extremities have a unique dermatomal map that correlates
with embryologic development whereby the limb starts in a
supinated position and pronates with longitudinal growth.
581
Table 39-5  Strength Grading in Motor
Examination
0 No function with total paralysis
1 Trace movement with palpable or visible contraction
2 Full range of joint motion with gravity eliminated
3 Active movement against gravity
4 Active movement against slight resistance
5 Normal strength
Each segment contributes innervation from superolateral to
inferomedial, mirroring this twisting motion that occurred
during development.
L1 and L2 contribute innervation below the inguinal
ligament to the medial thigh, L3 provides sensation to the
anterior midthigh, L4 provides sensation to the knee region
and medial calf, L5 provides sensation to the lateral calf
and first web space, and S1 provides sensation to the lateral
aspect and sole of foot. The perineum region receives con-
tributions from S3-S5. Perineal sensation and rectal tone
are important to examine because an abnormality may indi-
cate compression of the spinal cord or cauda equina, requir-
ing immediate surgical intervention. Isolating the level of
pathology can be challenging sometimes. Nerve roots with
proximal compression are more susceptible to distal com-
pression in a phenomenon termed “double crush.” The
cervical spine should always be considered as the potential
etiology in patients who present with symptoms of carpal
or cubital tunnel syndrome and peripheral neuropathy.
Ancillary imaging and nerve conduction studies can help
elucidate the etiology.
After palpation, range of motion testing, and assessment
of sensation, muscle strength testing is continued for localiza-
tion of any positive findings. Lower motor neuron disease is
indicated by weakness, hypotonia, and fasciculations. Upper
motor neuron disease is indicated by spasticity. Motor ­function
should be graded using the standard 0-to-5 nomenclature,
grade 0 having no function, 1 having trace, 2 having full range
of joint motion with gravity eliminated, 3 having antigrav-
ity function, 4 having function against slight resistance, and
5 having normal strength against resistance (Table 39-5). A
cursory examination can be performed assessing C5 with elbow
flexion, C6 with wrist extension, C7 with elbow extensors or
wrist flexion, C8 with finger flexion of the middle finger, and
T1 with finger abduction of the fifth finger. If weakness is pres-
ent, a more focused examination should be performed looking
at other muscles innervated by the same nerve root.
Deep tendon stretch reflexes should be performed and
graded 0 to 3 with 0 being no response, 1 being ­hyporeflexive,
2 being normal, and 3 being hyperreflexive. C5 is tested by
striking the biceps tendon; C6, brachioradialis; C7, triceps;
L4, patellar tendon; and S1, Achilles tendon. To facilitate
reflex testing, it may be helpful to use muscle loading or
Jendrassik’s maneuver (performed by having the patient flex
both sets of fingers into a hooklike form, interlocking the
hands, and pulling apart). This maneuver creates a diversion
to help relax the patient and assess lower extremity reflexes.
If difficulty with reflex testing persists, the clinician should
ensure that no peripheral neuropathy is present.
In addition to deep tendon reflex testing, the abdominal
reflexes, Babinski’s test, and bulbocavernosus test should
 582 DEVIN  |  Neck Pain
be assessed. The abdominal reflex is performed by divid-
ing the umbilicus and immediate surrounding area into
four quadrants, with the umbilicus being the center. The
umbilicus should deviate in the direction it is being stimu-
lated with light scratching. Absence of a reflex may signify
an upper motor neuron lesion, whereas asymmetrical loss
of the reflex may indicate a localized lower motor neuron
lesion. Babinski’s test is performed by stroking the lateral
plantar aspect of the foot with a pathologic response indi-
cated by an up-going great toe indicating an upper motor
neuron lesion. The bulbocavernosus reflex is an important
barometer in the setting of spinal shock in patients pre-
senting with paralysis. This reflex typically occurs within
24 hours of injury owing to traumatic swelling around the
neural tissues. In the presence of spinal shock, it cannot be
predicted whether a lesion is complete or incomplete, and
it cannot be determined whether decompression should be
performed at the time of surgical intervention. The bulbo-
cavernosus reflex is performed by pressure stimulation of
the penis or clitoris or by placing traction on an indwelling
Foley catheter while the examiner’s other finger is in the
rectum. Presence of the reflex is indicated by concurrent
contraction of the anal sphincter, and this indicates an
incomplete paralysis. The bulbocavernosus reflex is unreli-
able with injuries around the conus medullaris (i.e., T12-L2)
because the afferent nerve fibers synapse within the conus
medullaris.
Provocative tests that can be helpful in confirming com-
pressive extradural monoradiculopathy include Spurling’s
test, the arm abduction test, and the axial compression and
traction test. All of these tests are meant to change the
diameter of the neural foramen, increasing or decreasing
the symptoms. Spurling’s test is performed by the patient
extending his or her neck and rotating toward the side of
pain. The test is positive if the radicular pain worsens in
this position and indicates foraminal stenosis with poten-
tial compression of a nerve root. The arm abduction sign is
positive if the patient’s pain is relieved by placing the hand
on the affected side, on top of the head.43 The axial com-
pression test is performed by pressing on top of the patient’s
head with the neck in neutral position with a positive result
if the radicular symptoms are exacerbated by this maneuver
and relieved by placing traction on the head and opening
up the foramina.
Provocative tests that are helpful in diagnosing myelop-
athy include Hoffmann’s sign, finger escape sign, abnor-
mal grip-release test, and Lhermitte’s sign. Hoffmann’s
sign is obtained by holding the middle finger extended
and suddenly extending the distal interphalangeal joint,
resulting in flexion of the index finger and thumb if
pathologic. A positive finger escape sign occurs when an
individual cannot hold all of the fingers in an adducted
and extended position without the ulnar two digits fall-
ing into flexion and abduction over time. The grip-release
test is an inability to open and close a fist rapidly because
of weakness and spasticity of the hand. Lhermitte’s sign
evaluates for changes in the spinal cord itself and occurs
when the patient’s neck is forcefully flexed resulting in
electric-like shocks that travel down the arms and legs.
This indicates changes in the white matter of the spinal
cord and may be secondary to cervical myelopathy or
multiple sclerosis.
DIAGNOSTIC EVALUATION
After completing the history and clinical examination, indi­
cated imaging studies, neurophysiologic procedures, and
laboratory studies may aid in completing the differential
diagnosis and building a treatment plan for the patient’s
neck pain. Imaging modalities useful in the diagnosis of neck
pain include cervical radiographs, computed tomography
(CT), nuclear bone scans, and magnetic resonance imag-
ing (MRI). Cervical radiographs often show degenerative
changes in asymptomatic individuals by age 60.44 In the
absence of trauma, constitutional symptoms, or worsening
neurologic deficit, 4 weeks of conservative care is indicated
before obtaining radiographs.45 Complete static cervical
spine imaging includes anteroposterior, lateral, oblique,
and open mouth odontoid radiographs with attention to
sagittal alignment, which has a normal lordosis of 21 ± 13
degrees. Flexion and extension views allow for evaluation
of the dynamic properties of the cervical spine, but should
not be performed in the acute trauma setting because neck
muscle spasm can result in a false-negative test. Dynamic
radiographs also should be used for screening patients with
rheumatoid arthritis before endotracheal intubation, given
the risk of cervical instability at C1-C2. One study showed
that 61% of patients with rheumatoid arthritis had evidence
of instability, defined by at least 3 mm of atlantoaxial sub-
luxation on preoperative screening x-rays.46
In the presence of significant degenerative changes
and end plate osteophytes, CT myelography can be help-
ful in characterizing the bony involvement further. CT
­myelography should be thought of as a complementary test to
MRI.47 It should be used as the primary test to evaluate neu-
ral involvement only when MRI is contraindicated because
MRI is superior for evaluating spinal cord changes, such as
syringomyelia, myelomalacia, or neoplasm.48 MRI is indi-
cated for progressive neurologic deficit, disabling weakness,
and long tract signs and is recommended for patients with
persistent cervical radiculopathy after 6 weeks of conserva-
tive care.45 The addition of gadolinium contrast enhance-
ment is helpful in evaluating infection and neoplasm and
in differentiating scar from recurrent disk herniation in
patients who have undergone previous spinal surgery. MRI
results must be correlated with physical examination find-
ings given that asymptomatic volunteers have been found
to have abnormal cervical spine MRI.49 If the MRI findings
do not correlate with the history and physical examina-
tion findings, further studies may be required, such as CT
myelography. Nuclear bone scanning techniques, includ-
ing single photon emission CT, have been used to identify
and characterize acuity in occult fractures, periosteal injury,
and post-traumatic osteoarthritis in the absence of positive
radiograph findings.50
Neurophysiologic procedures are indicated when the
clinical examination and imaging studies fail to correlate,
or when there is conflicting information. Electromyogra-
phy, nerve conduction studies, and somatosensory evoked
responses help differentiate cervical spine disorders from
peripheral nerve entrapment syndromes and help in
­differentiating intrinsic joint pathology from a radicu-
lopathy.
Neck pain is typically secondary to mechanical causes,
and laboratory studies generally are not helpful in its
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
d­ iagnosis. Laboratory studies can be crucial, however, in
ruling out infection, neoplasm, and systemic arthritides
and should be obtained in patients with constitutional
symptoms and for suspicion of systemic arthritides. Eryth-
rocyte sedimentation rate may be elevated greater than
100 mm/hr with infection and neoplasm; less dramatic ele-
vations are seen with rheumatoid arthritis and after surgery.51
C-reactive protein peaks by day 2 of an acute inciting event
and returns to normal within 3 to 7 days of removing the
insult.52 Complete blood count with differential and lum-
bar puncture also can be helpful if meningitis is a concern.
If a systemic arthritis is suspected, the respective tests can
be ordered as indicated.
DIFFERENTIAL DIAGNOSIS
AND TREATMENT
Using the history, physical examination, and diagnostic
studies, it is helpful to divide the differential diagnosis into
benign axial neck pain, radiculopathy, myelopathy, infec-
tion, neoplasm, systemic arthritides, and referred pain. Most
axial neck pain is self-limiting and resolves with appropri-
ate conservative care.53 Patients with isolated neck pain
and a negative radiographic and laboratory workup are
best treated with a multimodal approach. During the acute
phase, patients can be treated with a soft collar to reduce
inflammation, but this should not be worn for more than
2 weeks so as to avoid deconditioning. Multimodal treat-
ments have been found to be the most effective at treating
axial neck pain, including proprioceptive training, exer-
cises with resisted strengthening, muscle relaxers during the
acute period, and nonsteroidal anti-inflammatory drugs.54-59
There is inconclusive evidence regarding the effectiveness
of radiofrequency denervation of facet joints, acupuncture,
transcutaneous electric nerve stimulation unit, iontopho-
resis, electromyography biofeedback, local injections, or
mechanical traction for treatment of axial neck pain.60-63 If
there is a history of trauma and radiographic findings, a rigid
cervical collar should remain in place with strict spine pre-
cautions and appropriate referral. A patient with acute neck
pain in the setting of trauma with negative radiographic
findings and no neurologic deficits should remain in a rigid
cervical collar with follow-up in 2 weeks for cervical spine
clearance.
In addition to degenerative changes to the cervical spine,
trauma, and acute disk herniations, there are more insidious
causes of neck pain, including schwannomas, Pancoast’s
tumor, brachial plexus neuritis, and reflex sympathetic dys-
trophy. Schwannomas, if intradural, may involve a sensory
nerve root causing dermatomal pain in addition to causing a
myelopathy or radiculopathy from compression. Pancoast’s
tumor involving the lung apex may cause caudal cervical
nerve root and sympathetic changes in addition to nerve
root or brachial plexus compression. Brachial plexus neuri-
tis (Parsonage-Turner syndrome) is of viral origin and causes
severe arm pain followed by weakness and then pain reso-
lution followed by return of arm strength. This condition
may progress to reflex sympathetic dystrophy in a few cases
associated with diffuse burning pain along with autonomic
changes, including discoloration of the skin.
Axial neck pain with associated radiculopathy also has
a fairly benign course with 75% of patients having only
583
one recurrence or mild symptoms at 19-year follow-up with
conservative treatment.64 A soft collar can be used to aid in
treating the associated muscle spasm, but this should not
be worn for more than 2 weeks. Cervical traction may be
prescribed when the acute muscle spasms subside, with a
typical regimen of 8 to 10 lb for 15- to 20-minute sessions
with the device at 20 to 25 degrees of flexion. Traction has
shown only short-term relief of radicular symptoms, how-
ever.65 Epidural steroids have been shown to be extremely
effective at treating lumbar radiculopathy, allowing 50% of
patients to avoid surgery, but a similar study has not been
undertaken in the cervical spine.66 Atlantoaxial facet joint
osteoarthritis may be treated successfully with a facet block
and nonsteroidal anti-inflammatory drugs. If conservative
therapy with nonsteroidal anti-inflammatory drugs fails, a
fusion may be indicated.
Follow-up and vigilance are in order because a progres-
sive neurologic deficit, segmental instability, and persistent
radicular symptoms for at least 6 weeks may be indications
for surgical intervention. In a prospective randomized study
comparing surgery, physical therapy, or cervical collar use
for long-standing cervical radiculopathy, no difference
was found between the three groups at 12 months.67 Cer-
vical myelopathy with very mild deficits can be followed
closely; however, the natural course of the illness is long
periods of stability with episodes of deterioration. Defini-
tive indications for surgery include presence of myelopathy
for 6 months or longer, progression of signs or symptoms,
difficulty walking, or change in bowel or bladder function.
Surgery is directed at decompressing the spinal cord and
preventing further deterioration, rather than improving
neurologic deficits.
Systemic arthritides, infection, and tumors can affect the
cervical spine with variable neurologic and constitutional
symptoms. Rheumatoid arthritis typically causes atlantoaxial
subluxation, atlantoaxial impaction, and subaxial sublux-
ation (Table 39-6). Surgical stabilization is indicated with
progressive neurologic deficit, persistent axial neck pain with
Table 39-6  Rheumatologic Disorders Causing
Neck Pain
Rheumatoid arthritis
Without disease of the C1-C2 joint
With structural cervical abnormalities
C1-C2 subluxation
C1-C2 facet involvement
Spondyloarthropathies
Ankylosing spondylitis
Reiter’s syndrome and reactive arthritis
Psoriatic arthritis
Enteropathic arthritis
Polymyalgia rheumatica
Osteoarthritis
Fibromyalgia
Nonspecific musculoskeletal pain
Miscellaneous spondyloarthropathies
Whipple’s disease
Behçet’s disease
Paget’s disease
Acromegaly
Ossification of the posterior longitudinal ligament
Diffuse idiopathic skeletal hyperostosis
 584 DEVIN  |  Neck Pain
radiographic evidence of instability, canal diameter of less
than or equal to 14 mm, and odontoid migration of greater
than or equal to 5 mm above McGregor’s line. In the setting
of ­atlantoaxial subluxation or subaxial subluxation, the in­
vol­ved levels are fused posteriorly, and atlantoaxial impaction
should be treated with occipitocervical fusion.9 Rheumatoid
arthritis patients with evidence of instability show radio-
graphic progression over time, but this correlates poorly with
neurologic outcome.68 Regardless, these patients require close
follow-up because when myelopathy develops, most patients
die within 1 year.69
Ankylosing spondylitis commonly affects the cervical
spine resulting in a kyphotic deformity over time with al­
te­red biomechanics, which has implications in the setting
of trauma. Even minor falls can create significant instabil-
ity owing to the lever arm effect. The kyphotic deformity
can have significant functional implications because the
individual’s gaze moves toward the floor, making interaction
with the surrounding world difficult. Corrective osteotomies
are available, but there is risk of neurologic deficit and intra-
operative bleeding. It is hoped that earlier diagnosis through
the use of MRI and treatment with tumor necrosis factor–
blocking agents will help make cervical deformity a thing
of the past.70
Infection and neoplastic processes can cause destruction
with mechanical or nonmechanical neck pain, constitu-
tional symptoms, and variable neurologic deficit. The goal
is similar with eradication of the infection or tumor, decom-
pression if a neurologic deficit exists, and stabilization of the
spinal column. A careful history, physical examination, and
ancillary studies help one arrive at a fairly narrow differen-
tial diagnosis. In the absence of spinal instability, neurologic
deficit, infectious process, or neoplastic process, the patient
may benefit from conservative treatment with expectant
recovery with “tincture of time.”
REFERENCES
  1. Nachemson AL: Disc pressure measurements. Spine 6:93-97, 1981.
  2. Makela M, Heliövaara M, Sievers K, et al: Prevalence, determinants,
and consequences of chronic neck pain in Finland. Am J Epidemiol
134:1356-1367, 1991.
  3. Andersson HI, Ejlertsson G, Leden I, et al: Chronic pain in a
geographically defined general population: Studies of differences
in age, gender, social class, and pain localization. Clin J Pain 9:
174-182, 1993.
  4. Hansson EK, Hansson TH: The costs for persons sick-listed more than
one month because of low back or neck problems: A two-year prospec-
tive study of Swedish patients. Eur Spine J 14:337-345, 2005.
  5. Freeman MD, Craft AC, Rossignol AM, et al: A review and meth-
odologic critique of the literature refuting whiplash syndrome. Spine
24:86-96, 1999.
  6. Honeyman PT, Jacobs EA: Effects of culture on back pain in Austra-
lian Aboriginals. Spine 21:841-843, 1996.
  7. Klekamp J, McCarty E, Spengler DM: Results of elective lumbar dis-
cectomy for patients involved in the workers’ compensation system.
J Spinal Disord 11:277-282, 1998.
  8. Daniels DL, Williams AL, Haughton VM: Computed tomography of
the articulations and ligaments at the occipito-atlantoaxial region.
Radiology 146:709-716, 1983.
  9. Kim DH, Hilibrand AS: Rheumatoid arthritis in the cervical spine.
J Am Acad Orthop Surg 13:463-474, 2005.
10. Bogduk N, Marsland A: The cervical zygapophyseal joints as a
source of neck pain. Spine 13:610-617, 1988.
11. Nachemson AL, Waddell G, Norhlund AI: Neck and Back Pain:
The Scientific Evidence of Causes, Diagnosis, and Treatment. Phil-
adelphia, Lippincott Williams & Wilkins, 2000, pp 172-173.
12. Dwyer A, Aprill C, Bogduk N: Cervical zygapophyseal joint pain pat-
terns, I: A study in normal volunteers. Spine 15:453-457, 1990.
13. Bogduk N, Windsor M, Inglis A: The innervation of the cervical
intervetebral discs. Spine 13:2-8, 1988.
14. Aprill C, Dwyer A, Bogduck N: Cervical zygapophyseal joint pain
patterns, II: A clinical evaluation. Spine 15:458-461, 1990.
15. Cavanaugh JM, Ying L, Chen C, et al: Pain generation in lumbar and
cervical facet joints. J Bone Joint Surg 88A:63-67, 2006.
16. Dreyfuss P, Michaelsen M, Fletcher D: Atlantooccipital and lateral
atlanto-axial joint pain patterns. Spine 1125-1131, 1994.
17. Kuklo TR, Riew KD, Orchowski JR, et al: Management of recalci-
trant osteoarthritis of the atlanto-axial joint. Orthopedics 29:633-638,
2006.
18. Bogduk N, Aprill C: On the nature of neck pain, discography and
cervical zygapophysial joint pain. Pain 54:213-217, 1993.
19. Schellhas KP, Smith MD, Gundry CR, et al: Cervical discogenic
pain: Prospective correlation of magnetic resonance imaging and
discography in asymptomatic subjects and pain sufferers. Spine
21:300-312, 1996.
20. Grubb SA, Kelly CK, Cervical discography: Clinical implications
from 12 years of experience. Spine 25:1382-1389, 2000.
21. Bengtsson A, Henriksson KG, Larsson J: Reduced high-energy phos-
phate levels in the painful muscles of patients with primary fibromyal-
gia. Arthritis Rheumatol 29:817-821, 1986.
22. Dryer SJ, Boden S: Laboratory evaluation in neck pain. Physical Med
Rehabil Clin N Am 14:589-604, 2003.
23. Crockard HA: Surgical management of cervical rheumatoid prob-
lems. Spine 20:2584-2590, 1995.
24. Cooper RG, Freemont AS, Hoyland JA, et al: Herniated interverte-
bral disc-associated periradicular fibrosis and vascular abnormalities
occur ­without inflammatory cell infiltration. Spine 20:591-598, 1995.
25. Chabot MC, Montgomery DM: The pathophysiology of axial and
radicular neck pain. Semin Spine Surg 7:2-8, 1995.
26. An HS, Riley L (eds): An Atlas of Surgery of the Spine: Anterior
Cervical Spine Procedures. London, Martin Dunitz Ltd, 1998.
27. Truumees E, Herkowitz H: Cervical spondylotic myelopathy and
radiculopathy. Instr Course Lect 29:339-360, 2000.
28. Houser OW, Onofrio B, Miller GM, et al: Cervical spondylotic steno-
sis and myelopathy: Evaluation with computed tomographic myelog-
raphy. Mayo Clin Proc 69:557-563, 1994.
29. Ono K, Otai H, Tada K, et al: Cervical myelopathy secondary to
multiple spondylotic protrusions: A clinicopathologic study. Spine 2:
109-125, 1977.
30. Breig A, Turnbull I, Hassler O: Effects of mechanical stresses on the
spinal cord in cervical spondylosis. J Neurosurg 25:45-56, 1966.
31. Mihara H, Ohnari K, Hachiya M, et al: Cervical myelopathy caused
by C3-C4 spondylosis in elderly patients: A radiographic analysis of
pathogenesis. Spine 25:796-800, 2000.
32. Ferguson RJ, Caplan LR: Cervical spondylotic myelopathy. Neurol
Clin 3:373-382, 1985.
33. Gooding MR, Wilson CB, Hoff ST: Experimental cervical myelopa-
thy: Effect of ischemia and compression of the canine cervical spinal
cord. J Neurosurg 43:9-17, 1975.
34. Nurick S: The pathogenesis of the spinal cord disorder associated
with cervical spondylosis. Brain 95:87-100, 1972.
35. Sampath P, Bendebba M, Davis JD, et al: Outcome of patients treated
for cervical myelopathy: A prospective, multicenter study with inde-
pendent clinical review. Spine 25:670-676, 2000.
36. Romanelli P, Esposito V: The functional anatomy of neuropathic
pain. Neurosurg Clin N Am 15:257-268, 2004.
37. Mackley RJ: Role of trigger points in the management of head, neck,
and face pain. Funct Orthod 7:4-14, 1990.
38. Henderson CM, Hennessy RG, Shuey HM, et al: Posterior-lateral
foraminotomy as an exclusive operative technique for cervical radi­
culopathy: A review of 846 consecutively operated cases. Neuro-
surgery 13:504-512, 1983.
39. Frobert O, Fossgreen J, Sondergaard-Peterson J, et al: Musculo-skeletal
pathology in patients with angina pectoris and normal coronary angio-
grams. J Intern Med 245:237-246, 1999.
40. Hoffman JR, Mower WR, Wolfson AB, et al: National Emergency
X-Radiography Utilization Study Group: Validity of a set of clinical
criteria to rule out injury to the cervical spine in patients with blunt
trauma. N Engl J Med 343:94-99, 2000.
41. Sforza C, Grassi G, Fragnito N, et al: Three-dimensional analysis of
active head and cervical spine range of motion: Effect of age in healthy
male subjects. Clin Biomech (Bristol, Avon) 17:611-614, 2002.
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
42. Weinfeld RM, Olson PN, Maki DD, et al: The prevalence of diffuse
idiopathic skeletal hyperostosis (DISH) in two large American Midwest
metropolitan hospital populations. Skeletal Radiol 26:222-225, 1997.
43. Davidson RI, Dunn EJ, Metzmaker JN: The shoulder abduction test
in the diagnosis of radicular pain in cervical extradural compressive
monoradiculopathies. Spine 6:441-446, 1981.
44. Gore DR, Sepic SB, Gardner GM: Roentgenographic findings of the
cervical spine in asymptomatic people. Spine 11:521-524, 1986.
45. Levine MJ, Albert T, Smith MD: Cervical radiculopathy: Diagnosis and
nonoperative management. J Am Acad Orthop Surg 4:305-316, 1996.
46. Collins DN, Barnes CL, Fitz Randolph RL: Cervical spine instabil-
ity in rheumatoid patients having total hip or knee arthroplasty. Clin
Orthop 272:127-135, 1991.
47. Modic MT, Masaryk T, Mulopulos GP, et al: Cervical radiculopathy:
Prospective evaluation with surface coil MR imaging, CT with metri-
zamide, and metrizamide myelography. Radiology 161:753-759, 1986.
48. Modic MT, Ross J, Masaryk TJ: Imaging of degenerative disease of the
cervical spine. Clin Orthop 239:109-120, 1989.
49. Boden SD, McCowin PR, Davis DO, Dina TS, et al: Abnormal
­magnetic-resonance scans of the cervical spine in asymptomatic
subjects. J Bone Joint Surg 72:1178-1184, 1990.
50. Seitz JP, Unguez CE, Corbus H, et al: SPECT of the cervical spine in
the evaluation of neck pain after trauma. Clin Nucl Med 20:667-673,
1995.
51. Waddell G: An approach to backache. Br J Hosp Med 28:187-190,
1982.
52. Kushner HL: Acute phase response. Clin Aspects Autoimmun 3:
20-30, 1989.
53. Gore DR, Sepic SB, Gardner GM, et al: Neck pain: A long-term
­follow-up of 205 patients. Spine 12:1-5, 1987.
54. Taimela S, Takala EP, Asklof T, et al: Active treatment of chronic
neck pain. Spine 25:1021-1027, 2000.
55. Waling K, Sundelin G, Ahlgren C, et al: Perceived pain before and
after three exercise programs: A controlled clinical trial of women
with work-related trapezius myalgia. Pain 85:201-207, 2000.
56. Bronfort G, Evans R, Nelson B, et al: A randomized clinical trial of
exercise and spinal manipulation for patients with chronic neck pain.
Spine 26:788-799, 2001.
585
57. Gross AR, Kay T, Hondras M, et al: Manual therapy for mechanical
neck disorders: A systematic review. Manual Ther 7:131-149, 2002.
58. Beebe FA, Barkin RL, Barkin S: A clinical and pharmacologic review
of skeletal muscle relaxants for musculoskeletal conditions. Am J Ther
12:151-171, 2005.
59. Kay TM, Gross A, Goldsmith C, et al: Exercises for mechanical neck
disorders. Cochrane Database Syst Rev 3:CD004250, 2005.
60. Irnich D, Behrens N, Molzen H, et al: Randomised trial of acupunc-
ture compared with conventional massage and “sham” laser acupunc-
ture for treatment of chronic neck pain. BMJ 322:1574-1578, 2001.
61. Niemisto L, Kalso E, Malmivaara A, et al: Radiofrequency denerva-
tion for neck and back pain: A systematic review of randomized con-
trolled trials. Cochrane Database Syst Rev 1:CD004058, 2003.
62. Dagenais S, Haldeman S, Wooley JR: Intraligamentous injection of
sclerosing solutions (prolotherapy) for spinal pain: A critical review of
the literature. Spine J 5:310-328, 2005.
63. Kroeling P, Gross A, Goldsmith CH: A Cochrane review of electro-
therapy for mechanical neck disorders. Spine 30:E641-E648, 2005.
64. Saal JS, Saal JA, Yurth EF: Nonoperative management of herniated
cervical intervertebral disc with radiculopathy. Spine 21:1877-1883,
1996.
65. Carette S, FM: Clinical practice: Cervical radiculopathy. N Engl J
Med 353:392-399, 2005.
66. Riew KD, Yuming Y, Gilula L: The effect of nerve-root injections on
the need for operative treatment of lumbar radicular pain: A prospec-
tive, randomized, controlled, double-blind study. J Bone Joint Surg
82:1589-1593, 2000.
67. Persson LC, Carlsson CA, Carlsson JY: Long-lasting cervical radicular
pain managed with surgery, physiotherapy, or cervical collar. Spine 22:
751-758, 1997.
68. Pellicci PM, Ranawat CS, Tsairis P, et al: A prospective study of the
­progression of rheumatoid arthritis of the cervical spine. J Bone Joint
Surg 63:342-350, 1981.
69. Marks JS, Sharp J: Rheumatoid cervical myelopathy. QJM 50:301-319,
1989.
70. Mansour M, Cheemu GS, Naquwa SM, et al: Ankylosing spondyli-
tis: A contemporary perspective on diagnosis and treatment. Semin
Arthritis Rheum 36:210-223, 2007.
 40
Key Points
Understand the anatomy and function of the shoulder.
Perform a thorough history and physical examination of the
shoulder.
Formulate a differential diagnosis of shoulder pain.
Know appropriate diagnostic tests to assist in diagnosis.
Develop an appropriate treatment plan.
Shoulder pain is one of the most common musculoskeletal
complaints that may require evaluation in the office set-
ting. Accurate diagnosis of shoulder pain is made difficult
by the unique anatomy and position of the shoulder as a
link between the upper extremity and thorax. One of the
most complex and mobile joints of the body, the shoulder
is traversed by muscle, tendon, and bone, and is surrounded
by major neurovascular structures, all of which may serve as
potential sources of local and referred pain.
Determining the source of shoulder pain is essential
in recommending the proper method of treatment. The
examining physician must be able to differentiate the
occurrence of shoulder pain caused by intrinsic, or local,
factors and extrinsic, or remote, factors, or a combination
of the two. Intrinsic factors originate from the shoulder
girdle and include glenohumeral and periarticular disor-
ders, whereas extrinsic factors occur outside of the shoulder
girdle with secondary referral pain to the shoulder (Table
40-1). Examples of extrinsic factors include left shoulder
pain as the initial presentation of coronary artery disease.
Hepatic or splenic disease also may initally manifest as
shoulder pain.
Accurate evaluation, diagnosis, and treatment require
a thorough understanding of shoulder anatomy, including
pain referral patterns. A complete and systematic physi-
cal examination is crucial to an accurate diagnosis. Dur-
ing the initial evaluation, care must be taken to discern
all possible causes of the shoulder pain. Final diagnosis
may require repeated office examinations and correlation
of diagnostic tests with symptoms and response to selec-
tive injections. Improvements in diagnostic tests, such as
magnetic resonance imaging (MRI), computed tomography
(CT)–arthrography, ultrasonography, and electromyog-
raphy (EMG), have facilitated early diagnosis of shoulder
pain and have provided a better understanding of shoulder
pathology.
This chapter provides practical guidelines for the diag-
nosis and treatment of painful shoulder disorders that may
be encountered in a rheumatology or general practice.
  Video available on the Expert Consult Premium Edition website.
Shoulder Pain
Scott David Martin  • 
Thomas S. Thornhill
A detailed analysis of shoulder problems and the treatment
of major trauma are beyond the scope of this chapter and
have been addressed by other authors.1-5 Figure 40-1 is a
useful algorithm that can direct the clinician in the initial
treatment of traumatic and nontraumatic causes of shoulder
pain.
ANATOMY AND FUNCTION
Because of its complexity, an understanding of the struc-
tural and functional anatomy of the shoulder is required
for the clinician treating shoulder pain. The shoulder
joint is the most mobile joint of the body, although mobil-
ity is gained at the sacrifice of stability. Only 25% of the
humeral head surface has contact with the glenoid at any
time. The labrum increases the contact area of the articu-
lar surface and confers stability to the joint.6 Lesions of
the labrum may result from instability, and the type of
lesion may indicate the type of instability. Labral tears
also may be a source of pain from internal derangement
of the shoulder.7 Joint stability also is provided by a thin
capsule and the glenohumeral ligaments, which are thick-
enings of the capsule anteriorly, posteriorly, and inferi-
orly.6 Anterior stability is predominantly conferred by the
anterior band of the inferior glenohumeral ligament. The
rotator cuff provides dynamic stability of the joint. It is
composed of three musculotendinous units—the supra-
spinatus, infraspinatus, and teres minor—posteriorly and
the subscapularis anteriorly. The shoulder consists of three
joints—acromioclavicular (AC), sternoclavicular, and
glenohumeral—and two gliding planes—the scapulotho-
racic and subacromial surfaces.
Figure 40-2 shows the musculoskeletal and topographic
localization of pain associated with common shoulder dis-
orders. Figure 40-3 shows the relationship of the three
posterior rotator cuff muscles coursing anteriorly under-
neath the acromion to insert on the greater tuberosity.
The subscapularis, the only anterior rotator cuff muscle,
inserts on the lesser tuberosity. By understanding the rela-
tionship between the rotator cuff and the subacromial
region, bounded inferiorly by the humeral head and supe-
riorly by the undersurface of the acromion, the clinician
can visualize the problems of impingement syndrome and
can accurately inject this space. Knowledge of the route
of the tendon of the long head of the biceps through the
bicipital groove and onto the superior aspect of the gle-
noid helps in understanding bicipital tendinitis. Before
attempting to diagnose and treat shoulder pain, the cli-
nician should review in detail one of the many sources
describing the structural and functional relationships of
the shoulder girdle.2,3
587
 588 Martin  |  Shoulder Pain

Table 40-1  Common Causes of Shoulder Pain Pain intensity, character, location, and periodicity and
Intrinsic Causes aggravating or alleviating factors should be assessed. Pain
should be graded on a visual analog scale of 0 to 10—0 indi-
Periarticular disorders
Rotator cuff tendinitis or impingement syndrome cates no pain, and 10 indicates the worst pain the patient has
Calcific tendinitis ever experienced. Another indication of the severity of pain is
Rotator cuff tear disruption of sleep. The patient should be asked whether the
Bicipital tendinitis pain prevents sleep, if pain awakens the patient, and whether
Acromioclavicular arthritis the patient can lie on the affected shoulder. Is the pain sharp
Glenohumeral disorders
Inflammatory arthritis or dull? Sharp, burning pain indicates a neurogenic origin,
Osteoarthritis whereas a dull, aching pain suggests rotator cuff pathology
Osteonecrosis with impingement. Location or distribution of the pain should
Cuff arthropathy be identified. Is it local around the shoulder girdle, or does the
Septic arthritis
Glenoid labral tears
pain radiate down the arm? Is there concomitant sensory loss
Adhesive capsulitis or weakness? Periodicity of the pain as constant or intermit-
Glenohumeral instability tent should be determined, as should factors that aggravate or
Extrinsic Causes alleviate the pain. Pain caused by rotator cuff tendinopathy
usually is exacerbated by repetitive activities that involve the
Regional disorders
Cervical radiculopathy
elbow away from the side of the body.
Brachial neuritis Any history of neck pain should be considered, along
Nerve entrapment syndromes with history of radicular pain. Radicular-type pain frequently
Sternoclavicular arthritis extends below the elbow and is associated with sensory loss
Reflex sympathetic dystrophy and ­ weakness. Pain located in the paracervical region may
Fibrositis
Neoplasms indicate a cervical origin, or it can be localized to the trape-
Miscellaneous zius. Trapezial pain often is associated with shoulder pain and
Gallbladder disease results from the patient trying to favor the shoulder. Assuming
Splenic trauma a military brace position may produce fatiguing and spasm of
Subphrenic abscess
Myocardial infarction
the trapezius.
Thyroid disease Any pertinent medical history, such as a history of malig-
Diabetes mellitus nancy, should be considered. Neurologic, visceral, and vas-
Renal osteodystrophy cular disease can produce referred pain to the shoulder and
should always be kept in mind, especially in a patient with a
painless range of motion.

DIAGNOSIS
CLINICAL EVALUATION OF THE SHOULDER
Accurate diagnosis and successful treatment of a shoulder
disorder begins with a thorough history and physical exami-
nation. Most of the information needed to make a correct
diagnosis can be elicited with basic clinical skills, rather
than relying on expensive and highly technologic investiga-
tive aids. Diagnostic tests should be used only to confirm an
established diagnosis or to assist in cases with a challenging
presentation.
HISTORY
In establishing a diagnosis, it is important to consider the
patient’s age and chief complaint. The differential diagno-
sis of shoulder pain in a 70-year-old sedentary individual
is entirely different from shoulder pain in a 20-year-old
pitcher. Did the pain occur slowly over time or suddenly
with a particular event? The gradual onset of pain over the
anterolateral or deltoid region that is increased with forward
elevation of the shoulder suggests impingement with rotator
cuff tendinitis. The presence of significant weakness with
pain on overhead activities suggests impingement with rota-
tor cuff tear. Initiating factors relative to the onset of the
symptoms should be elicited, and any history of shoulder
pain or trauma should be carefully documented.
PHYSICAL EXAMINATION
Proper physical examination of the shoulder includes close
inspection of the shoulder girdle from the front and back.
The evaluation is started by standing behind the patient,
with both shoulders exposed. The normal shoulder is always
inspected and compared with the injured shoulder. Exami-
nation can be started with the patient in the sitting or
standing position. Contour and symmetry are observed and
compared between shoulders, assessing any atrophy or asym-
metry in shoulder position or level. Spinatus muscle atrophy
may result from disuse, chronic cuff tear, or suprascapular
or brachial neuropathy.8 If evidence of scapular winging is
evident, the patient should be asked to do a wall push-up,
which accentuates winging.
Range of motion should be carefully recorded, notic-
ing any absence of rhythmic shoulder motion or excessive
scapulothoracic motion that may compensate for the lack
of glenohumeral motion. Internal rotation of the shoulder
is checked by having the patient reach behind the back
with the thumb while the examiner notices the vertebral
level. Loss of internal rotation is seen early with shoulder
pain and usually indicates some tightness of the posterior
shoulder capsule. Palpation of the biceps tendon, coracoid,
lesser and greater tuberosities, and posterior cuff is done,
and any tenderness is gauged (Fig. 40-4A). Tenderness on
palpation of the long head of the biceps frequently is associ-
ated with rotator cuff tendinopathy and tenderness of the
greater tuberosity. Any spasm or tenderness of the trapezius
 HISTORY AND PHYSICAL EXAM

Glenohumeral joint pain

Hx trauma NONTRAUMATIC

AC joint path
+ Pain palpation ? Radiographs
Shoulder radiographs Positive impingement signs Negative impingment signs + Cross chest adductor test True AP
True AP Positive impingement test Primary C-spine pain + Lidocaine injection test Scapular Y
Scapular Y (Lidocaine injection) Radicular symptoms Axillary
Axillary No instability R/O Pancoast tumor Instability ±
Neuro exam Good strength/mild weakness impingement
Good ROM ? Radiographs
Zanca view + GH arthridities
(AP or AC joint) Physical therapy
Cervical x-rays - Joint changes
Physical therapy 10-15 degree cephalic Injection
AP NSAIDs
- X-ray + X-ray Periscapular, cuff, and tilt 50% penetrance
PT Lateral
Rest/ice + Fracture NSAIDs Obliques subscapularis strengthening
+ Dislocation NSAIDs MRI
NSAIDs Ice
PT Restrict overhead activity Avoidance of above Reevaluate 3 months
shoulder activity
Reevaluate 3 months + Findings - Findings
- X-ray + X-ray
Refer orthopedics
Reevaluate or rheumatology Reevaluate 4-6 weeks Improvement No improvement
Reevaluate 3 months
3-4 weeks Improvement PT
PT Cervical
No improvement NSAIDs
spondylosis
PART 6 

Improvement Cervical trx Continue home Refer Ice

No improvement Cervical collar Improvement
| 

PT and NSAIDs orthopedics or

Improvement NSAIDs Gradual resumption of rheumatology
No improvement
X-ray overhead activity
Continued Home PT Flex/ext views
pain/weakness Gradual resumption Reevaluate 3 months
Home PT Refer orthopedics of overhead activity Reevaluate 6 weeks Home PT Refer
Gradual or rheumatology? NSAIDs PRN Gradually increase orthopedics or
resumption of Refer activities rheumatology
shoulder activity orthopedics or Improvement No improvement
Refer No improvement rheumatology
orthopedics or
Impingement series?
rheumatology
- AP of shoulder with 30-degree caudal tilt
Continue home Refer
- Scapular Y with 10-degree caudal tilt
+ Radicular Sx - Radicular Sx PT and NSAIDs orthopedics or
- Axillary view
rheumatology
Subacromial injection
Continued PT
NSAIDs
MRI Continue conservative Tx
PT
NSAIDs
Cervical traction
+ Findings - Findings
Reevaluate 3 months

Reevaluate 3 months
Improvement No improvement Refer
orthopedics or
rheumatology
Continued
pain
DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN

Home PT
Resume overhead activity
Figure 40-1  Algorithmic evaluation of shoulder pain. AC, acromioclavicular; AP, anteroposterior; GH, glenohumeral; Hx, history; MRI, magnetic resonance imaging; NSAID, nonsteroidal anti-­inflammatory
drug; PRN, as required; PT, physical therapy; R/O, rule out; ROM, range of motion; Sx, symptoms; trx, traction; Tx, therapy.
589
 590 Martin  |  Shoulder Pain

4 7
3
1
5
6
2

Figure 40-2  A-D, Musculoskeletal

(A and B) and topographic (C and D)
areas localizing pain and tenderness
associated with specific shoulder
problems. 1, Subacromial space (ro-­
tator cuff tendinitis/impingement
syndrome, calcific tendinitis, rotator A B
cuff tear). 2, Bicipital groove (bicipi-­
tal tendinitis, biceps tendon sublux-­
ation and tear). 3, Acromioclavicular 3 6
2 1
joint. 4, Anterior glenohumeral joint 5 7
(glenohumeral arthritis, osteonecro-­
sis, glenoid labrum tears, adhesive
capsulitis). 5, Sternoclavicular joint.
6, Posterior edge of acromion (rota-­
tor cuff tendinitis, calcific tendinitis, 8
rotator cuff tear). 7, Suprascapular
notch (suprascapular nerve entrap-­
ment). 8, Quadrilateral space (axil-­ 4
lary nerve entrapment). These areas
of pain and tenderness frequently C D
overlap.

or levator scapulae may be associated with rotator cuff dis-
ease or cervical spine disease. Cervical range of motion and
palpation of the paracervical muscles are carried out. Para-
cervical tenderness and limited range of motion of the neck
may indicate cervical spondylosis or neurogenic disease.
A Spurling test is done by flexing the neck laterally while
applying axial compression to the skull. The production of
pain that radiates to the ipsilateral shoulder is considered a
positive test result and indicates radiculopathy.
To elicit the impingement sign, the shoulder is elevated
passively in forward flexion, while depressing the scapula
with the opposite hand, forcing the greater tuberosity
against the anterior acromion and producing pain in cases
of impingement (Fig. 40-4B).9 This maneuver also may be
painful in conditions such as adhesive capsulitis, glenohu-
meral and AC arthritis, glenohumeral instability, and calcific
tendinitis. A dynamic impingement test, the circumduc-
tion-adduction shoulder maneuver, also called the Clancy
test, is 95% sensitive and 95% specific for diagnosing rotator
cuff tendinopathy, including partial tears.10 The test is done
with the patient in the standing position and with the head
turned to the contralateral shoulder. The affected shoulder
is circumducted and adducted across the body to shoulder
level, keeping the elbow in extension, the shoulder in inter-
nal rotation, and the thumb pointing toward the floor (Fig.
40-4C). In this position, the patient is instructed to resist
maximally as a uniform downward force is applied to the
extended arm by the examiner. The test result is considered
positive if pain or weakness is elicited during the maneuver,
with pain being localized to the anterolateral aspect of the
shoulder. There is a strong positive correlation of pain and
weakness with complete cuff tear.10
The sternoclavicular and AC joints should be observed for
prominences and palpated for stability and tenderness. Many
patients with impingement have tenderness on direct down-
ward palpation of the AC joint owing to impingement on the
cuff from undersurface osteophytes of the distal clavicle.2,8
AC joint tenderness also may result from primary AC
joint arthrosis and should be differentiated by physical
examination, including the cross-chest adduction test and
O’Brien’s test.11 Radiographic evidence of AC joint arthro-
sis is common in patients older than 40 years, but is not
usually painful.12
The cross-chest adduction test or horizontal adduc-
tion test is performed by forward flexion of the shoulder
90 degrees with subsequent cross-chest adduction of the arm
(Fig. 40-4D). Pain localized to the AC joint is considered a
positive test result. If pain occurs posteriorly over the shoul-
der, a tight posterior capsule with impingement is suspected.
O’Brien’s test is performed by forward flexing the arm
90 degrees and adducting the arm 10 degrees out of the sag-
ittal plane of the body. The first part of the test is ­performed
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN 591

A
Coracoclavicular ligament: Subscapularis tendon
Trapezoid ligament Coracoid
Supraspinatus tendon
Conoid ligament process
Infraspinatus tendon

Teres minor tendon

Clavicle

Subscapularis
muscle

Acromion of scapula

Coracoacromial ligament

Spine of scapula
Superior margin of scapula
Infraspinatus muscle
Supraspinatus
muscle Coracoid
B process
Acromioclavicular joint
Clavicle Acromion
Coracoacromial ligament
Supraspinatus tendon
Subscapularis tendon
Greater tuberosity
Lesser tuberosity
Humerus

Figure 40-3  A, Superior view of the rotator cuff musculature as

Bicipital tendon groove
it courses anteriorly underneath the coracoacromial arch to insert
on the greater tuberosity. B, Anterior view of the shoulder reveals
the subscapularis, which is the only anterior rotator cuff muscle
inserting on the lesser tuberosity. It internally rotates the humerus
and provides dynamic anterior stability to the shoulder. (A from the
Ciba Collection of Medical Illustrations, Volume 8, Part I. Reproduced
by permission of Ciba-Geigy; B from Martin TL, Martin SD: Rotator cuff
tendinopathy. Hosp Med 12:23-31, 1998.)

with the hand maximally pronated with the thumb pointed
down. In this position, the patient is asked to resist as the
examiner applies a downward force on the arm. If the test
elicits pain, the patient is asked if the pain is on top of the
shoulder or deep inside. Pain localized to the top of the shoul-
der indicates AC joint pain, and pain deep inside the shoul-
der indicates a superior labrum anterior posterior (SLAP)
lesion. In the second part of the test, the patient is asked to
supinate the hand maximally, while the examiner applies
a downward force to the arm. If the patient notices signifi-
cantly less pain, the test result is positive for a SLAP lesion.
If the pain is unchanged and located on top of the shoulder,
the test result is positive for AC joint pathology.11
If the cause of AC joint tenderness is still in question, a
lidocaine injection should be administered, carefully avoid-
ing injecting the subacromial space by advancing the needle
too far inferiorly through the AC joint, which can lead to
false interpretation. Painful degenerative changes of the AC
joint may exist concomitantly with subacromial impingement
and should be evaluated thoroughly when surgical treatment
(i.e., distal clavicle excision) is being considered.13
In patients with pain out of proportion to objective
findings, other causes of shoulder pain should be sought,
including calcific tendinitis, infection, reflex sympathetic
dystrophy, and fracture. Patients with significant wasting of
the supraspinatus and infraspinatus muscles and posterior
shoulder pain, especially younger patients, may have supra-
scapular neuropathy or brachial plexopathy.8,14
Patients with chronic cuff disease frequently have vari-
able disuse atrophy of the supraspinatus and infraspinatus
fossae; in cases of chronic massive cuff tears, atrophy and
weakness can be severe. Strength testing of external ­rotation
should be done with the elbow at the side and supported by
the examiner; the patient is asked to attempt external rota-
tion of the shoulder from a neutral position (0 degrees of
adduction), while the examiner applies resistance (see Fig.
40-4E).15 Weakness in this position may suggest a tear of
the infraspinatus tendon. Abduction strength testing against
 592 Martin  |  Shoulder Pain

Sternoclavicular Acromioclavicular joint

joint
Subdeltoid bursa Examiner elevates
shoulder here and...
Bicipital
tendon
groove
Greater
tuberosity

Lesser ... depresses

tuberosity scapula
Glenohumeral here (from
A joint space B back)

Pain on palpation =
acromioclavicular
joint
Examiner Arm impingement
applies force adducted
here across
chest

Pain on palpation
= tight posterior
Patient
capsule
resists
force
here
C D

Patient attempts
external rotation
of the shoulder Examiner
applies
30 force
here

90

Examiner
applies Patient resists
E force here F force here
Figure 40-4  A, Tenderness on palpation of trigger points may help localize the site of pathology. Tenderness on palpation of the long head of the biceps
and greater tuberosity suggests impingement with possible cuff tendinopathy. B, To elicit the impingement sign, the shoulder is elevated in forward
flexion while the scapula is depressed with the opposite hand, forcing the greater tuberosity and rotator cuff against the anterior acromion and produc-­
ing pain when impingement exists. Relief of pain after injection of local anesthetics (i.e., impingement test) provides additional evidence of subacromial
pathology. C, The Clancy test is performed with the patient standing and with the head turned toward the contralateral shoulder. The affected shoulder
is circumducted and adducted across the body to shoulder level, keeping the elbow in extension with the arm internally rotated with the thumb pointed
toward the floor. In this position, the patient is asked to resist maximally as a uniform downward force is applied to the extended arm by the examiner. The
production of pain or weakness localized to the anterior lateral portion of the shoulder is considered a positive test result. D, The test is performed by for-­
ward flexion of the arm at 90 degrees and subsequent cross-chest adduction of the arm. Pain localized to the acromioclavicular joint is considered a posi-­
tive test result. E, The test is performed with the patient’s elbow flexed at 90 degrees and held at the patient’s side by the examiner. The patient is asked
to attempt external rotation of the shoulder from a neutral position (0 degrees of adduction) as the examiner applies resistance to the forearm. Strength
is compared with that of the contralateral arm. F, Abduction strength testing is performed with the patient’s shoulder in 30 degrees of forward flexion
and 90 degrees of abduction and with the thumb pointed toward the floor. The patient is asked to resist as the examiner exerts a downward force on the
abducted arm. Strength is compared with the contralateral shoulder. (From Martin TL, Martin SD: Rotator cuff tendinopathy. Hosp Med 12:23-31, 1998.)

resistance is done with the shoulder in 30 degrees of forward
flexion and 90 degrees of abduction, and the thumb pointed
toward the floor (see Fig. 40-4F).16,17 Weakness in this posi-
tion may suggest a tear of the supraspinatus tendon. A lift-
off test should be performed with the shoulder in internal
r­ otation; the patient is asked to try to hold the hand away from
the back. Inability to do so indicates a subscapularis tear.
If after a thorough physical examination impingement is
suspected, an impingement test should be performed with
injection of 5 mL of local anesthetic into the subacromial
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN 593

space.18,19 Before performing the test, the patient is asked to

grade the pain during the impingement signs on a visual ana-
log scale of 0 to 10, with 0 equal to no pain and 10 equal to
the most severe pain the patient has ever experienced. The
injection may be done anteriorly or posteriorly, depending on
physician’s preference. Ten minutes after injection of local
anesthetic into the subacromial space, the patient should be
re-examined and asked to regrade the pain on the same visual
analog scale. A 50% or more reduction in pain is thought to
be a positive test result for impingement; otherwise, an alter-
native cause of shoulder pain should be sought, or inadequate
placement of the anesthetic should be suspected. If the AC
joint is thought to be contributing to the shoulder pain, 1 to X-ray
2 mL of local anesthetic should be injected into the joint,
and the shoulder should be re-­examined. When subacromial 10°
impingement and the AC joint are thought to be contribut-
ing to shoulder pain, serial injections during separate office
visits may be needed to evaluate the shoulder while minimiz-
ing discomfort to the patient.12
In cases of suspected bicipital tendinitis, Speed’s test
is performed by having the patient flex the shoulder and
extend the elbow while a downward force is applied to the
arm. The production of pain over the long head of the biceps
is a positive test result and suggests bicipital tendinitis.
Upper extremity strength testing should be performed
and compared with the contralateral side so that any atro-
phy is detected. Grip strength is checked, and the hands are
examined carefully for evidence of intrinsic atrophy. The Figure 40-5  Zanca view of the acromioclavicular joint is obtained with a
biceps (C5), triceps (C7), and brachioradialis (C6) reflexes 10-degree cephalic tilt and 50% penetrance. (From Rockwood CA Jr, Young
are checked for symmetry and briskness. DC: Disorders of the acromioclavicular joint. In Rockwood CA Jr, Matsen TA III
Light touch sensory testing should be conducted, and the [eds]: The Shoulder. Philadelphia, WB Saunders, 1985, pp 413-476.)
dermatomal distribution of any deficits that may suggest cervi-
cal radiculopathy should be identified. The cervical, supracla- views of the AC joint may be obtained by strapping 5 to
vicular, axillary, and epitrochlear regions should be palpated 10 lb of weight to the patient’s forearms and determining
for enlarged lymph nodes, which may suggest malignancy. AC separation. Comparing the coracoclavicular distance of
both shoulders may be helpful. When clinically indicated,
RADIOGRAPHIC ASSESSMENT cervical spine radiographs should be obtained to exclude
cervical spondylosis as a cause of shoulder pain.
For nontraumatic painful shoulder evaluation, standard radio-
graphic profiles are used. An impingement series should
SCINTIGRAPHY
be obtained, which includes anteroposterior views with a
30-degree caudal tilt (Rockwood view), outlet view ­(scapular Tc 99m MDP or gallium may be of diagnostic help in evalu-
Y with 10- to 15-degree caudal tilt), and axillary view. Inter- ating skeletal lesions around the shoulder joint. Bone scans
nal and external rotational views may be obtained if calcific generally are not helpful in the diagnosis of non-neoplastic
tendinitis or instability is suspected. The Rockwood view or noninfectious shoulder disease.
can reveal any osteophytes off the anterior acromion and Scintigraphy may have a role in identifying patients with
AC joint.20 In cases of traumatic injury, a trauma series is complete rotator cuff tears that proceed to cuff-tear arthropa-
obtained that includes a true anteroposterior view, scapular thy. This is an important distinction because patients with
Y view, and an axillary view. The axillary view is useful in complete rotator cuff tears may do well, whereas patients who
assessing posterior or anterior subluxation of the humeral develop progressive changes of cuff-tear arthropathy have pro-
head. Additional views, such as the West Point view, which gressive arthritis, pain, and significant functional impairment.
evaluates the glenoid for evidence of a bony Bankart lesion, Synovitis or calcium pyrophosphate deposition disease may be
or the Styker notch view, which assesses the humeral head an important factor in the pathogenesis of cuff-tear arthropa-
for a Hill-Sachs lesion, may be obtained to assist the evalu- thy. In such cases, scintigraphy may show the increased blood
ation if the diagnosis of instability is in doubt. Secondary flow and blood pooling associated with chronic synovitis.
impingement-type rotator cuff tendinitis may be caused
by increased anterior translation with subluxation of the
ARTHROGRAPHY
humeral head. In such cases, an axillary view or fluoroscopy
can help show the subluxation.21,22 Double-contrast arthrotomography (DCAT) can be used
When AC joint pathology is suspected, a 10-degree, to evaluate problems of the rotator cuff, glenoid labrum,
cephalic-tilt view of the AC joint at 50% penetrance, as biceps tendon, and shoulder capsule.24-27 Figure 40-6 shows
described by Zanca,23 should be obtained (Fig. 40-5). Stress normal DCAT of the shoulder. Rotator cuff tears can be
 594 Martin  |  Shoulder Pain
RC
AC
BT
Figure 40-6  Normal double-contrast arthrography shows the inferior
edge of the rotator cuff (RC) as it courses through the subacromial space
to the greater tuberosity, the tendon of the long head of the biceps (BT),
and the articular cartilage of the humeral head (AC).
Figure 40-7  Multidetector CT revealing a superior labral tear of shoulder.
shown by single-contrast or double-contrast studies. The
proponents of double-contrast arthrography believe that
the extent of the tear, the preferred surgical approach, and
the quality of the rotator cuff tissue are best determined
by double-contrast studies.24-29 Arthrography without
MRI or CT can be misleading and result in underestimat-
ing the extent of a rotator cuff tear. Multidetector CT can
increase the accuracy of diagnosing labral and rotator cuff
tears (Fig. 40-7).
Tears of the glenoid labrum without shoulder disloca-
tion are sources of anterior shoulder pain in athletes.7 Gle-
noid labrum tears (Fig. 40-8), with or without associated
glenohumeral subluxation, frequently can be identified by
DCAT.27,28 Kneisl and colleagues30 described 55 patients
who underwent DCAT followed by diagnostic shoulder
arthroscopy. DCAT predicted the arthroscopic findings in
76% of ­anterior labrum studies and 96% of posterior labrum
studies. This test was 100% sensitive and 94% specific in
Figure 40-8  Double-contrast arthrotomography shows a tear of the
anterior-inferior portion of the glenoid labrum (arrow).
Figure 40-9  Double-contrast arthrography of a patient with calcific ten-­
dinitis (arrow) and adhesive capsulitis. Notice the contracted capsule with
diminution of the synovial space and obliteration of the axillary recess.
diagnosing complete rotator cuff tears. Partial rotator cuff
tears identified at arthroscopy were missed in 83% of patients
undergoing DCAT. The investigators believed DCAT was
better in diagnosing intra-articular and cuff pathology in
cases of instability than when pain alone was the presenting
diagnosis.30
Shoulder arthrography can confirm a diagnosis of adhe-
sive capsulitis by showing a contracted capsule with an
obliterated axillary recess (Fig. 40-9). The use of sub-
acromial bursography has been beneficial in visualizing
the outer surface of the rotator cuff and the subacromial
space in cases of impingement.31,32 Fukuda and associ-
ates33 reported a small series of younger patients (average
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN 595

A B

C D
Figure 40-10  CT-arthrography of shoulder. A, Normal findings. B, Tear of the anterior glenoid labrum. C, A large defect of the articular surface of the
posterior portion of the humeral head (Hill-Sachs lesion) (arrow). D, Loose body in the posterior recess (arrow).

age 41.8 years) who underwent subacromial bursography
after a negative glenohumeral arthrographic result. These
patients showed pooling of contrast medium on the bursal
side of a tear, which was confirmed at the time of surgery.
Subacromial bursography is not routinely used diagnosti-
cally, and in our opinion, it is of little value in planning
surgical procedures.
COMPUTED TOMOGRAPHY
CT is helpful in evaluating the musculoskeletal system, and
CT combined with contrast arthrography (CT-arthrography)
has become a major diagnostic tool for the evaluation of gle-
noid labrum tears, loose bodies, and chondral lesions (Fig.
40-10). Rafii and coworkers34 reported using CT-arthrogra-
phy in the evaluation of shoulder derangement. This study
found a 95% accuracy of CT-­arthrography for investigating
lesions of the labrum and articular surface.34 More recently,
multidetector CT-arthrography scans have been used to
evaluate partial cuff tears (Fig. 40-11A), cystic lesions (Fig.
40-11B), and calcific tendinopathy (Fig. 40-11C).
ULTRASONOGRAPHY
The technologic improvement of ultrasound equipment has
allowed improved ultrasound study of the rotor cuff. The
technique is noninvasive, is rapid, and involves no radiation
exposure.30-32,35 The cuff is examined in the horizontal and
transverse planes with the arm in different positions to visu-
alize various areas of the cuff. These techniques generally
provide visualization of the distal cuff, where most rotator
cuff tears are located. Figure 40-12 shows normal and abnor-
mal ultrasound images of the rotator cuff in longitudinal and
transverse planes.
Several studies report a high sensitivity and specificity
for the diagnosis of a rotator cuff tear by ultrasound.32-35
The specificity and sensitivity of the procedure are
reported to be greater than 90% as determined by arthro-
graphic and surgical correlations.34,35 This technique also
has been used for the postoperative evaluation of a rota-
tor cuff repair and for evaluation of abnormalities of the
biceps tendon.36-40
Gardelin and Perin41 reported ultrasound to be 96%
sensitive in determining rotator cuff and biceps tendon
pathology. Mack and associates36 found ultrasound to be
valuable in evaluating postoperative patients with recur-
rent shoulder symptoms. In a prospective study, Hodler and
colleagues39 compared ultrasound with MRI and arthrog-
raphy in evaluating rotator cuff lesions in 24 shoulders.
Ultrasound identified 14 of 15 torn cuffs, MRI identified 10
of 15, and arthrography identified 15 of 15.39 Ultrasound
identified seven of nine intact rotator cuffs, whereas MRI
was accurate in eight of nine intact cuffs.39 Vestring and
colleagues42 found ultrasound to be as accurate as MRI in
 596 Martin  |  Shoulder Pain
B C
Figure 40-11  Multidetector CT-arthrography. A, Partial rotator cuff tear (coronal view). B, Cystic humeral head erosions with calcification (axial view).
C, Calcification within rotator cuff tendon (coronal view).
the diagnosis of humeral head defects and joint effusions,
but inferior to MRI in the diagnosis of labrum lesions, rota-
tor cuff lesions, subacromial spurs, and synovial inflamma-
tory disease. In the hands of an experienced sonographer,
ultrasound may be the most cost-effective test for the ini-
tial evaluation of a rotator cuff injury, but most surgeons
require CT-arthrography or MRI confirmation before surgi-
cal exploration.36,39,41-43
ARTHROSCOPY
The use of arthroscopy for the diagnosis of shoulder pathol-
ogy increased in the 1980s, partially because of its accuracy,
which was far greater than clinical examination and better
than other diagnostic modalities of the time. With techno-
logic advances of fiberoptics, video output, and arthroscopic
instrumentation, the use of arthroscopy to diagnose and
treat shoulder problems exponentially increased to include
procedures previously considered reserved only for open
techniques.44
Compared with DCAT, arthroscopy is more accurate
in the diagnosis of intra-articular lesions associated with a
painful shoulder.30 An additional benefit is that ­arthroscopy
can be used to diagnose and treat shoulder problems of
the glenohumeral joint and the subacromial region. With
the increased accuracy of MRI-arthrography in detecting
partial cuff tears and labral lesions, diagnostic shoulder
arthroscopy has become less common in the absence of
clear indications and specific treatment plans. In combi-
nation with a detailed history and physical examination,
and along with examination under anesthesia, shoulder
arthroscopy has been helpful in the diagnosis of chronic
instability patterns of the glenohumeral joint.44-47
The indications and usefulness of shoulder arthroscopy
in the treatment of common pathologic conditions have
continued to increase as the technology improves, and the
understanding of the pathophysiology of shoulder prob-
lems grows. Shoulder arthroscopy has been routinely used
to confirm and treat SLAP lesions, labral tears, partial cuff
tears, refractory adhesive capsulitis, partial biceps tendon
tears, and multidirectional instability. Other conditions
that are routinely treated arthroscopically are rotator cuff
tears, glenohumeral instability, AC joint pathology, loose
bodies, sepsis, osteochondritis dissecans, synovitis, chon-
dral lesions, subacromial impingement, and calcific tendi-
nitis.7,13,44,47
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN 597

4
4

3 3
5 2
2
1 1

A B

C D
Figure 40-12  A, Normal longitudinal view of rotator cuff by ultrasound shows the humeral head (1), the superior articular surface (2), the rotator cuff
(3), the deltoid tendon (4), and tapering of the cuff to its insertion on the greater tuberosity (5). B, Transverse view of a normal intact rotator cuff cover-­
ing the humeral head. C, Rotator cuff tear, showing a hypoechoic area (arrow) on a longitudinal view. D, Rotator cuff tear, showing hypoechoic area
(arrows) on a transverse view.

MAGNETIC RESONANCE IMAGING
MRI has been used to diagnose partial-thickness and full-
thickness rotator cuff tears, biceps tendon tears, impinge-
ment of the rotator cuff, synovitis, articular cartilage damage,
and labral pathology associated with glenohumeral instabil-
ity.48-50 In rheumatoid arthritis, MRI is reported to be more
sensitive than plain radiographs in determining soft tissue
abnormalities and osseous abnormalities of the glenoid and
humeral head.51
One of the most useful diagnostic uses of MRI is for rota-
tor cuff pathology. Morrison and Offstein52 studied 100
patients with chronic subacromial impingement syndrome
using arthrography and MRI. MRI was 100% sensitive, but
only 88% specific in confirming arthrography-proven rota-
tor cuff tears. Nelson and associates53 studied 21 patients
with shoulder pain and found MRI to be more accurate
than CT-arthrography or ultrasound in identifying partial-
­thickness cuff tears. These investigators also reported MRI
to be as accurate as CT-arthrography in the diagnosis of
abnormalities of the glenoid labrum.53
The characteristic MRI findings in rotator cuff tears
in­clude a hypointense gap within the supraspinatus muscle
tendon complex on T1-weighted films, absence of a demon-
strable supraspinatus tendon with narrowing of the subacro-
mial space, and an increased signal within the supraspinatus
tendon on T2-weighted images.54 Seeger and colleagues,55
reporting the results of 170 MRI studies, found that T1-
weighted images were highly sensitive for identifying abnor-
malities within the supraspinatus tendon, but T2-weighted
images were required to differentiate tendinitis from a small
supraspinatus tendon tear. Large full-thickness tears could
be identified, however, on T1-weighted and T2-weighted
images. Figure 40-13 depicts common shoulder pathology as
seen by MRI. MRI is almost as sensitive as and more specific
than scintigraphy in the diagnosis of osteonecrosis and neo-
plastic lesions around the shoulder.
ELECTROMYOGRAPHY AND NERVE CONDUCTION
VELOCITY STUDIES
EMG and nerve conduction velocity studies can help dif-
ferentiate shoulder pain from pain of neurogenic origin.
They also may be beneficial in determining the localization
of neurogenic pain to a particular cervical root, the brachial
plexus, or a peripheral nerve.56,57
INJECTION
Injection of local anesthetics and glucocorticoids is a useful
technique for the diagnosis and treatment of shoulder pain.
The physician must have a thorough understanding of the
anatomy of the shoulder girdle and a presumptive diagnosis
to direct the injection properly. Injection of referred pain
 598 Martin  |  Shoulder Pain

A
B

A B

A
A
B
B

Figure 40-13  A, MRI proton densi-­

ty–weighted coronal view shows su-­
praspinatus tendon as a black band C D
(A) that has an increased signal as
it nears insertion on the greater tu-­
berosity (B). B, Similar view in a T2-
weighted image shows increased
signal as gray (arrow), indicating
partial-thickness tear or tendinitis.
C, MRI proton density–weighted
coronal view shows abrupt end of
supraspinatus tendon as it courses
right to left (A). From A to B is an area
of increased signal followed by a
short portion of tendon (B) inserting
at the greater tuberosity. D, Similar
view in a T2-weighted image shows
increased signal as white (fluid den-­
sity), indicating fluid in the gap of
a complete rotator cuff tear. E, MR
arthrography shows normal rotator
cuff. F, MR arthrography shows a E F
chronic cuff tear with retraction.

areas may be misleading. In a patient with lateral arm pain
secondary to deltoid bursal involvement from calcific tendi-
nitis of the supraspinatus tendon, injection should be in the
subacromial space, rather than in the area of referred pain in
the deltoid muscle. It is often better to use a posterior sub-
acromial approach when injecting a rotator cuff tendinitis
in a patient with anterior impingement symptoms because it
is easier to enter the subacromial region posteriorly, and this
approach is less traumatic to contracted anterior structures.
The instillation of rapidly acting local anesthetics can be
beneficial in determining the source of shoulder pain. Oblit-
eration of pain by injection of a local anesthetic along the
bicipital groove can confirm a diagnosis of bicipital tendini-
tis. The use of local anesthetics is less helpful when injecting
the subacromial space because of its extensive communica-
tion with the rest of the shoulder girdle, but relief of symptoms
by such an injection can exclude pain from conditions such
as cervical radiculopathy or entrapment neuropathy.
AUTHORS’ PREFERRED DIAGNOSTIC TESTS
Table 40-2 lists the reimbursement and charges for various
shoulder diagnostic tests based on 2006 Medicare fee sched-
ules and 2006 charges at a single institution. Choice of a
specific test depends on its sensitivity, specificity, and cost-
benefit analysis. History and physical examination are the
most important factors in establishing diagnosis of the pain-
ful shoulder. Plain radiographs (three views) should be the
first radiographic test performed. Although not as sensitive
as the more sophisticated tests, plain radiographs can iden-
tify arthritic change, calcific tendinitis, established osteone-
crosis, and most neoplasms.
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
Table 40-2  Relative Costs of Shoulder Diagnostic Procedure in 2006
Procedure Initial Fee ($)
Medicare B Fee Schedule
Initial office visit (30 min) 154.00
Plain radiography (3 views)
Arthrography
Ultrasonography
Magnetic resonance imaging
Computed tomography
Tomography
Institutional Charges
Initial office visit (30 min) 196.00
Plain radiography (3 views)
Arthrography
Ultrasonography
Magnetic resonance imaging
Computed tomography
If intra-articular pathology (e.g., labrum tear, capsular
tear, loose body, or chondral defect) is suspected, MRI-
arthrography is preferable to CT-arthrography. In diagnosing
acute rotator cuff tears in a younger patient, ultrasound is
the most cost-effective test to confirm a clinical suspicion.
In cases of impingement syndrome, MRI is sensitive, but
it is difficult to differentiate tendinitis, partial tears, and
small complete tears without MRI-arthrography. Ortho-
paedic surgeons prefer MRI-arthrography for verification
of labral tears or partial rotator cuff tears. In the case of a
suspected full-thickness rotator cuff tear, MRI is preferred
to determine the size of the tear, amount of muscle atro-
phy and tendon retraction, and quality of the remaining
tissue for repair.
INTRINSIC FACTORS CAUSING
SHOULDER PAIN
PERIARTICULAR DISORDERS
Shoulder Impingement and
Rotator Cuff Tendinopathy
One of the most common nontraumatic causes of shoulder
pain is impingement with rotator cuff tendinopathy. In 1972,
Neer9 described his results of 100 anatomic shoulder dissec-
tions and coined the term impingement syndrome. Impinge-
ment may be defined as the encroachment of the acromion,
coracoacromial ligament, coracoid process, or AC joint on
the rotator cuff as it passes beneath them during glenohu-
meral motion. The function of the posterior rotator cuff is to
abduct and externally rotate the humerus. The cuff with the
biceps tendon serves as a humeral head depressor to main-
tain the head centered within the glenoid fossa as the cuff
and deltoid elevate the arm.58-60
Controversy continues, however, as to the exact cause
of impingement—whether it is a primary, intrinsic, degen-
erative event within the tendon with superior migration
of the head on arm elevation and secondary impingement
on the acromion or a purely mechanical attrition of the
tendon with primary impingement against the acromion.
The mechanical impingement of the rotator cuff may
be influenced by variations in the shape and slope of the
599
Technical Fee ($) Interpretation Fee ($)
36.28 23.09
156.08 34.07
74.70 41.62
531.28 81.42
233.06 66.77
78.53 39.69
164.00 68.00
455.00 190.00
239.00 131.00
2081.00 228.00
973.00 163.00
a­ cromion.61,62 The supraspinatus outlet may become nar-
rowed from proliferative spur formation of the acromion or
degenerative changes of the AC joint. These changes, along
with intrinsic degenerative changes of the rotator cuff, may
lead to rotator cuff tear, but the exact pathogenesis remains
controversial. Many studies have found a strong correlation
between degenerative hypertrophic spur formation, with its
resulting narrowing of the supraspinatus outlet, and the pres-
ence of full-thickness cuff tears,9,19,63-70 but clinical studies
have failed to resolve whether the hypertrophic changes of
the coracoacromial arch are caused by the cuff lesions or are
a cause of the lesions themselves.
Neer9 developed a staging system for description of im­­
pingement lesions of the shoulder. A stage I lesion involves
edema and hemorrhage of the rotator cuff and is typically
found in individuals younger than 25 years who are active
in overhead athletics. The condition usually reponds to
conservative treatment of rest, anti-inflammatory medica-
tion, and physical therapy. Stage II lesions usually occur in
the 30s or 40s and represent the biologic response of fibro-
sis and thickening of the tendon after repeated episodes
of mechanical impingement over time. The lesion also is
treated conservatively, as in stage I, but attacks may recur.
If symptoms persist despite adequate conservative manage-
ment for more than 6 to 12 months, surgical intervention is
warranted. Stage III lesions involve rotator cuff tears, biceps
tendon rupture, and bone changes, and they rarely occur
before age 40. Patients may present with pain, weakness, or
supraspinatus atrophy, depending on the chronicity of the
tear. Surgical treatment depends on the patient’s age, loss of
function, weakness, and pain.
Patients usually present to the clinician with a complaint
of pain that has failed to resolve after a variable period. Pain
can be sudden and incapacitating in cases of traumatic cuff
tears, or more commonly it manifests as a dull ache in cases of
chronic impingement. Pain usually is located over the ante-
rior and lateral aspects of the shoulder and may radiate into
the lateral deltoid. The pain may worsen with sleeping on
the affected extremity and is exacerbated by overhead activ-
ity. Tenderness on palpation may be elicited over the greater
tuberosity and long head of the biceps within the bicipital
groove, indicating an associated biceps tendinitis. In cases
with concomitant degenerative changes of the AC joint,
 600 Martin  |  Shoulder Pain
Figure 40-14  The impingement sign is elicited by forced forward  with stage II disease. Job modification for individuals with
elevation of the arm. Pain results as the greater tuberosity impinges
on the acromion. The examiner’s hand prevents scapular rotation. This 
maneuver may be positive in other periarticular disorders. (From Neer
CS II: Impingement lesions. Clin Orthop 173:70, 1983.)
there may be tenderness on palpation over the AC joint as an
offending osteophyte impinges on the rotator cuff beneath.
The impingement sign as described by Neer9 (Fig. 40-14)
is useful in the diagnosis of rotator cuff tendinopathy. The
patient often describes a catch as the arm is brought into the
overhead position. The patient may be observed to raise the
arm by abduction and external rotation to clear the greater
tuberosity of the acromion, bypassing the painful area. A
typical painful arc usually occurs between 70 degrees and
110 degrees of abduction. Neer9 also described an impinge-
ment test that involves injection of lidocaine into the sub-
acromial bursa. Relief of pain is a positive impingement test
result and usually indicates rotator cuff origin of the shoul-
der pain.
Radiographs in the early stages of cuff tendinopathy may
be normal or may reveal a hooked acromion. As the disease
progresses, there may be some sclerosis, cyst formation, and
sclerosis of the anterior third of the acromion and the greater
tuberosity. An anterior acromial traction spur may appear
on the undersurface of the acromion lateral to the AC joint
and represents contracture of the coracoacromial ligament.
Late radiographic findings include narrowing of the acro-
miohumeral gap, superior subluxation of the humeral head
in relation to the glenoid, and erosive changes of the ante-
rior acromion.70 Arthrography, MRI, and ultrasound may be
helpful in diagnosing a full-thickness tear of the rotator cuff
in association with stage III disease. In some cases of chronic
large rotator cuff tears, proximal migration of the humeral
head leads to a pattern of degenerative arthritis termed cuff-
tear arthropathy.
The choice of treatment and frequently its result are
functions of the stage of the impingement and response to
pain. In stage I disease, in which there is little mechanical
impingement, most patients respond to rest. It is important
not to immobilize the shoulder for any period because con-
traction of the shoulder capsule and periarticular structures
can produce an adhesive capsulitis. After a period of rest, a
progressive program of stretching and strengthening exer-
cises generally restores the shoulder to normal function. Use
of aspirin and other nonsteroidal anti-inflammatory drugs
(NSAIDs) may shorten the symptomatic period. Modalities
such as ultrasound, neuroprobe, and transcutaneous elec-
trical nerve stimulation generally are not helpful. Patients
with stage I and II disease may have a dramatic response
to local injection of glucocorticosteroids and local anes-
thetic agents. For stage II disease in which there is fibrosis
and thickening anteriorly, it is frequently better to inject
through a posterior approach. We prefer a combination of
3 mL of 1% lidocaine (Xylocaine), 3 mL of 0.5% bupiva-
caine, and 20 mg of triamcinolone. This injection combines
a short-acting anesthetic to help confirm the diagnosis, a
longer acting anesthetic for analgesic purposes, and a steroid
preparation in a depot form.
An integrated program of occupational and physical
therapy often precludes the need for surgery in patients
impingement syndrome caused by overuse may alleviate
symptoms. More businesses are becoming aware of the cost
savings associated with proper job ergonomics.71,72
The initial rehabilitation in stage II impingement is
the cessation of repetitive overhand activity. Ice, NSAIDs,
and local injections also may be beneficial. Initial physical
therapy includes passive, active assisted, and active range
of motion combined with stretching and mobilization exer-
cises to prevent contracture. As pain and inflammation sub-
side, isometric or isotonic exercises are used to strengthen
the rotator cuff musculature. Isokinetic training at variable
speeds and in variable positions is instituted before return-
ing the patient to full activity. For patients with a job-related
injury, it is crucial to review and modify job mechanics to
prevent recurrent episodes that can cause further disability
and may precipitate the need for surgery.71
Neer19 suggested that a patient with refractory stage II dis-
ease may respond to division of the coracoacromial ligament
and bursectomy of the subacromial bursa. Neer’s descrip-
tion of open anterior acromioplasty has become accepted
as the procedure of choice for stage II and III impingement
lesions, with many investigators reporting high success rates
in treating impingement syndrome and rotator cuff tears.73-76
Reported results show good and excellent relief of symptoms
in 71% to 87% of patients treated by the open surgical pro-
cedure.77-80
In 1985, Ellman45 described the technique of arthroscopic
subacromial decompression. His initial results46 and the
results of others are comparable to those of open surgical
techniques.47,81 Arthroscopic subacromial decompression
has become a widely accepted treatment for refractory stage
II and III impingement lesions. The procedure can be done
as outpatient surgery, and because no deltoid is detached as
with the open technique, the procedure facilitates rehabili-
tation and overall recovery rates.
Calcific Tendinitis
Calcific tendinitis is a painful condition around the rotator
cuff and is associated with deposition of calcium salts, pri-
marily hydroxyapatite.82-84 The cause of calcific tendinitis
is unknown. The commonly accepted cause is degeneration
of the tendon, which leads to calcification through a dystro-
phic process.84 A common clinicopathologic correlation is
three distinct phases of the disease process: the ­precalcific or
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
f­ormative phase, which can be relatively painless; the calcific
phase, which tends to be quiescent and may last months to
years; and the resorptive or postcalcific phase, which tends
to be painful, as calcium crystals are resorbed.82 Although
it is more common in the right shoulder, there is at least a
6% incidence of bilaterality. Patients with bilateral shoulder
involvement often have the syndrome of calcific periarthri-
tis, in which calcium hydroxyapatite crystals are found at
multiple sites.85 The patients usually present with impinge-
ment-type pain in the affected shoulder during overhead
activity. The pain may seem to be out of proportion to any
objective physical findings. There may be difficulty sleeping
on the shoulder and in falling asleep. The symptoms may
last a few weeks or a few months.
The incidence of calcific tendinitis varies in the litera-
ture among asymptomatic individuals from 2.7% to 20%.
Most calcification occurs in the supraspinatus tendon, and
57% to 76.7% of patients are women. The average age of
patients is 40 to 50 years.82,86
Codman1 pointed out the localization of calcification
within the tendon of the supraspinatus. He provided a detailed
description of the symptoms and the natural history of this
condition. In describing the phases of pain, spasm, limita-
tion of motion, and atrophy, he noted the lack of correla-
tion between symptoms and the size of the calcific deposit.
According to Codman, the natural history includes degen-
eration of the supraspinatus tendon, calcification, and even-
tual rupture into the subacromial bursa. During the latter
phase, pain and decreased motion can lead to adhesive cap-
sulitis (see Fig. 40-9).
Several factors may affect localization of the calcium
within the supraspinatus. Many of these patients have an
early stage of impingement, compressing the supraspinatus
tendon on the anterior portion of the acromion.9,19 This
long-standing impingement may lead to local degenera-
tion of the tendon fibers. In patients without impingement,
localization of the calcium within the supraspinatus may be
related to the blood supply of the rotator cuff, which nor-
mally is derived from an anastomotic network of vessels
from the greater tuberosity or the bellies of the short rotator
muscles.83 The watershed of these sources is just medial to
the tendinous attachment of the supraspinatus.87 Rathburn
and Macnab88 referred to this watershed as the critical zone
and pointed out that during abduction this area was ren-
dered ischemic.
Treatment of calcific tendinitis depends on the clinical
presentation and the presence of associated impingement.
These patients can have an acute inflammatory reaction
that may resemble gout. The acute inflammation can be
treated with local glucocorticoid injection, NSAIDs, or
both. Ultrasound may be beneficial. If there is associated
impingement, treatment depends on the stage at presenta-
tion. The radiographic appearance of the calcification can
direct and perhaps predict the response to therapy. In the
resorptive state, the deposits appear floccular, suggesting
that the process is in the phase of repair, and that a conser-
vative program is indicated.
Patients with discrete calcification and perhaps associ-
ated adhesive capsulitis (see Fig. 40-9) may be at a stable
phase in which the calcium produces a mechanical block
and is unlikely to be resorbed. For these patients, mechanical
removal of the calcific deposits and correction of ­associated
601
pathologic lesions may be necessary.89-91 Percutaneous dis-
ruption of the calcified areas may be performed using a nee­
dle directed by fluoroscopy. This technique allows lavage
and injection, but does not treat associated impingement.
Subacromial arthroscopy allows the mechanical débride-
ment of calcific deposits under direct visualization. This
technique can be combined with arthroscopic removal of
the inflamed bursa and decompression of associated impinge-
ment. In many cases of refractory calcific tendinitis associ-
ated with impingement, open or arthroscopic acromioplasty,
subacromial bursectomy, and decompression are indicated.
ROTATOR CUFF TEAR
Pathophysiology
Spontaneous tear of the rotator cuff in an otherwise normal
individual is rare.19 It can occur in patients with rheuma-
toid arthritis or systemic lupus erythematosus as part of the
pathologic process with invasion from underlying pannus.
Metabolic conditions such as renal osteodystrophy or agents
such as glucocorticoids occasionally are associated with cuff
tears. Most patients report a traumatic episode, such as fall-
ing on an outstretched arm or lifting a heavy object. The
usual presenting symptoms are pain and weakness of abduc-
tion and external rotation. There may be associated crepitus
and even a palpable defect. Long-standing tears generally
are associated with atrophy of the supraspinatus and infra-
spinatus muscles. It may be difficult to differentiate a pain-
ful tendinitis from a partial-thickness or small full-thickness
cuff tear.
Controversy exists about the exact cause of cuff tendi-
nopathy.87,91-93 Most likely, the pathophysiology involves a
combination of factors, including decreased vascularity and
cellularity of the tendon along with changes in the collagen
fibers of the tendon that occur with aging.
Loss of motion with subsequent capsular tightness, partic-
ularly the posterior capsule, may lead to cephalad migration
of the humeral head, with subsequent impingement of the
cuff under the coracoacromial arch.94 Rehabilitation exer-
cises stress regaining a normal range of motion. To achieve
full, painless motion, the normal relationship of glenohu-
meral-to-scapulothoracic motion must be achieved.16,17,95
Diagnosis
History. Patients with nontraumatic tears of the rotator cuff
report symptoms of chronic impingement. There is often a
loss of motion and feeling of stiffness with extremes of mo-
tion and difficulty during activities of daily living, such as
combing the hair, hooking a bra strap, putting on a shirt or
coat, and reaching into the back pocket. In chronic cases of
cuff tendinopathy, there is usually a loss of motion. Limita-
tion of internal rotation occurs initially, is caused by poste-
rior capsular contracture, and is often associated with poste-
rior shoulder pain with adduction of the ipsilateral shoulder.
Further shoulder impingement occurs with forward flexion
because of superior migration of the humeral head against
the anterior inferior acromion. This upward translation is
analogous to the action of a yo-yo climbing on a string.94,96
In time, loss of forward flexion, abduction, and external rota-
tion occurs with passive and active motion of the ­shoulder.
 602 Martin  |  Shoulder Pain
Imaging. In acute cases, there may be a history of trauma,
such as a fall onto the affected shoulder. In cases involving
an anterior shoulder dislocation with subsequent profound
weakness of the rotator cuff, a large tear on greater tuber-
osity avulsion should be suspected in addition to axillary
nerve palsy. In younger patients, traumatic failure of the cuff
under tensile overload may result in cuff failure because of
forced adduction of the affected shoulder or active abduc-
tion against resistance, and it may occur with traumatic
dislocation. Repetitive tensile overload also can result in
partial rotator cuff tears in an overhead athlete.
Plain radiographs are used in initial evaluation of
impingement-type shoulder pain with cuff tendinopathy.
An impingement series should be ordered, including an
anteroposterior radiograph with a 30-degree cephalic tilt
(Rockwood view), which can reveal osteophytes of the
anterior os acromion and AC joint; a scapular Y view with
a 10-degree cephalic tilt (supraspinatus outlet view), which
can evaluate the type of acromion and reveal anterior and
AC osteophytes; and an axillary view, which can evaluate
the acromion for possible os acromionale. Calcific depos-
its within the rotator cuff tendon can be viewed best with
rotational anteroposterior radiographs. Cuff arthropathy
should be suspected if the acromial-humeral distance is less
than 7 mm, or there is cyst formation within the greater
tuberosity, humeral head osteopenia, sclerosis around the
greater tuberosity, or humeral head collapse. In advanced
stages of cuff arthropathy, there may be complete loss of
glenohumeral joint space with superior migration and
abutment of the humeral head against the undersurface of
the acromion.58
In the past, shoulder arthrography was considered the
“gold standard” for diagnosing full-thickness and partial-
thickness rotator cuff tears, with greater than 90% sensitiv-
ity and specificity.33,97 Currently, arthrography with CT or
MRI is routinely used to diagnose rotator cuff pathology,
including full-thickness and partial-thickness tears.
Ultrasonography has been accurate in the diagnosis of
full-thickness rotator cuff tears.39,98-101 Ultrasonography has
advantages of being inexpensive and noninvasive, but dis-
advantages include unproven effectiveness in determining
subacromial impingement, capsular and labral abnormali-
ties, and partial cuff tears. The procedure and its results are
technician dependent. Ultrasonography may have a use-
ful role in determining postoperative integrity of the cuff
repair.38
MRI has been invaluable in evaluating rotator cuff tears.
The sensitivity and specificity for diagnosing full-thickness
cuff tears are 100% and 95%.102 Through the use of gado-
linium or saline, partial tears that are otherwise difficult to
detect with conventional imaging also can be detected.
Diagnosing cuff tears with MRI usually is based on dis­
continuity of the tendon on T1-weighted images and con-
sistency with fluid signal on T2-weighted images. Ancillary
findings include fluid in the subacromial space on T2-
weighted images, loss of the subacromial fat plane on T1-
weighted images, and proliferative spur formation of the
acromion or AC joint. Large, chronic cuff tears also may
be associated with cephalad migration of the humeral head
and fatty atrophy of the spinatus muscle. Periarticular soft
tissues, including the capsulolabral complex and biceps ten-
don, and the rotator cuff can be thoroughly examined. The
degree of tear, tendon retraction, and evidence of muscle
atrophy can be evaluated, all of which are crucial in preop-
erative planning for possible cuff repair.
Treatment
Nonsurgical Treatment. Codman and Akerson63 recom-
mended early operative repair for acute full-thickness rota-
tor cuff tears and reported the first documented repair in
1911. McLaughlin65 recommended early repair in cases
of grossly displaced tuberosity fractures or massive tears.
Several other clinical studies also supported the concept
that a full-thickness tear does not preclude good shoulder
function. DePalma103 reported that 90% of patients with
rotator cuff tears responded to conservative measures, such
as rest, analgesics, anti-inflammatory agents, and physio-
therapy.
The reported percentage of patients responding to non-
surgical treatment in the literature varies from 33% to
90%.3,18,104 Conservative treatment includes pain control
with NSAIDs, ultrasound, heat before shoulder stretching
and exercise, and ice after overhead activity. Deep mas-
sage therapy is employed to reduce trigger point tenderness
within the trapezius, levator scapulae, and periscapular mus-
cles. Patients on long-term anti-inflammatory medications
are monitored periodically for evidence of gastrointestinal
bleeding and for hepatic or renal toxicity. Opiate-based
drugs are used only in the acute setting, such as after a fall,
or in the perioperative period.
Steroid and local anesthetic injections are used when the
patient has significant pain that prohibits rehabilitation.
Injections may be repeated once every 3 months if needed;
injection into the cuff tendon is to be avoided. If the patient
fails to improve after 3 months of conservative treatment, or
does not continue to improve after three sequential injec-
tions, surgical options should be discussed.
The mainstay of conservative therapy is exercise. Reha-
bilitation stresses pain relief with exercises aimed at restor-
ing shoulder motion and strengthening the remaining cuff
muscles, deltoid, and scapular stabilizers. Therapy can be
divided into three phases. The goals of the initial phase
of therapy are to relieve pain and restore shoulder motion.
Motion therapy includes pendulum exercises, passive
motion with use of a wand with the assistance of the unin-
volved shoulder, an overhead pulley system, and posterior
capsular stretching. The arc of motion is gradually increased
and is guided by the patient’s discomfort to avoid painful
impingement arcs.
The second phase of therapy is entered after the patient
has return of motion and little discomfort with overhead
activity. Emphasis is placed on strengthening the remain-
ing rotator cuff musculature and deltoid and periscapular
muscles. Strengthening is done with elastic surgical tubing
that provides variable degrees of resistance, depending on
the size of the tubing. Initial strengthening is performed
out of the impingement arc (70 to 120 degrees of shoulder
flexion). The goal of this phase is to strengthen the shoul-
der to prevent dynamic proximal humeral migration with
impingement during active shoulder elevation.58,60 Normal
shoulder kinematics relies on combined and synchronous
glenohumeral flexion and scapular rotation.59,91 In addition
to strengthening the cuff and deltoid, the scapular rotators,
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
including the trapezius and serratus anterior muscles, are
emphasized.105
After the patient has successfully completed phase two
of the rehabilitation program with minimal symptoms and
good shoulder function, the final phase is entered. Phase
three is characterized by a gradual return to normal over-
head activities, including work and sporting activities. This
part of the rehabilitation program should be tailored to the
individual patient’s needs and the demands placed on the
shoulder.
Surgical Treatment. Severity and duration of pain are the
primary indications for surgical intervention in a rotator
cuff tear. Other factors important in surgical decision mak-
ing include shoulder dominance, activity level, physiologic
age, acuteness of the tear, degree of tear, loss of function,
amount of tendon retraction, and fatty atrophy of the re-
maining cuff musculature.
Acute Tears. Acute tears of the rotator cuff can be treat-
ed with conservative measures of periscapular and cuff
strengthening along with capsular stretching to restore mo-
tion. Early surgical intervention should be considered in
a young patient, however, especially an overhead athlete.
Conservative shoulder rehabilitation should be maintained
for 3 to 6 months before deciding on surgery for an older sed-
entary patient, in whom functional results without surgery
may be acceptable. Many older patients may function well
with chronic cuff tears, but they may become debilitated if
an acute tear is superimposed on chronic changes. Surgical
intervention may be required in these cases to return the
patient to baseline function by repairing the acute tear and
attempting to repair the chronic tear if possible.
Chronic Tears. For elderly patients whose pain and weak-
ness do not create a functional problem, a conservative pro-
gram is preferable for chronic tears. Pain unresponsive to
conservative management is the main indication for surgery
in an older patient with a chronic rotator cuff tear. In these
cases, surgery should be considered on an individual basis
after at least 3 months of conservative treatment, including
subacromial steroid injection. If the cuff tear is massive and
irreparable, débridement and subacromial decompression
may provide good pain relief without extensive surgery and
prolonged immobilization.46,81,106-110 In a younger patient
with a chronic tear and weakness, surgery to repair the cuff
may be indicated to improve strength and prevent further
extension of the tear.108
In cases of rotator cuff arthropathy with glenohumeral
joint degeneration, a reverse total shoulder replacement may
be indicated. This type of total shoulder replacement reverses
the normal relationship between scapular and humeral com-
ponents, moving the center of rotation ­medially and distally
to increase the lever arm length of the deltoid muscle. The
deltoid compensates for the deficient rotator cuff, allowing
as near-normal function as possible (Fig. 40-15).
BICIPITAL TENDINITIS AND RUPTURE
The long head of the biceps passes through the bicipital
groove, crosses over the head of the humeral, and inserts
on the superior rim of the glenoid (see Fig. 40-2A).111 The
603
biceps tendon aids in flexion of the forearm, supination of
the pronated forearm if the elbow is flexed, and forward ele-
vation of the shoulder.3 Bicipital tendinitis, subluxation or
dislocation of the biceps tendon within the bicipital groove,
and rupture of the long head of the biceps generally are asso-
ciated with anterior shoulder pain.
Bicipital tendinitis is sometimes an associated feature of
a rotator cuff tear. The rotator cuff tear compromises center-
ing of the humeral head on the glenoid. This compromise
results in increased mechanical loading of the long head of
the biceps, which initiates a hypertrophic tendinitis.112
Dislocation of the long head of the biceps usually is
combined with a lesion of the subscapularis tendon.12 Iso-
lated rupture of the long head of the biceps tendon is rare
when the rotator cuff is intact. Rupture of the long head of
the biceps is common, however, when there is a coexist-
ing rotator cuff tear.113 The effect of rotator cuff tear and
concomitant biceps tendon rupture on strength can be sub-
stantial.12
The early phases of bicipital tendinitis are associated with
hypervascularity, edema of the tendon, and tenosynovi-
tis.114 Persistence of this process leads to adhesions between
the tendon and its sheath, with impairment of the normal
gliding mechanism in the groove. Stretching of the adhe-
sions may be associated with chronic bicipital tendinitis.115
The diagnosis of bicipital tendinitis is based on the localiza-
tion of tenderness. It is often confused with impingement
symptoms and is frequently seen with an impingement syn-
drome.24 Isolated bicipital tendinitis can be differentiated
by the fact that the tender area migrates with the bicipi-
tal groove as the arm is abducted and externally rotated.
Many eponyms are associated with tests to identify bicipital
tendinitis.3 Yergason’s supination sign refers to pain in the
bicipital groove when the examiner resists supination of the
pronated forearm with the elbow at 90 degrees. Ludington’s
Figure 40-15  Reverse total shoulder replacement in a 72-year-old man
who had severe cuff arthropathy.
 604 Martin  |  Shoulder Pain
sign refers to pain in the bicipital groove when the patient
interlocks the fingers on top of the head and actively abducts
the arms.
Biceps tendon ruptures can occur in some patients who
report no history of shoulder pain. The patients often com-
plain of an acute onset of pain and ecchymosis around the
anterior shoulder and sagging of the biceps muscle belly.
In these cases, a concomitant rotator cuff injury should be
excluded by clinical examination. More often, the biceps
tendon rupture is preceded by painful shoulder symptoms
that often improve or disappear after the rupture.115,116
Treatment generally is conservative and consists of rest,
analgesics, NSAIDs, and local injection of glucocorticoids.
The use of ultrasound and a neuroprobe is more beneficial
in this condition than in isolated rotator cuff tendinitis.
Patients with refractory bicipital tendinitis and recurrent
symptoms of subluxation are treated by arthroscopic biceps
tenodesis or open tenodesis, opening the bicipital groove
and resecting the proximal portion of the tendon with teno-
desis of the distal portion into the groove or beneath the
pectoralis tendon.
ACROMIOCLAVICULAR DISORDERS
The AC joint is a common source of shoulder pain. Acute
causes of AC joint pain are often related to direct trauma
of the affected shoulder that may result in a distal clavicle
injury with an intra-articular chondral fracture or in AC
joint instability from ligamentous disruption.117
Post-traumatic distal clavicle osteolysis, with resorption
of the distal clavicle, may ensue 4 weeks after a shoulder
injury, leading to AC joint pain.118,119 Osteolysis may be
caused by microfractures of the subchondral bone and subse-
quent attempts at repair.120 Other authors believe the cause
to be an autonomic nerve dysfunction affecting the blood
supply to the clavicle. The increased blood supply leads
to resorption of bone from the distal clavicle.118,121 More
commonly, chronic osteolysis results from repetitive micro-
trauma to the AC joint from activities such as weightlifting,
gymnastics, and swimming.120,122,123
The underlying pathophysiology is believed to be an
inflammatory process from stress fractures of the subchondral
bone with hyperemic resorption of the distal clavicle.120,124
Other causes of osteolysis include rheumatoid arthrosis,
hyperparathyroidism, and sarcoidosis, which should be con-
sidered in the differential diagnosis, especially in bilateral
cases.118,119 Patients with atraumatic osteolysis of the distal
clavicle should be forewarned that bilateral involvement
may occur, with an incidence of 70% reported for one long-
term follow-up.125 Other chronic causes of AC pain include
idiopathic, intra-articular disk pathology, post-­traumatic
degenerative arthrosis from joint incongruity, primary degen­
erative arthrosis, and rheumatoid arthrosis.
Evaluation always should include a detailed history,
physical examination, and radiographic evaluation. There
may be a history of trauma to the AC joint from a direct fall
or blow to the ipsilateral shoulder. Less commonly, the AC
joint may be injured indirectly, such as during a fall on the
outstretched arm with the forces being transmitted through
the arm to the AC joint.117,126 Patients with osteolysis of
the distal clavicle sometimes give a history of acute trauma,
although the more common cause is repetitive microtrauma
to the AC joint from activities such as weightlifting or
gymnastics.118,119,122,123
Patients frequently complain of pain over the AC joint
when adducting the ipsilateral shoulder, such as during
a golf swing or when buckling a seat belt. Often, there is
pain when sleeping on the affected shoulder. Athletes may
experience AC joint pain on bench pressing, push-ups, and
dips.125,127,128 Pain and weakness of the affected shoulder
also may be experienced with forward flexion and adduction
of the arm.118
On physical examination, there may be a visible step-
off between the medial acromion and the distal clavicle,
indicating a probable AC separation. Pain usually can be
elicited on direct palpation of the AC joint and is made
worse by a cross-arm adduction maneuver. This test is per-
formed by internally rotating the arm, which is maximally
adducted across the chest and is considered positive if pain
is produced in the AC joint (see Fig. 40-4D). Pain also may
be elicited by moving the arm from a horizontally abducted
position to the extended position and on maximal internal
rotation of the shoulder.127,129 These tests cause rotation
and compression of the AC joint and are sensitive but less
specific. They also may be positive with other disorders of
the shoulder, such as posterior capsular stiffness.130
Frequently, AC joint pain coexists with subacromial
impingement and rotator cuff pathology. In these cases,
im­pingement signs are positive, and rotator cuff weakness
may be present. Otherwise, there should be no detectable
muscle weakness on manual resistance testing and no evi-
dence of muscle atrophy.125,130,131 The AC joint and sub-
acromial space may have to be injected on separate occasions
to determine the true source of the symptoms. Some physi-
cians have noticed an association of AC joint symptoms
with shoulder instability.125 Glenohumeral motion can vary,
depending on the chronicity and isolation of the problem
to the AC joint. In isolated cases, there may be some loss of
internal rotation of the affected shoulder because of pain.
Radiographs should include anteroposterior views of
the shoulder in the scapular plane in neutral, internal, and
external rotation; a transcapular Y view; an axillary view;
and a 15-degree cephalic tilt view of the AC joint at 50%
penetrance as described by Zanca (see Fig. 40-5).23 Stress
views may be obtained by strapping 5 to 10 lb of weight
to the forearms and determining AC separation. Compar-
ing the coracoclavicular distance of both shoulders also may
be helpful. When clinically indicated, cervical spine radio-
graphs should be obtained to exclude cervical spondylosis.
Radiographic evaluation may reveal AC joint arthrosis
with microcystic changes in the subchondral bone, sclerosis,
osteophytic lipping, and joint space narrowing.132 In cases
of osteolysis, radiographs may reveal a loss of subchondral
bone detail with microcystic appearances in the subchondral
region of the distal clavicle and osteopenia of the lateral one
third of the clavicle.119,120,122,123 In the late stages of oste-
olysis, resorption of the distal end of the clavicle results in
marked widening of the AC joint and sometimes complete
resorption of the distal clavicle. There may be evidence of
AC separation with widening of the coracoclavicular dis-
tance and post-traumatic ossification of the coracoclavicu-
lar ligaments.
The AC symptoms do not always correlate with the
radiographic appearance of the joint. DePalma133 found
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
AC joint degeneration to be an age-related process, with
symptoms not always correlating with radiographic findings
of AC joint arthrosis.23 AC joint pain may occur despite
normal radiographs.134
A technetium 99m phosphate bone scan may assist in the
diagnosis, revealing increased uptake in the distal clavicle
and medial acromion.120 In cases of atraumatic osteolysis of
the distal clavicle, increased uptake may be isolated to the
distal clavicle, but in approximately 50% of cases, there is
increased scintigraphic activity of the adjacent medial acro-
mion.125 The bone scan may reveal pathologic changes of
the AC joint when plain radiographs appear normal.
In selected cases, MRI can be valuable in determining
a diagnosis and evaluating the glenohumeral and subacro-
mial regions for coexisting pathology (Fig. 40-16). AC
joint involvement may reveal increased fluid with synovitis,
soft tissue enlargement, and periarticular ossifications with
encroachment on the underlying bursal and cuff tissue.
Patients with AC joint pain usually respond well to
nonoperative treatment; however, complete relief of symp-
toms may take an extended period. Conservative therapy
includes heat, NSAIDs, steroid injections, shoulder reha-
bilitation, and avoidance of painful positions and activities.
Steroid injections are repeated at 3-month intervals if pain-
ful conditions persist.
Open resection of the distal clavicle for chronic AC joint
pain was initially reported by Gurd135 and by Mumford,136
both with good results. Since then, other surgeons have
reported similar good results with open resection, but sig-
nificant morbidity, such as disruption of the deltotrapezial
fascia and anterior deltoid rupture, can occur.118,119,132,134,137
Arthroscopic resection of the distal clavicle has been described
with results similar to open resection.13,124,129-131,138-141
Figure 40-16  Sagittal section MRI of the shoulder in a 32-year-old
weightlifter complaining of shoulder pain. Fat-suppressed proton den-­
sity fast spin echo images of bursal-side high-grade partial cuff tear (small
arrowheads).
605
GLENOHUMERAL DISORDERS
The various arthritides that affect the shoulder joint are
discussed in detail in other chapters. They are presented
here to address aspects that are unique to the glenohumeral
joint. The usual presentation of intra-articular disorders
is pain with motion and symptoms of internal derange-
ment, such as locking and clicking. The pain is generalized
throughout the shoulder girdle and sometimes referred to
the neck, back, and upper arm. The usual response to pain
is to decrease glenohumeral motion and substitution with
increased scapulothoracic mobility. Patients with adequate
elbow and scapulothoracic motion require little glenohu-
meral motion for activities of daily living; patients with gle-
nohumeral arthrodesis can achieve adequate function.142,143
The response to pain is diminution of motion and secondary
soft tissue contractures with muscle atrophy. With increasing
weakness and involvement of adjacent joints, pain, limita-
tion of motion, and weakness cause a substantial functional
deficit.
INFLAMMATORY ARTHRITIS
Although the most common inflammatory arthritis involv-
ing the shoulder joint is rheumatoid arthritis, other systemic
disorders, such as systemic lupus erythematosus, psori-
atic arthritis, ankylosing spondylitis, Reiter’s syndrome,
and scleroderma, may cause glenohumeral degeneration.
Motion is limited by splinting of the joint with secondary
soft tissue contractures or by primary soft tissue involvement
with scarring or rupture. Plain radiographs confirm glenohu-
meral involvement (Fig. 40-17A). There is narrowing of the
glenohumeral joint space, with erosion and cyst formation
without significant sclerosis or osteophytes. As the disease
progresses, superior and posterior erosion of the glenoid
with proximal subluxation of the humeral head may occur.
Eventually, there may be secondary degenerative changes
and even osteonecrosis of the humeral head.
Treatment is initially conservative and directed toward
controlling pain, inducing a systemic remission, and main-
taining joint motion by physical therapy. The use of intra-
articular glucocorticoids may be beneficial in controlling
local synovitis. In rheumatoid arthritis, the involvement of
periarticular structures with subacromial bursitis and rupture
of the rotator cuff magnifies the functional deficit. When
the synovial cartilage interactions produce significant symp-
toms and radiographic changes that cannot be controlled
by conventional therapy, glenohumeral resurfacing should
be considered.
When following a rheumatoid patient with shoulder
involvement, the rheumatologist should assess range of
motion carefully and obtain periodic radiographs. Patients
with progressive loss of motion or radiographic destruction
should be referred for evaluation for possible surgical treat-
ment. The treatment of choice is an unconstrained total
shoulder arthroplasty.144,145 Total shoulder arthroplasty is
best performed in patients with rheumatoid arthritis before
end-stage bony erosion and soft tissue contractions have
occurred.146,147 Acute inflammatory arthritis of the gleno-
humeral joint also may be associated with gout, pseudo­gout,
hydroxyapatite deposition of renal osteodystrophy, and
recurrent hemophilic hemarthrosis.
 606 Martin  |  Shoulder Pain
A B
Figure 40-17  Plain radiographs. A, Rheumatoid arthritis with loss of joint space, cyst formation, glenohumeral erosion, and early proximal subluxation
of the humerus, indicating a rotator cuff tear. B, Osteoarthritis with narrowing of the glenohumeral joint space, sclerosis, and osteophyte formation.
Notice the preservation of the subacromial space, suggesting an intact rotator cuff.
OSTEOARTHRITIS
Osteoarthritis of the glenohumeral joint is less common
than that in the hip, its counterpart in the lower extremity;
this is the result of the non–weight-bearing characteristics of
the shoulder joint and the distribution of forces throughout
the shoulder girdle. Osteoarthritis is divided into conditions
associated with high unit loading of articular cartilage and
conditions in which there is an intrinsic abnormality within
the cartilage that causes abnormal wear at normal loads.
Because the shoulder is normally a non–weight-bearing joint
and is less susceptible to repeated high loading, the presence
of osteoarthritis of the glenohumeral joint should alert the
physician to consider other factors. Has the patient engaged
in unusual activities, such as boxing, heavy construction, or
chronic use of a pneumatic hammer? Is there some disorder,
such as epiphyseal dysplasia, that has created joint incongru-
ity with high unit loading of the articular cartilage? Is this
a neuropathic process caused by diabetes, syringomyelia, or
leprosy? Is there associated hemochromatosis, hemophilia,
or gout that may have altered the ability of articular carti-
lage to withstand normal loading? Is unrecognized chronic
dislocation responsible?
Pain is the usual presentation, but it is generally not
as acute or associated with the spasm seen in inflamma-
tory conditions. Plain radiographs show narrowing of the
glenohumeral joint, osteophyte formation, sclerosis, and
some cyst formation (see Fig. 40-17B). Because the rota-
tor cuff usually is intact, there is less bone erosion of the
glenoid and proximal subluxation of the humerus. Patients
with osteoarthritis of the glenohumeral joint frequently do
well with functional adjustments and conservative ther-
apy. Analgesics and NSAIDs may provide sympto­matic
relief. The use of glucocorticoid injections is less ben-
eficial, unless there is evidence of synovitis. Patients with
severe involvement who fail to respond are best treated by
­shoulder arthroplasty.144-147
OSTEONECROSIS
Osteonecrosis of the shoulder refers to necrosis of the humer­
­al head seen in association with a variety of conditions.
Symptoms are due to synovitis and joint incongruity ­resulting
from resorption, repair, and remodeling. The pathogenesis
and various causes are discussed in Chapter 94.
The most common cause of osteonecrosis of the shoulder
is avascularity resulting from a fracture through the anatomic
neck of the humerus.148 Fracture through this area disrupts
the intramedullary and capsular blood supplies to the humer­
­al head.149 Another common cause of osteonecrosis of the
shoulder is steroid therapy in conjunction with organ trans-
plantation, systemic lupus erythematosus, or asthma. Other
conditions associated with osteonecrosis of the humeral head
include hemoglobinopathies, pancreatitis, and hyperbarism.
Early diagnosis is difficult because there is frequently a
considerable delay until symptoms are present. Bone scans
may be helpful in early cases before radiographic changes
are evident. MRI is highly sensitive and more specific than
scintigraphy. Plain radiographs show progressive phases of
necrosis and repair (as discussed in Chapter 94). In the early
stages, the films may be normal or show osteopenia or bone
sclerosis. A crescent sign representing subchondral fracture
or demarcation of the necrotic segment appears during the
reparative process. Patients who fail to remodel show collapse
of the humeral head with secondary degenerative changes.
There is often a considerable discrepancy between symptoms
and radiographic involvement. Patients with extensive bone
changes may be asymptomatic. ­Treatment should be directed
by the patient’s symptoms rather than the radiographs and is
similar to that for osteoarthritis. Arthroscopy occasionally is
helpful by removing loose chondral fragments and débriding
chondral incongruities.150 Patients with severe symptoms
that cannot be controlled by conservative means are best
treated with an unconstrained shoulder arthroplasty, hemi-
arthroplasty, or resurfacing arthroplasty.144
CUFF-TEAR ARTHROPATHY
In 1873, Adams described the pathologic changes in rheuma-
toid arthritis of the shoulder and a condition that has since
been referred to as Milwaukee shoulder or cuff-tear arthropa-
thy.151 McCarty called the condition Milwaukee shoulder
and reported that the factors predisposing to this syndrome
included deposition of calcium pyrophosphate dihydrate
crystals, direct trauma, chronic joint overuse, chronic renal
­failure, and denervation.152 Patients with Milwaukee shoulder
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
have elevated levels of synovial fluid 5-nucleotidase activity
and elevated levels of synovial fluid inorganic pyrophosphate
and nucleotide pyrophosphohydrolase activity.153
Neer and colleagues154 reported a similar condition in
which untreated massive tears of the rotator cuff with proxi-
mal migration of the humeral head are associated with ero-
sion of the humeral head. The erosion of the humeral head
differs from that seen in other arthritides and is presumed to
be caused by a combination of mechanical and nutritional
factors acting on the superior glenohumeral cartilage.
Patients with cuff-tear arthropathy present a difficult
therapeutic problem because the bone erosion and disruption
of the cuff jeopardize the functional result from an uncon-
strained prosthesis.146 Hemiarthroplasty or a reverse total
shoulder arthroplasty may be indicated.155,156 The major
question in treating cuff-tear arthropathy is to determine
which patients with massive rotator cuff tears will proceed
to the syndrome of cuff-tear arthropathy. Patients with mas-
sive rotator cuff tears who develop localized calcium pyro-
phosphate disease may be predisposed to further proximal
migration and further joint destruction. This situation poses
a dilemma for the treating physician. Many patients with
massive rotator cuff tears remain stable and require little or
no treatment. Occasionally, symptomatic patients can be
treated by arthroscopic débridement of the cuff tear. It is cru-
cial to define the patient who will proceed to the syndrome
of cuff-tear arthropathy. If crystal deposition disease predis-
poses patients to proximal migration and joint destruction,
joint aspiration with crystal analysis or scintigraphy to deter-
mine synovial reaction may be helpful diagnostic tools.
Hamada and coworkers157 followed 22 patients with
massive rotator cuff tears treated conservatively. The radio-
graphic findings included narrowing of the acromiohumeral
interval and degenerative changes of the humeral head,
tuberosities, acromion, AC joint, and glenohumeral joint.
Five of seven patients followed for more than 8 years pro-
gressed to cuff-tear arthropathy. The investigators concluded
that progressive radiographic changes were associated with
repetitive use of the arm in elevation, rupture of the long
head of the biceps, impingement of the humeral head against
the acromion, and weakness of external rotation.157
SEPTIC ARTHRITIS
Septic arthritis can masquerade as any of the conditions clas-
sified as periarticular or glenohumeral disorders (see Chap-
ters 90 and 100). Sepsis must be included in any differential
diagnosis of shoulder pain because early recognition and
prompt treatment are necessary to achieve a good functional
result. The diagnosis is confirmed by joint aspiration with
synovial fluid analysis and culture. Cultures should include
aerobic, anaerobic, mycobacterial, and fungal studies.
LABRAL TEARS
The glenoid labrum increases the depth of the glenoid and
serves as an anchor for the attachment of the glenohumeral
ligaments. Historically, labral tears have been difficult to
diagnose. Findings on physical examination can be con-
fused with impingement and rotator cuff tendinopathy and
bicipital tendinitis. Diagnosis can be confirmed with MRI-
arthrography, CT-arthrography, and DCAT.27 Arthroscopy
607
has greatly increased the knowledge of the glenoid labrum
in normal and pathologic situations and has aided the diag-
nosis and treatment of labral lesions.
Labral tears can be divided into tears associated with
symptoms of internal derangement and tears associated with
anterior or posterior instability. A soft tissue Bankart lesion
is associated with a tear of the anterior band of the infe-
rior glenohumeral ligament and is associated with anterior
instability. Isolated labral tears that do not involve detach-
ment of the ligaments can cause internal derangement and
may have an arthroscopic appearance similar to a meniscal
tear of the knee.
Andrews and associates7 first described lesions of the
anterior superior labrum in throwing athletes; these lesions
were often associated with biceps tendon tears (10%). The
tear results from traction of the biceps tendon. Snyder and
coworkers158 introduced the term SLAP lesion in 1990 to
describe an injury involving the long head of the biceps ten-
don and the superior portion of the glenoid labrum.
The long head of the biceps tendon originates at the
supraglenoid tubercle and glenoid labrum in the superior-
most portion of the glenoid. The major portion of the ten-
don blends with the posterior superior aspect of the labrum.
The most common mechanism of a SLAP injury is a fall
onto the outstretched arm with the shoulder in abduction
and slight forward flexion.158 The lesion also can result from
an acute traction on the arm and from an abduction and
external rotation mechanism.160
Patients usually complain of pain with overhead activi-
ties and a frequent catching or popping sensation in the
shoulder. The most reliable diagnostic test is the O’Brien
test. The test is performed against resistance with the arm
in forward flexion with the elbow extended and the forearm
pronated. In the second part of the test, the arm is supi-
nated. Less pain occurring during the latter part of the test
suggests a SLAP lesion.158 The most accurate diagnostic
test is MRI-arthrography with gadolinium.161 Treatment for
symptomatic SLAP lesions is surgical.
ADHESIVE CAPSULITIS
Adhesive capsulitis, or frozen shoulder syndrome (FSS), is a
condition characterized by limitation of motion of the shoul-
der joint with pain at the extremes of motion. It was first
described by Putman162 in 1882 and later by Codman.1 The
initial presentation is pain, which is generalized and referred
to the upper arm, back, and neck. As the pain increases, loss
of joint motion ensues. The process is generally self-limiting
and in most cases resolves spontaneously within 10 months,
unless there is an underlying problem.
The exact cause of FSS is unknown.91,163 It is frequently
associated with conditions such as diabetes mellitus, par-
kinsonism, thyroid disorders, and cardiovascular disease.
When one of these conditions exists, there is often a his-
tory of some mild trauma that initiated the frozen shoulder.
Major skeletal trauma and soft tissue injury may coexist with
FSS. It also may be seen with a variety of other conditions,
including apical lung tumors, pulmonary tuberculosis, cer-
vical radiculopathy, and post–myocardial infarction.164-166
In one review of FSS, 3 of 140 patients with this syn-
drome had local primary invasive neoplasms.167 Another
study reviewed three patients with adhesive capsulitis
 608 Martin  |  Shoulder Pain
who subsequently were found to have a neoplastic lesion
of the midshaft of the humerus.168 In a high-risk patient
with an underlying disorder, even minor surgery or trauma
in a remote location, such as the hand, can precipitate
FSS.91,169,170
The pathophysiology involves a diffuse inflammatory
synovitis with subsequent adherence of the capsule and a
loss of the normal axillary pouch and joint volume, which
leads to a significant loss of motion. Capsular contracture
is thought to result from adhesion of the capsular surfaces
or fibroblastic proliferation in response to cytokine produc-
tion.163,169,171 The condition is more common in women in
their 40s and 50s. Typically, the patient relays a history of
diffuse, dull aching around the shoulder, with weakness and
loss of motion occurring over a few months.
Usually, there are three distinct clinical stages of the syn-
drome. Stage one is the painful or freezing phase. During
this stage, the pain is severe, is exacerbated by any attempts
at movement, and usually lasts a few weeks or months. The
patient usually feels most comfortable with the arm at the
side in an adducted and internally rotated position. Phase
two is the adhesive or stiffening phase and generally lasts
4 to 12 months. Pain is usually minimal during this phase,
although periscapular symptoms may develop from compen-
satory motion to achieve elevation of the arm. The third
phase of the syndrome is the resolution or thawing phase
and may last 5 to 26 months. During this time, the pain
eases, and motion slowly improves, although some patients
may improve dramatically over a short period.172
In the early stages, any attempts at motion may produce
severe pain and associated weakness. The syndrome usually
is associated with a prolonged period of immobilization.173
Night pain is common, with an inability to sleep on the
associated shoulder, which is similar to findings of impinge-
ment syndrome.
In patients with a history of minimal or no trauma and FSS,
a metabolic cause should be excluded. A complete blood cell
count, erythrocyte sedimentation rate, serum chemistry, and
thyroid function tests are done as a screening panel. Further
testing is done if the results suggest the possibility that the
patient may have a systemic illness. Plain radiographs should
include true anteroposterior, axillary, and scapular Y views of
the shoulder. In patients with no underlying detectable ill-
nesses and a negative workup, a Tc 99m pertechnetate scan
may show increased uptake in FSS, but more importantly, it
is used to exclude occult lesions or metastasis.174
Treatment of FSS is mainly conservative using intra-
articular injections, heat, gentle stretching, NSAIDs, and
modalities such as transcutaneous electrical nerve stimula-
tion. The disease usually is self-limited and, after the painful
phase, is not severely disabling. Communication between
the physician and the patient, with a thorough explanation
of the condition, is essential because resolution of the syn-
drome occurs slowly over time. Closed manipulation and
surgery (open and arthroscopic) are reserved for patients
whose condition is recalcitrant to conservative measures
or for whom the diagnosis is in question. Paramount in
the prevention of FSS is avoiding overimmobilization in a
minor shoulder injury, in addition to careful identification
of patients at risk for FSS.
Fareed and Gallivan175 reported good results with hy­­
draulic distention of the glenohumeral joint using local
anesthetic agents. Rizk and associates176 conducted a pro-
spective, randomized study to assess the effect of steroid or
local anesthetic injection in 48 patients with FSS. There
was no significant difference in outcome between individu-
als who received intrabursal or intra-articular injection. Ste-
roid with lidocaine had no advantage over lidocaine alone
in restoring shoulder motion. Transient pain relief occurred,
however, in two thirds of the steroid-treated patients.176
General anesthesia occasionally is indicated for closed
manipulation. Hill and Bogumill177 reported the results of
manipulation of 17 frozen shoulders in 15 patients who did
not respond to physical therapy. An average 2.6 months
after manipulation, 78% of individuals working before their
shoulder problems returned to work. The investigators con-
cluded that manipulation allowed patients to return to a nor-
mal lifestyle and to work sooner than the reported natural
history of the condition.177 Surgical intervention for adhe-
sive capsulitis should be limited to treatment of an underly-
ing problem, such as calcific tendinitis or an impingement
syndrome.
GLENOHUMERAL INSTABILITY
Glenohumeral instability is a pathologic condition that
manifests as pain associated with excessive translation of
the humeral head on the glenoid during shoulder motion.
Instability can range from excessive laxity with episodes of
subluxation to frank dislocation of the joint. Traumatic dis-
location of the glenohumeral joint has characteristic clini-
cal and radiographic findings that are beyond the scope of
this chapter and have been reviewed in detail elsewhere.178
The most common type of instability is anterior, although
posterior and multidirectional laxity of the shoulder are
increasingly recognized as causes of shoulder pain. Ante-
rior dislocation usually occurs with the arm in an abducted
and externally rotated position, and the diagnosis is usually
obvious. Posterior dislocation is frequently associated with
convulsive disorders or unusual trauma with the arm in a
forward flexed and internally rotated position. The diagnosis
is often missed and should always be suspected in the patient
who is unable to rotate the arm externally after trauma.
Recurrent subluxation without dislocation may be diffi-
cult to diagnose and may be mistakenly identified as impinge-
ment with chronic cuff tendinitis. An overhead athlete
may experience repetitive stresses to the shoulder, causing
microtrauma to the static stabilizers. Jobe and ­colleagues21
described the syndrome of shoulder pain in overhead or
throwing athletes that manifests as impingement, but is
caused by anterior subluxation of the joint with the humeral
head impinging on the anterior aspect of the coracoacro-
mial arch. Fu and coworkers179 underscored this distinction
by dividing the cause of rotator cuff tendinitis into primary
impingement of the tendon on the coracoacromial arch and
anterior subluxation with secondary impingement in young
athletes performing overhead movements. Walch and col-
leagues180 described intra-articular impingement between
the undersurface of the rotator cuff (supraspinatus and infra-
spinatus) and the posterior superior glenoid rim and labrum.
This “internal impingement” usually is observed in over-
head athletes with subtle anterior glenohumeral instability
and results in tendinitis or partial tears of the rotator cuff
(see Fig. 40-17).
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
The diagnosis of glenohumeral instability with sublux-
ation in one or multiple directions is made by the combi-
nation of a detailed history and physical examination and
the use of adjuncts, such as arthrography, CT, MRI, and
arthroscopy with examination under anesthesia. The syn-
drome of multidirectional instability has been recognized in
patients with symptomatic inferior instability in addition
to anterior or posterior instability. Approximately 50% of
affected patients have evidence of generalized laxity. Fre-
quently, the syndrome occurs in young athletic patients
who are loose jointed, in particular, in the dominant arm of
pitchers, racket sports players, and swimmers. In this type of
athlete, repetitive microtrauma may cause stretching of the
shoulder, resulting in a large capsular pouch without labral
detachment. A traumatic event may damage the shoulder,
resulting in the syndrome of multidirectional instability and
a Bankart lesion.181
The most common manifestation in these patients is
pain, often mistakenly considered to be rotator cuff ten-
dinitis. The patient may relate a history of minor trauma
causing acute pain and a “dead arm” syndrome lasting min-
utes or hours. Other associated symptoms include a sense of
instability, weakness, and radicular symptoms suggestive of
neuropathy. There may be few or no positive physical find-
ings associated with chronic subluxation or multidirectional
instability. The patient may have signs of generalized liga-
mentous laxity, and pain may be reproduced by subluxating
the glenohumeral joint in multiple directions. One particu-
larly helpful sign for inferior laxity is the sulcus sign, which
refers to the subacromial indentation that occurs when lon-
gitudinal traction is applied to the humerus with the arm
at the side. The sign occurs with inferior translation of the
humeral head. Because this syndrome frequently occurs
in athletes with highly developed musculature around the
shoulder girdle, these physical findings of subluxation may
be difficult to reproduce in the office setting.
Plain radiographs are generally normal, although some
inferior subluxation may be shown by obtaining stress radio-
graphs with weights. Special radiographs, as discussed previ-
ously, may show a Bankart lesion (i.e., avulsion of the anterior
inferior glenoid rim) or a Hill-Sachs lesion (i.e., osteochon-
dral defect of the posterior humeral head) occurring with
subluxation of the humeral head in front of the anterior
glenoid rim. CT-arthrography or MRI-arthrography may
show increased capsular volume, a labral detachment, or a
Hill-Sachs lesion (see Fig. 40-10). When surgery is indicated,
examination under anesthesia and shoulder arthroscopy may
assist in diagnosing the primary direction of instability in the
syndrome of multidirectional instability. In selected patients
with traumatic anterior dislocation without a history of mul-
tidirectional instability, arthroscopic stabilization may be
done with stabilization of the capsulolabral complex.
Treatment of chronic subluxation or the syndrome of
multidirectional instability is first directed toward prolonged
rehabilitation. Activities that stress the shoulder and produce
symptoms are avoided. Strengthening exercises of the shoulder
girdle may control symptoms, dynamically stabilizing the gleno-
humeral joint, and obviate the need for surgical intervention.
If a conservative treatment program fails, surgery is performed
on the side associated with the greatest amount of clinical
instability. Stabilization is directed toward tightening of the
capsular structures to stabilize the glenohumeral joint.181,182
609
EXTRINSIC OR REGIONAL FACTORS
CAUSING SHOULDER PAIN
Because the shoulder girdle connects the thorax with the
upper extremity, and the major neurovascular structures
pass in proximity to the joint, shoulder pain is a hallmark of
many nonarticular conditions.
CERVICAL RADICULOPATHY
Cervical pathology may manifest with associated shoulder
pain. The area of referred pain is along a dermatomal pat-
tern consistent with the distribution of the dermatomal nerve
roots. Isolation of the pain usually defines the exact location of
the associated cervical pathology. It can be differentiated from
shoulder pain on the basis of history, physical examination,
EMG, cervical radiographs, and myelography or MRI when
indicated. Because conditions causing cervical neck pain and
conditions causing shoulder pain, such as calcific tendinitis
and cervical radiculopathy, may coexist, it is often difficult
to distinguish which lesion is responsible for the symptoms.
These conditions often can be differentiated by injection of
local anesthetics to block certain components of the pain.
The thoracic outlet is an interval created by the anterior
and middle scalene muscles and the first rib through which
the brachial plexus and vessels pass to the arm. In thoracic
outlet syndrome, compression of these nerves and vessels
often manifests as a vague shoulder pain with numbness of
the ipsilateral fourth and fifth digits. Cervical rib or hyper-
trophy of the scalene muscles can be related to the onset
of pain.183-185 Occurrence of pain also has been related to
scapular ptosis, poor posture, and clavicular fracture with
malunion or copious callus formation.
BRACHIAL NEURITIS
In the 1940s, Spillane186 and Parsonage and Turner187,188
described a painful condition of the shoulder associated
with limitation of motion. As the pain subsided and motion
improved, muscle weakness and atrophy became apparent.
The deltoid, supraspinatus, infraspinatus, biceps, and triceps
are the most frequently involved muscles,189 although dia-
phragmatic paralysis also has been reported.188,190 The cause
is unclear, but the clustering of cases suggests a viral or post-
viral syndrome.187,188 Occasionally, an associated influenza-
like syndrome or previous vaccination has been reported.189
Hershman and colleagues191 described acute brachial
neuropathy in athletes. The findings that suggest an acute
brachial neuropathy include acute onset of pain without
trauma; persistent, severe pain that continues despite rest;
and patchy neurologic signs. The diagnosis is confirmed by
EMG and nerve conduction studies.191 The prognosis for
recovery is excellent, although full recovery may take 2 to 3
years. Tsairis and associates192 reported 80% recovery within
2 years and more than 90% recovery by the end of 3 years.
NERVE ENTRAPMENT SYNDROMES
Peripheral compression neuropathies of the upper extremi-
ties also may produce referral pain to the shoulder. Distant
compression neuropathies, such as carpal tunnel (median
nerve) and cubital tunnel (ulnar nerve) syndromes, may
 610 Martin  |  Shoulder Pain
manifest with concomitant and separate shoulder impinge-
ment with rotator cuff disease. Associated numbness and
paresthesias with mapping of the dermatomal distribution
and with peripheral neuropathy often direct the examiner
to the appropriate diagnosis. Patients often give a history
of dropping objects and a feeling of clumsiness with the
affected hand. A Tinel sign may be elicited over the region
of entrapment at the elbow or wrist. Provocative maneuvers
such as Phalen’s test may be positive and usually indicate
median nerve compression at the wrist. Diminished vibra-
tory sensation is an early finding in the disease and is easily
reproducible,193,194 whereas decreased two-point discrimi-
nation and intrinsic atrophy are late findings of peripheral
compression neuropathy.193
The diagnosis usually can be made by clinical examina-
tion with exclusion of other possible causes. EMG and nerve
conduction velocity tests may reveal slowed conduction
and latency at the appropriate compression points to aid
in diagnosis. Spinal accessory nerve injury with subsequent
denervation of the trapezius may cause weakness and pain in
the shoulder consistent with impingement. The injury can
occur from traction injury to the neck or a direct blow or
pressure to the base of the neck. Iatrogenic nerve injury may
occur from surgical procedures on the neck such as lymph
node biopsy.195 The injury produces weakness in shoul-
der abduction with associated pain that radiates from the
neck into the trapezius and shoulder. Subsequent atrophy
of the trapezius may lead to dyssymmetry and ptosis of the
involved shoulder, with narrowing of the supraspinatus out-
let and secondary impingement with shoulder pain. Defini-
tive diagnosis can be made by EMG examination.
Early treatment is conservative. If return of function is
not evident at 6 months, surgical exploration of the nerve
with possible tendon transfers may be indicated.196
Injury to the long thoracic nerve (cervical fifth, sixth,
and seventh roots) can lead to scapular winging. The
resultant scapular dysrhythmia and weakness can lead to
a painful shoulder that may mimic rotator cuff disease.195
Patients also complain of pain and discomfort with active
forward flexion of the shoulder. Patients who remain sympto­
matic after conservative treatment may require surgery for
­scapulothoracic fusion or tendon transfer using the pectora-
lis major or minor to stabilize the scapula.197,198
In quadrilateral space syndrome, the axillary nerve is com-
pressed by fibrous bands in the quadrilateral space.195,199,200
This syndrome typically occurs when the arm is held in
abduction and external rotation, with subsequent tightening
of the fibrous bands across the nerve.201 It is most commonly
seen in the dominant shoulder of young athletic individuals
such as pitchers, tennis players, and swimmers who func-
tion with excessive overhead activity. The pain may occur
throughout the shoulder girdle and radiate down the arm in
a nondermatomal pattern. Neurologic and EMG testing may
be normal. Diagnosis often is made by an arteriogram of the
subclavian artery. A positive arteriogram reveals compres-
sion of the posterior humeral circumflex artery as it traverses
the quadrangular space when the arm is in the abducted and
externally rotated position. Surgical intervention may be
required to release the fibrotic bands or tendon of the teres
minor if the patient fails conservative treatment.195,202
Suprascapular nerve entrapment syndrome can result
from a traction lesion, compression lesion, or both to the
nerve caused by tethering of the nerve at the suprascapu-
lar notch by the suprascapular ligament or the spinoglenoid
notch by the transverse ligament. It also can result from
direct compression of a space-occupying lesion, such as a
ganglion or lipoma. Rengachary and coworkers203 described
variations in the size and shape of the suprascapular notch
that may predispose the nerve to entrapment.
The resulting suprascapular neuropathy produces pain in
the posterolateral aspect of the shoulder that may radiate
into the ipsilateral extremity, shoulder, or side of the neck.
Although uncommon, undiagnosed, it can have a prolonged
and disabling course. Because the suprascapular nerve has
no cutaneous innervation, there is no associated numb-
ness, tingling, or paresthesias. There is usually weakness in
abduction and external rotation, and significant atrophy is
often noticed at diagnosis. The pain frequently is described
as a deep burning or aching that can be well localized and
often can be elicited by palpation over the region of the
suprascapular notch. Any activity that brings the scapula
forward, such as reaching across the chest, may aggravate
the pain.204 The location of the pain and other symptoms
can mimic more common entities, such as impingement,
rotator cuff disease, cervical disk disease, brachial neuropa-
thy, biceps tendinitis, thoracic outlet syndrome, AC disease,
and instability of the shoulder.205
Fritz and colleagues206 reported the efficacy of MRI in the
diagnosis of suprascapular nerve entrapment secondary to
space-occupying lesions. Definitive diagnosis is made with
EMG and nerve conduction studies. EMG changes usually
reveal spontaneous activity in the muscle at rest and fibril-
lations indicating motor atrophy and denervation. Nerve
conduction studies may reveal slowing across the site of
entrapment. As with axillary nerve entrapment, the syn-
drome is often associated with young, athletic individuals
with excessive overhead activity.207 It also has been associ-
ated with trauma.205,208,209
There has been a lack of consensus regarding the opti-
mal treatment of suprascapular neuropathy.6,205,207,210,211
Post and Grinblat210 reported 25 of 26 cases to have good-
to-excellent results with surgical treatment. No difference
in residual atrophy and strength deficits has been shown,
however, for operative and nonoperative treatments. Fer-
retti and coworkers207 evaluated 96 top-level volleyball
players from the 1985 European Championships and
found that 12 had isolated suprascapular neuropathy with
atrophy of the infraspinatus of the dominant shoulder. All
the players were unaware of any impairments, however,
and played without limitations. After a space-occupying
lesion has been excluded, a 6-month trial of conservative
treatment may be indicated for some individuals. If the
entrapment does not improve, or symptoms worsen with
conservative treatment, surgical decompression is war-
ranted for pain relief; however, resolution of atrophy and
strength gains can vary.205
STERNOCLAVICULAR ARTHRITIS
Occasionally, traumatic, nontraumatic, or infectious con-
ditions can cause pain around the sternoclavicular joint
(see Fig. 40-2). The most common problem is ligamentous
injury and painful subluxation or dislocation. This can
be diagnosed by palpable instability and crepitus over the
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
s­ ternoclavicular joint. Sternoclavicular views may radio-
graphically show dislocation.212
Inflammatory arthritis of the sternoclavicular joint has
been associated with rheumatoid arthritis, ankylosing spon-
dylitis, and septic arthritis. The association of palmoplantar
pustulosis and sternoclavicular arthritis has been reported.213
Seven of 15 patients who underwent biopsies for this condi-
tion had cultures positive for Propionibacterium acnes, sug-
gesting an infectious origin of the condition.213
Two other conditions involving the sternoclavicular joint
are Tietze’s syndrome, a painful, nonsuppurative swelling
of the joint and adjacent sternochondral junctions, and
Friedrich’s syndrome, a painful osteonecrosis of the sternal
end of the clavicle.3 Condensing osteitis of the clavicle is a
rare benign idiopathic lesion of the medial one third of the
clavicle. This condition, better described as aseptic enlarg-
ing osteosclerosis of the clavicle, is most commonly seen in
middle-aged women and manifests as a tender swelling over
the medial one third of the clavicle.214
REFLEX SYMPATHETIC DYSTROPHY
Since its original description by Mitchell215 in 1864, reflex
sympathetic dystrophy (RSD) has remained a poorly under-
stood and frequently overlooked condition. Its cause is
unknown, but may be related to sympathetic overflow or
short-circuiting of impulses through the sympathetic sys-
tem. Any clinician dealing with painful disorders must be
familiar with the diagnosis and treatment of this condition.
Bonica’s216 excellent review covers the clinical presenta-
tion, various stages of the disease, and importance of early
intervention to ensure a successful outcome.
RSD has been confusingly called causalgia, shoulder-
hand syndrome, and Sudeck’s atrophy. It is generally asso-
ciated with minor trauma and is to be differentiated from
causalgia, which involves trauma to major nerve roots.215
RSD is divided into three phases, which are important in
determining treatment.216 Phase one is characterized by
sympathetic overflow with diffuse swelling, pain, increased
vascularity, and radiographic evidence of demineralization.
If left untreated for 3 to 6 months, this may progress to phase
two, which is characterized by atrophy. The extremity may
now be cold and shiny, with atrophy of the skin and muscles.
Phase three refers to progression of the trophic changes,
with irreversible flexion contracture and a pale, cold, painful
extremity. It has been speculated that phase one is related
to a peripheral short-circuiting of nerve impulses, phase two
represents short-circuiting through the internuncial pool in
the spinal cord, and phase three is controlled by higher tha-
lamic centers.216,217
Steinbrocker218 reported that recovery is possible as
long as there is evidence of vasomotor activity with swell-
ing and hyperemia. After trophic phase two or three is
established, the prognosis for recovery is poor. Prompt rec-
ognition of the syndrome is important because early inter-
vention to control pain is mandatory. Careful supervision
and reassurance are crucial because many of these patients
are emotionally labile as a result of the pain or an underly-
ing problem. The syndrome may be remarkably reversed by
a sympathetic block. Patients who receive transient relief
from sympathetic blockade may be helped by surgical sym-
pathectomy.
611
NEOPLASMS
Primary and metastatic neoplasms may cause shoulder
pain by direct invasion of the musculoskeletal system or
by compression with referred pain.2,219 Primary tumors are
more likely to occur in younger individuals. More common
lesions have a typical distribution, such as the predilection
of a chondroblastoma for the proximal humeral epiphysis or
an osteogenic sarcoma for the metaphysis.220 The differen-
tial diagnosis of spontaneous onset of shoulder pain in older
individuals should include metastatic lesions and myeloma.
Neoplasms are best identified by plain radiographs, MRI, Tc
99m MDP scintigraphy, and CT.
Neoplasms also may involve the shoulder region by me­­
tastases to the region. An associated history of carcinomas
should alert the examiner to the possibility of a bone tumor,
especially in patients who had malignancies with a predilec-
tion for metastasis to bone (e.g., thyroid, renal, lung, pros-
tate, breast). Pain is often present at rest and exacerbated
at night. Atypical pain distribution that is not relieved
by injection without specific dermatomal distribution also
should alert the examiner to the other underlying possibili-
ties. Plain radiographs should be evaluated thoroughly for
any cortical destruction or lytic lesions.
Pancoast syndrome or apical lung tumor may manifest as
shoulder pain or cervical radiculitis because of invasion of
the brachial plexus or invasion of the C8 or T1 roots.221-223
With invasion of the cervical sympathetic chain, the patient
also may develop Homer’s syndrome.
MISCELLANEOUS CONDITIONS
With the increasing numbers of patients undergoing long-
term maintainence hemodialysis, a shoulder pain syndrome
known as dialysis shoulder arthropathy has been described.
It consists of shoulder pain, weakness, loss of motion, and
functional limitation. The cause and pathogenesis of this
syndrome are unclear, although rotator cuff disease, patho-
logic fracture, bursitis, and local amyloid deposition have
been implicated as causative factors.224 There are insuffi-
cient surgical or necropsy data to confirm a specific diagnosis.
These patients generally respond poorly to local measures of
injection, heat, and NSAIDs, but they may improve with
correction of underlying metabolic disorders, such as osteo-
malacia and secondary hyperparathyroidism.
REFERENCES
  1. Codman EA: The Shoulder: Rupture of the Supraspinatus Tendon
and Other Lesions in or About the Subacromial Bursa. Boston,
Thomas Todd, 1934.
  2. Bateman E: The Shoulder and Neck, 2nd ed. Philadelphia, WB
Saunders, 1978.
  3. Post M: The Shoulder: Surgical and Non-Surgical Management.
Philadelphia, Lea & Febiger, 1988.
  4. Caillet R: Shoulder Pain, 2nd ed. Philadelphia, FA Davis, 1981.
  5. Greep JM, Lemmens HAJ, Roos DB, et al: Pain in the Shoulder and
Arm: An Integrated View. The Hague, Martinus Nijhoff, 1979.
  6. O’Brien SJ, Arnoczky SP, Warren RF: Developmental anatomy of
the shoulder and anatomy of the glenohumeral joint. In Rockwood
CA, Matsen FA III (eds): The Shoulder. Philadelphia, WB Saunders,
1990.
  7. Andrews JR, Carson W, McLeod W: Glenoid labrum tears related
to the long head of the biceps. Am J Sports Med 13:337, 1985.
 612 Martin  |  Shoulder Pain
  8. Martin SD, Warren RF, Martin TL, et al: The non-operative man-
agement of suprascapular neuropathy. J Bone Joint Surg Am 79:1159,
1997.
  9. Neer CS II: Anterior acromioplasty for the chronic impingement
syndrome in the shoulder: A preliminary report. J Bone Joint Surg
Am 54:41, 1972.
10. Martin SD, Al-Zahrani SM, Andrews JR, et al: The circumduction
adduction shoulder test. Presented at Sixty-third Annual Meeting of
the American Academy of Orthopedic Surgeons, Atlanta, February
22, 1996.
11. O’Brien SJ, Pagnani MJ, McGlynn SR, et al: A new and effective
test for diagnosing labral tears and AC joint pathology. Presented at
Sixty-third Annual Meeting of the American Academy of Orthope-
dic Surgeons, Atlanta, February 22, 1996.
12. DePalma AF: Surgical anatomy of the acromioclavicular and sterno-
clavicular joints. Surg Clin North Am 43:1540, 1963.
13. Martin SD, Baumgarten T, Andrews JR: Arthroscopic subacromial
decompression with concomitant distal clavicle resection. J Bone
Joint Surg Am 85:328, 2001.
14. Dillin L, Hoaglund FT, Scheck M: Brachial neuritis. J Bone Joint
Surg Am 67:878, 1985.
15. Kelly BT, Kadrmas WR, Speer KP: The manual muscle examination
for rotator cuff strength. Am J Sports Med 24:581, 1996.
16. Saha AK: Theory of Shoulder Mechanism. Springfield, Ill, Charles C
Thomas, 1961.
17. Saha AK: Mechanics of elevation of glenohumeral joint: Its applica-
tion in rehabilitation of flail shoulder in upper brachial plexus inju-
ries and poliomyelitis and in replacement of the upper humerus by
prosthesis. Acta Orthop Scand 44:668, 1973.
18. Brown JT: Early assessment of supraspinatus tears: Procaine infiltra-
tion as a guide to treatment. J Bone Joint Surg Br 31:423, 1949.
19. Neer CS: Impingement lesions. Clin Orthop 173:70, 1983.
20. Rockwood CA: The role of anterior impingement in lesions of the
rotator cuff. J Bone Joint Surg 62:274, 1980.
21. Jobe FW, Kvitne RS, Giangarra CE: Shoulder pain in the overhead
or throwing athlete: The relationship of anterior instability and rota-
tor cuff impingment. Orthop Rev 18:963, 1989.
22. Dalton SE, Snyder SJ: Glenohumeral instability. Baillieres Clin
Rheumatol 3:511, 1989.
23. Zanca P: Shoulder pain: Involvement of the acromioclavicular joint
(analysis of 1000 cases). AJR Am J Roentgenol 112:493, 1971.
24. Goldman AB: Shoulder Arthrography. Boston, Little, Brown, 1982.
25. Goldman AB, Ghelman B: The double contrast shoulder arthrogram:
A review of 158 studies. Radiology 127:655, 1978.
26. Mink J, Harris E: Double contrast shoulder arthrography: Its use in
evaluation of rotator cuff tears. Orthop Trans 7:71, 1983.
27. Braunstein EM, O’Connor G: Double-contrast arthrotomography of
the shoulder. J Bone Joint Surg Am 64:192, 1982.
28. Ghelman B, Goldman AB: The double contrast shoulder arthrogram:
Evaluation of rotator cuff tears. Radiology 124:251, 1977.
29. Goldman AB, Ghelman B: The double contrast shoulder arthrogram.
Cardiology 127:665, 1978.
30. Kneisl JS, Sweeney HJ, Paige ML: Correlation of pathology observed
in double contrast arthrotomography. Arthroscopy 4:21, 1988.
31. Strizak AM, Danzig L, Jackson DW, et al: Subacromial bursography.
J Bone Joint Surg 64:196, 1982.
32. Lie S: Subacromial bursography. Radiology 144:626, 1982.
33. Fukuda H, Mikasa M, Yamanaka K: Incomplete thickness rotator cuff
tears diagnosed by subacromial bursography. Clin Orthop 223:51, 1987.
34. Rafii M, Minkoff J, Bonana J, et al: Computed tomography (CT)
arthrography of shoulder instabilities in athletes. Am J Sports Med
16:352, 1988.
35. el-Khoury GY, Kathol MH, Chandler JB, et al: Shoulder instability:
Impact of glenohumeral arthrotomography on treatment. Radiology
160:669, 1986.
36. Mack LA, Matsen FA, Kilcoyne RF, et al: US evaluation of the rota-
tor cuff. Radiology 157:206, 1985.
37. Crass JR, Craig EV, Bretzke C, et al: Ultrasonography of the rotator
cuff. Radiographics 5:941, 1985.
38. Harryman DT, Mack LA, Wang KA, et al: Integrity of the post-
­operative cuff: Ultrasonography and function. Presented at Fifty-
seventh Annual Meeting of the American Academy of Orthopedic
Surgeons, New Orleans, February 9, 1990.
39. Hodler J, Fretz CJ, Terrier F, et al: Rotator cuff tears: Correlation of
sonic and surgical findings. Radiology 169:791, 1988.
40. Middleton WD, Edelstein G, Reinus WR, et al: Ultrasonography of
the rotator cuff: Technique and normal anatomy. J Ultrasound Med
3:549, 1984.
41. Gardelin G, Perin B: Ultrasonics of the shoulder: Diagnostic possibili-
ties in lesions of the rotator cuff. Radiol Med (Torino) 74:404, 1987.
42. Vestring T, Bongartz G, Konermann W, et al: The place of magnetic
resonance tomography in the diagnosis of diseases of the shoulder
joint. ROFO. Fortschr Geb Rontgenstr Neuen Bildgeb Verfahr
154:143, 1991.
43. Ahovuo J, Paavolainen P, Slatis P: Diagnostic value of sonography in
lesions of the biceps tendon. Clin Orthop 202:184, 1986.
44. Ogilvie-Harris DJ, D’Angelo G: Arthroscopic surgery of the shoulder.
Sports Med 9:120, 1990.
45. Ellman H: Arthroscopic subacromial decompression. Orthop Trans
9:49, 1985.
46. Ellman H: Arthroscopic subacromial decompression: Analysis of
one- to three-year results. Arthroscopy 3:173, 1987.
47. Paulos LE, Franklin JL: Arthroscopic shoulder decompression devel-
opment and application: A five year experience. Am J Sports Med
17:235, 1990.
48. Zlatkin MB, Reicher MA, Kellerhouse LE, et al: The painful shoul-
der: MRI imaging of the glenohumeral joint. J Comput Assist Tomogr
12:995, 1988.
49. Meyer SJ, Dalinka MK: Magnetic resonance imaging of the shoulder.
Orthop Clin North Am 21:497, 1990.
50. Seeger LL, Gold RH, Bassett LW: Shoulder instability: Evaluation
with MR imaging. Radiology 168:696, 1988.
51. Kieft GJ, Dijkmans BA, Bioem JL, et al: Magnetic resonance imaging
of the shoulder in patients with rheumatoid arthritis. Ann Rheum
Dis 49:7, 1990.
52. Morrison DS, Offstein R: The use of magnetic resonance imaging in
the diagnosis of rotator cuff tears. Orthopedics 13:633, 1990.
53. Nelson MC, Leather GP, Nirschl RP, et al: Evaluation of the painful
shoulder: A prospective comparision of magnetic resonance imag-
ing, computerized tomographic arthrography. J Bone Joint Surg Am
73:707, 1991.
54. Reeder JD, Andelman S: The rotator cuff tear: MR evaluation. Magn
Reson Imaging 5:331, 1987.
55. Seeger LL, Gold RH, Bassett LW, et al: Shoulder impingement syndrome:
MR findings in 53 shoulders. AJR Am J Roentgenol 150:343, 1988.
56. Nakano KK: Neurology of Musculoskeletal and Rheumatic Disorders.
Boston, Houghton Mifflin, 1979.
57. Leffert RD: Brachial plexus injuries. N Engl J Med 291:1059, 1974.
58. Altchek DW, Schwartz E, Warren RF, et al: Radiologic measurement
of superior migration of the humeral head in impingement syndrome.
Presented at Sixth Open Meeting of the American Shoulder and
Elbow Surgeons, New Orleans, February 11, 1990.
59. Inman VT, Saunders JB, Abbott LC: Observations on the functions
of the shoulder joint. J Bone Joint Surg 26:1, 1944.
60. Poppen NK, Walker PS: Normal and abnormal motion of the shoul-
der. J Bone Joint Surg Am 58:195, 1976.
61. Bigliani LU, Morrison D, April EW: The morphology of the acro-
mion and its relationship to rotator cuff tears. Orthop Trans 10:228,
1986.
62. Morrison DS, Bigliani LU: The clinical significance of variations in
acromial morphology. Orthop Trans 11:234, 1987.
63. Codman E, Akerson TB: The pathology associated with rupture of
the supraspinatus tendon. Ann Surg 93:354, 1911.
64. Fukuda H, Hamada K, Yamanaka K: Pathology and pathogenisis
of bursal side rotator cuff tears: Views from en-bloc histological
sections. Clin Orthop 254:75, 1990.
65. McLaughlin HL: Lesions of the musculotendinous cuff of the shoul-
der, I: The exposure and treatment of tears with retraction. J Bone
Joint Surg 26:31, 1944.
66. Neer CS II, Flatow EL, Lech O: Tears of the rotator cuff: Long term
results of anterior acromioplasty and repair. Presented at Fourth Open
Meeting of the American Shoulder and Elbow Surgeons, Atlanta,
February 1988.
67. Ogata S, Uhthoff HK: Acromial enthesopathy and rotator cuff tears:
A radiographic and histologic postmortem investigation of the cora-
coacromial arch. Clin Orthop 254:39, 1990.
68. Olsson O: Degenerative changes in the shoulder joint and their con-
nection with shoulder pain: A morphological and clinical investiga-
tion with special attention to the cuff and biceps tendon. Acta Chir
Scand 181(Suppl):1, 1953.
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
69. Ozaki J, Fujimoto S, Nakagawa Y, et al: Tears of the rotator cuff of
the shoulder associated with pathologic changes of the acromion:
A study in cadavers. J Bone Joint Surg 70:1224, 1988.
70. Skinner HA: Anatomical consideration relative to ruptures of the
supraspinatus tendon. J Bone Joint Surg Br 19:137, 1937.
71. Ellman H: Occupational supraspinatus tendinitis: The rotator cuff
syndrome. Ugeskr Laeger 151:2355, 1989.
72. Scheib JS: Diagnosis and rehabilitation of the shoulder impingement
syndrome in the overhand and throwing athlete. Rheum Dis Clin
North Am 16:971, 1990.
73. Jackson DW: Chronic rotator cuff impingement in the throwing ath-
lete. Am J Sports Med 4:231, 1976.
74. Hawkins RJ, Kennedy JC: Impingement syndrome in athletes. Am J
Sports Med 8:151, 1980.
75. McShane RB, Leinberry CF, Fenlin JM: Conservative open anterior
acromioplasty. Clin Orthop 223:137, 1987.
76. Rockwood CA, Lyons FA: Shoulder impingement syndrome: Diag-
nosis, radiographic evaluation, and treatment with a modified Neer
acromioplasty. J Bone Joint Surg Am 75:1593, 1993.
77. Hawkins RJ, Brock RM, Abrams JS, et al: Acromioplasty for
impingement with an intact rotator cuff. J Bone Joint Surg Br
70:797, 1988.
78. Stuart MJ, Azevedo AJ, Cofield RH: Anterior acromioplasty for treat-
ment of the shoulder impingement syndrome. Clin Orthop 260:195,
1990.
79. Bigliani LU, D’Alesandro DF, Dduralde XA, et al: Anterior acromio-
plasty for subacromial impingement in patients younger than 40 years
of age. Clin Orthop 246:111, 1988.
80. Bjorkheim JM, Paavolainen P, Ahovuo J, et al: Surgical repair of
the rotator cuff and the surrounding tissues: Factors influencing the
results. Clin Orthop 236:148, 1988.
81. Levy HJ, Gardner RD, Lemak LJ: Arthroscopic subacromial decom-
pression in the treatment of full-thickness rotator cuff tears. Arthros-
copy 7:8, 1991.
82. McKendry RJR, Uhthoff HK, Sarkar K, et al: Calcifying tendinitis of
the shoulder: Prognostic value of clinical, histologic, and radiographic
features in 57 surgically treated cases. J Rheumatol 9:75, 1982.
83. Uhthoff HK, Sarkar K, Maynard JA: Calcifying tendinitis, a new
concept of its pathogenesis. Clin Orthop 118:164, 1976.
84. Sarkar K, Uhthoff HK: Ultrastructure localization of calcium in cal-
cifying tendinitis. Arch Pathol Lab Med 102:266, 1978.
85. Hayes CW, Conway WF: Calcium hydroxyapatite deposition disease.
Radiographics 10:1031, 1990.
86. Vebostad A: Calcific tendinitis in the shoulder region: A review of 43
operated shoulders. Acta Orthop Scand 46:205, 1975.
87. Moseley HF, Goldie I: The arterial pattern of the rotator cuff of the
shoulder. J Bone Joint Surg Br 45:780, 1963.
88. Rathburn JB, Macnab I: The microvascular pattern of the rotator
cuff. J Bone Joint Surg Br 52:540, 1970.
89. Bosworth BM: Calcium deposits in the shoulder and subacromial bur-
sitis: A survey of 12,122 shoulders. JAMA 116:2477, 1941.
90. Bosworth BM: Examination of the shoulder for calcium deposits.
J Bone Joint Surg 23:567, 1941.
91. DePalma AF, Kruper JS: Long term study of shoulder joints afflicted
with and treated for calcified tendonitis. Clin Orthop 20:61, 1961.
92. Linblom K: Arthrography and roentgenography in ruptures of the
tendon of the shoulder joint. Acta Radiol 20:548, 1939.
93. Swiontkowski M, Iannotti JP, Herrmann HJ, et al: Intraoperative
assessment of rotator cuff vascularity using laser Doppler flowmetry.
In Post M, Morrey BE, Hawkins RJ (eds): Surgery of the Shoulder. St.
Louis, Mosby, 1990, pp 208-212.
94. Matsen FA III, Arntz CT: Subacromial impingment. In Rockwood
CA Jr, Matsen FA III (eds): The Shoulder. Philadelphia, WB Saun-
ders, 1990, pp 623-646.
95. Neer CS II, Poppen NK: Supraspinatus outlet. Orthop Trans 11:234,
1987.
96. Clark J, Sidles JA, Matsen FA: The relationship of the glenohumeral
joint capsule to the rotator cuff. Clin Orthop 254:29, 1990.
97. Samilson RL, Raphael RL, Post L, et al: Arthrography of the shoul-
der. Clin Orthop 20:21, 1961.
98. Collins RA, Gristina AG, Carter RE, et al: Ultrasonography of the
shoulder. Orthop Clin North Am 18:351, 1987.
99. Crass JR, Craig EV, Feinberg SB: Ultrasonography of rotator cuff
tears: A review of 500 diagnostic studies. J Clin Ultrasound 16:313,
1988.
613
100. Crass JR, Craig EV, Feinberg SB: The hyperextended internal rota-
tion view in rotator cuff ultrasonography. J Clin Ultrasound 15:415,
1987.
101. Mack LA, Gannon MK, Kilcoyne RF, et al: Sonographic evaluation
of the rotator cuff: Accuracy in patients without prior surgery. Clin
Orthop 234:21, 1988.
102. Iannotti JP, Zlatkin MB, Esterhai JL, et al: Magnetic resonance imag-
ing of the shoulder: Sensitivity, specificity, and predictive value. J
Bone Joint Surg Am 73:17, 1991.
103. DePalma AF: Surgery of the Shoulder, 2nd ed. Philadelphia, JB Lip-
pincott, 1973.
104. Wolfgang GL: Surgical repair of tears of the rotator cuff of the shoul-
der: Factors influencing the result. J Bone Joint Surg Am 56:14,
1974.
105. Jobe FW, Moynes DR: Delineation of diagnostic criteria and a rehabili-
tation program for rotator cuff injuries. Am J Sports Med 10:336, 1982.
106. Earnshaw P, Desjardins D, Sakar K, et al: Rotator cuff tears: The role
of surgery. Can J Surg 25:60, 1982.
107. Rockwood CA, Williams GR, Burkhead WZ: Debridement of mas-
sive, degenerative lesions of the rotator cuff. Presented at Seventh
open meeting of the American Shoulder and Elbow Society Sur-
geons, Anaheim, Calif, March 10, 1991.
108. Martin SD, Andrews JR: The rotator cuff: Open and mini-open
repairs. Presented at American Orthopaedic Society for Sports Medi-
cine 20th Annual Meeting, Palm Desert, June 26, 1994.
109. Rockwood CA Jr: The shoulder: Facts, confusion and myths. Int
Orthop 15:401, 1991.
110. Steffens K, Konermann H: Rupture of the rotator cuff in the elderly.
Z Gerontol 20:95, 1987.
111. Goss CM: Gray’s Anatomy of the Human Body, 28th ed. Philadel-
phia, Lea & Febiger, 1966.
112. Neer CS, Craig EV, Fukuda H: Cuff tear arthropathy. J Bone Joint
Surg Am 65:1232, 1983.
113. Neer CS II, Bigliani LU, Hawkins RJ: Rupture of the long head of the
biceps related to subacromial impingement. Orthop Trans 1:111, 1977.
114. Crenshaw AH, Kilgore WE: Surgical treatment of bicipital tendon-
itis. J Bone Joint Surg Am 48:1496, 1966.
115. Hitchcock HH, Bechtol CO: Painful shoulder: Observations on the
role of the tendon of the long head of the biceps brachii in its causa-
tion. J Bone Joint Surg Am 30:263, 1948.
116. Meyer AW: Spontaneous dislocation and destruction of the tendon
of the long head of the biceps brachii, fifty-nine instances. Arch Surg
17:493, 1928.
117. Rockwood RA Jr, Williams GR, Young CD: Injuries to the acro-
mioclavicular joint. In Rockwood CA Jr, Green DP, Bucholz RW
(eds): Fractures in Adults. Philadelphia, JB Lippincott, 1991, pp
1181-1251.
118. Jacobs P: Post-traumatic osteolysis of the outer end of the clavicle.
J Bone Joint Surg Br 46:705, 1964.
119. Murphy OB, Bellamy R, Wheeler W, et al: Post-traumatic osteolysis
of the distal clavicle. Clin Orthop 109:108, 1975.
120. Cahill RB: Osteolysis of the distal part of the clavicle in male ath-
letes. J Bone Joint Surg Am 64:1053, 1982.
121. Maden B: Osteolysis of the acromial end of the clavicle following
trauma. Br J Radiol 36:822, 1963.
122. Scavenius M, Iversen BF: Nontraumatic clavicular osteolysis in
weight lifters. Am J Sports Med 20:463, 1992.
123. Slawski DP, Cahill BR: Atraumatic osteolysis of the distal clavicle.
Am J Sports Med 22:267, 1994.
124. Meyers JF: Arthroscopic debridement of the acromioclavicular joint
and distal clavicle resection. In McGinty JB, Caspari RB, Jackson
RW, et al (eds): Operative Arthroscopy. New York, Raven Press,
1991, pp 557-560.
125. Cahill BR, Lee MT: Atraumatic osteolysis of the distal clavicle. In
Torg JS, Shephard RJ (eds): Current Therapy in Sports Medicine.
St Louis, Mosby, 1995, pp 177-181.
126. Rockwood CA Jr: Disorders of the acromioclavicular joint. In Rock-
wood CA Jr, Matsen FA III (eds): The Shoulder. Philadelphia, WB
Saunders, 1985, p 449.
127. Gartsman GM, Combs AH, Davis PF, et al: Arthroscopic acromio-
clavicular joint resection: An anatomic study. Am J Sports Med 19:2-
5, 1991.
128. Fink EP: Injuries to the acromioclavicular joint. In Torg JS, Sheph-
ard RJ (eds): Current Therapy in Sports Medicine. St Louis, Mosby,
1995, pp 174-177.
 614 Martin  |  Shoulder Pain
129. Gartsman GM: Arthroscopic resection of the acromioclavicular
joint. Am J Sports Med 21:71, 1993.
130. Flatow EL, Cordasco FA, Bigliani LU: Arthroscopic resection of the
outer end of the clavicle from a superior approach: A critical, quan-
titative, radiographic assessment of bone removal. Arthroscopy 8:55,
1992.
131. Flatow EL, Duralde XA, Nicholson GP, et al: Arthroscopic resection
of the distal clavicle with a superior approach. J Shoulder Elbow Surg
4:41, 1995.
132. Worcester JN, Green DP: Osteoarthritis of the acromioclavicular
joint. Clin Orthop 58:69, 1987.
133. DePalma AF: The role of the disks of the sternoclavicular and acro-
mioclavicular joints. Clin Orthop 13:222, 1959.
134. Novack PJ, Bach BB, Romeo AA, et al: Surgical resection of the
distal clavicle. J Shoulder Elbow Surg 4:35, 1995.
135. Gurd FB: The treatment of complete dislocation of the outer end of the
clavicle: A hitherto undescribed operation. Ann Surg 63:1094, 1941.
136. Mumford EB: Acromioclavicular dislocation: A new operative
treatment. J Bone Joint Surg Am 23:799, 1941.
137. Cook FF, Tibone JE: The Mumford procedure in athletes: An objec-
tive analysis of function. Am J Sports Med 16:97, 1988.
138. Bigliani LU, Nicholson GP, Flatow EL: Arthroscopic resection of the
distal clavicle. Orthop Clin North Am 24:133, 1993.
139. Gartsman GM, Combs AH, Davis PF, et al: Arthroscopic acromio-
clavicular joint resection: An anatomic study. Am J Sports Med 19:2,
1991.
140. Kay SP, Ellman H, Harris E: Arthroscopic distal clavicle resection:
Technique and early results. Clin Orthop 301:181, 1994.
141. Tolin BS, Snyder SJ: Our technique for the arthroscopic Mumford
procedure. Orthop Clin North Am 24:143, 1993.
142. Cofield RH, Briggs BT: Glenohumeral arthrodesis. J Bone Joint Surg
Am 61:668, 1979.
143. Rowe CR: Arthrodesis of the shoulder used in treating painful condi-
tions. Clin Orthop 173:92, 1983.
144. Neer CS II, Watson KC, Stanton FJ: Recent experience in total
shoulder replacement. J Bone Joint Surg Am 64:319, 1982.
145. Cofield RH: Unconstrained total shoulder prosthesis. Clin Orthop
173:97, 1983.
146. Thornhill TS, Karr MJ, Averill RM, et al: Total shoulder arthroplasty,
the Brigham experience. Presented at Fiftieth Annual Meeting of the
American Academy of Orthopedic Surgeons, Anaheim, Calif, 1983.
147. Thornhill TS, Barrett WP: Total shoulder arthroplasty. In Rowe CR
(ed): The Shoulder. New York, Churchill Livingstone, 1988.
148. Neer CS II: Fractures and dislocations of the shoulder. In Rockwood
CA, Green DP (eds): Fractures. Philadelphia, JB Lippincott, 1975.
149. Ficat RP, Arlet J: Ischemia and Necrosis of the Bone. Baltimore,
Williams & Wilkins, 1980.
150. Hayes JM: Arthroscopic treatment of steroid-induced osteonecrosis
of the humeral head. Arthroscopy 5:218, 1989.
151. McCarty DJ: Robert Adams’ rheumatic arthritis of the shoulder:
“Milwaukee shoulder” revisited. J Rheumatol 32:668, 1989.
152. Halverson PB, Carrera GF, McCarty DJ: Milwaukee shoulder syn-
drome: Fifteen additional cases and a description of contributing fac-
tors. Arch Intern Med 150:677, 1990.
153. Wortmann RL, Veum JA, Rachow JW: Synovial fluid 5-­nucleotidase
activity: Relationship to other purine catabolic enzymes and to
arthropathies associated to calcium deposition disease. Arthritis
Rheum 34:1014, 1991.
154. Neer CS II, Craig EV, Fukada H, et al: Cuff tear arthropathy. Exhibit
at the Annual Meeting of the American Academy of Orthopedic
Surgeons, New Orleans, 1982.
155. Post M, Haskell SS, Jablon M: Total shoulder replacement with a
constrained prosthesis. J Bone Joint Surg Am 62:327, 1980.
156. Post M, Jablon M: Constrained total shoulder arthroplasty: Long
term follow-up observations. Clin Orthop 173:109, 1983.
157. Hamada K, Fukuda H, Mikasa M, et al: Roentgenographic findings
in rotator cuff tears: A long-term observation. Clin Orthop 254:92,
1990.
158. Snyder SJ, Karzel RP, Del Pizzo W, et al: SLAP lesions of the
shoulder. Arthroscopy 6:274, 1990.
159. Burkhart SS, Fox DL: SLAP lesions in association with complete
tears of the long head of the biceps tendon: A report of two cases.
Arthroscopy 8:31, 1992.
160. Maffet MW, Gartsman GM, Moseley B: Superior labrum-biceps ten-
don complex lesions of the shoulder. Am J Sports Med 23:93, 1995.
161. Grauer JD, Paulos LE, Smutz WP: Biceps tendon and superior labral
injuries. Arthroscopy 8:488, 1992.
162. Putman JJ: The treatment of a form of painful periarthritis of the
shoulder. Boston Med Surg J 107:536, 1882.
163. Neviaser JS: Adhesive capsulitis of the shoulder: Study of the patho-
logic findings in periarthritis of the shoulder. J Bone Joint Surg
27:211, 1945.
164. McLaughlin HL: The “frozen shoulder.” Clin Orthop 20:126,
1961.
165. Johnson JTH: Frozen shoulder syndrome in patients with pulmonary
tuberculosis. J Bone Joint Surg Am 41:877, 1959.
166. Dee PE, Smith RG, Gullickson MJ, et al: The orthopedist and apical
lung carcinoma. J Bone Joint Surg Am 42:605, 1960.
167. Demaziere A, Wiley AM: Primary chest wall tumor appearing as fro-
zen shoulder: Review and case presentations. J Rheumatol 18:911,
1991.
168. Smith CR, Binder AI, Paice EW: Lesions of the mid-shaft of the
humerus presenting as shoulder capsulitis. Br J Rheumatol 29:386,
1990.
169. Dickson JA, Crosby EH: Periarthritis of the shoulder: An analysis of
two hundred cases. JAMA 99:2252, 1932.
170. McLaughlin HL: On the frozen shoulder. Bull Hosp Joint Dis 12:383,
1951.
171. Lundberg BJ: The frozen shoulder. Acta Orthop Scand 119:59,
1969.
172. Rowe CR, Leffert RD: Idiopathic chronic adhesive capsulitis. In
Rowe CR (ed): The Shoulder. New York, Churchhill Livingstone,
1988, pp 155-163.
173. Coventry MB: Problem of the painful shoulder. JAMA 151:177,
1953.
174. Waldburger M, Meier JL, Gobelet C: The frozen shoulder: Diagnosis
and treatment. Clin Rheumatol 11:364, 1992.
175. Fareed DO, Gallivan WR Jr: Office management of frozen shoulder
syndrome: Treatment with hydraulic distention under local anesthe-
sia. Clin Orthop 242:177, 1989.
176. Rizk TE, Pinalls RA, Talaiver AS: Corticosteroid injections in adhe-
sive capsulitis: Investigation of their value and site. Arch Phys Med
Rehabil 72:20, 1991.
177. Hill JJ Jr, Bogumill H: Manipulation in the treatment of frozen shoul-
der. Orthopedics 11:1255, 1988.
178. Rowe CR: Dislocations of the shoulder. In Rowe CR (ed): The
Shoulder. New York, Churchill Livingstone, 1988.
179. Fu FH, Harner CD, Klein AH: Shoulder impingement syndrome:
A clinical review. Clin Orthop 269:162, 1991.
180. Walch G, Bioleau P, Noel E: Impingement of the deep surface of
the supraspinatus tendon on the posterosuperior glenoid rim: An
arthroscopic study. J Shoulder Elbow Surg 1:238, 1992.
181. Altchek DW, Warren RF, Ortiz G: T-plasty: A technique for treating
multidirectional instability in the athlete. Orthop Trans 13:560, 1989.
182. Neer CS, Foster CR: Inferior capsular shift for involuntary and
multidirectional instability of the shoulder: A preliminary report.
J Bone Joint Surg Am 62:897, 1980.
183. Roos DB: Congenital anomalies associated with thoracic outlet syn-
drome: Anatomy, symptoms, diagnosis and treatment. Am J Surg
132:771, 1976.
184. Leffert RD: Thoracic outlet syndrome. In Gelberman RH (ed): Oper-
ative Nerve Repair and Reconstruction. Philadelphia, JB Lippincott,
1991, pp 1177-1195.
185. Urschel HC, Paulson DL, MacNamara JJ: Thoracic outlet syndrome.
Ann Thorac Surg 6:1, 1968.
186. Spillane JD: Localized neuritis of the shoulder girdle: A report of 46
cases in the MEF. Lancet 2:532, 1943.
187. Parsonage MJ, Turner JWA: Neurologic amyotrophy: The shoulder
girdle syndrome. Lancet 1:1973, 1948.
188. Turner JWA, Parsonage MJ: Neurologic amyotrophy (paralytic bra-
chial neuritis): With special reference to prognosis. Lancet 2:209,
1957.
189. Bacevich BB: Paralytic brachial neuritis. J Bone Joint Surg Am
58:262, 1976.
190. Walsh NE, Dumitru D, Kalantri A, et al: Brachial neuritis involving
the bilateral phrenic nerves. Arch Phys Med Rehabil 68:46, 1987.
191. Hershman EB, Wilbourn AJ, Bergfield JA: Acute brachial neuropa-
thy in athletes. Am J Sports Med 17:655, 1989.
192. Tsairis P, Dyck PJ, Mulder DW: Natural history of brachial plexus
neuropathy. Arch Neurol 27:109, 1972.
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
193. Szabo RM: Carpal tunnel syndrome—general. In Gelberman RH
(ed): Operative Nerve Repair and Reconstruction. Philadelphia, JB
Lippincott, 1991, pp 869-888.
194. Slater RR, Bynum DK: Diagnosis and treatment of carpal tunnel syn-
drome. Orthop Rev 22:1095, 1993.
195. Narakas AO: Compression and traction neuropathies about the
shoulder and arm. In Gelberman RH (ed): Operative Nerve
Repair and Reconstruction. Philadelphia, JB Lippincott, 1991, pp
1147-1175.
196. Bigliani L, Perez-Sanz JR, Wolfe IN: Treatment of trapezius paralysis.
J Bone Joint Surg 67:871, 1985.
197. Chavez JP: Pectoralis minor transplanted for paralysis of the serratus
anterior. J Bone Joint Surg Br 33:21, 1951.
198. Marmor L, Bechtal CO: Paralysis of the serratus anterior due to elec-
tric shock relieved by transplantation of the pectoralis major muscle:
A case report. J Bone Joint Surg 45:156, 1983.
199. Cahill BR, Palmer RE: Quadrilateral space syndrome. J Hand Surg
8:65, 1983.
200. Jobe FW, Tibone JE: The shoulder in sports. In Rockwood CA, Mat-
sen FA (eds): The Shoulder. Philadelphia, WB Saunders, 1990, pp
961-990.
201. Cahill BR: Quadrilateral space syndrome. In Omer GE Jr, Spinner
MD (eds): Management of Peripheral Nerve Problems. Philadelphia,
WB Saunders, 1980, pp 56-65.
202. Bretzke CA, Crass JR, Craig EV, et al: Ultrasonography of the rotator
cuff: Normal and pathologic anatomy. Invest Radiol 20:311, 1985.
203. Rengachary SS, Neft JP, Singer PA, et al: Suprascapular entrapment
neuropathy: A clinical, anatomical and comparative study. Neurosur-
gery 5:441, 1979.
204. Habermeyer P, Rappaport D, Wiedermann E, et al: Incisura scapulae
syndrome. Handchir Mikrochir Plast Chir 22:120, 1990.
205. Martin SD, Warren RF, Martin TL, et al: Suprascapular neuropathy.
J Bone Joint Surg Am 79:1159, 1997.
206. Fritz RC, Helms CA, Steinbach LS, et al: Suprascapular nerve entrap-
ment: Evaluation with MR imaging. Radiology 182:437, 1992.
207. Ferretti A, Cerullo G, Russo G: Suprascapular neuropathy in
volleyball players. J Bone Joint Surg Am 69:260, 1987.
615
208. Rask MR: Suprascapular nerve entrapment: A report of two cases
treated with suprascapular notch resection. Clin Orthop 123:73,
1977.
209. Solheim LF, Roaas A: Compression of the suprascapular nerve after
fracture of the suprascapular notch. Acta Orthop Scand 49:338,
1978.
210. Post M, Grinblat E: Suprascapular nerve entrapment: Diagnosis and
results of treatment. J Shoulder Elbow Surg 2:197, 1993.
211. Hadley MN, Sonntag VK, Pittman HW: Suprascapular nerve entrap-
ment: A summary of seven cases. J Neurosurg 64:843, 1986.
212. Rockwood CA Jr: Fractures and dislocations of the shoulder, part II.
In Rockwood CA Jr, Green DP (eds): Fractures. Philadelphia, JB Lip-
pincott, 1975, pp 262-270.
213. Edlund E, Johnsson U, Lidgren L, et al: Palmoplantar pustulosis and
sternocostoclavicular arthro-osteitis. Ann Rheum Dis 47:809, 1988.
214. Lissens M, Bruyninckx F, Rossele N: Condensing osteitis of the clav-
icle: Report of two cases and review of the literature. Acta Belg Med
Phys 13:235, 1990.
215. Mitchell SW: Phantom limbs. Lippincott 8:563, 1871.
216. Bonica JJ: Causalgia and other reflex dystrophies. In Bonica JJ
(ed): Management of Pain. Philadelphia, Lea & Febiger, 1979, pp
139-142.
217. Evans JA: Reflex sympathetic dystrophy. Surg Gynecol Obstet 82:36,
1946.
218. Steinbrocker O: The shoulder-hand syndrome: Present perspective.
Arch Phys Med 49:388, 1968.
219. Brown C: Compressive invasive referred pain to the shoulder. Clin
Orthop 173:55, 1983.
220. Dahlin DC: Bone Tumors. Springfield, Ill, Charles C Thomas, 1978.
221. DePalma AF: Loss of scapulohumeral motion. Ann Surg 135:193,
1952.
222. Pancoast HK: Importance of careful roentgen ray investigation of
apical chest tumors. JAMA 83:1407, 1923.
223. Vargo MM, Flood KM: Pancoast tumor presenting as cervical radicu-
lopathy. Arch Phys Med Rehabil 71:606, 1990.
224. Brown EA, Arnold LR, Gower PE: Dialysis arthropathy: Complica-
tion of long term treatment with haemodialysis. BMJ 292:163, 1986.
 41
Key Points
In adult population studies, the point prevalence and 1-year
prevalence of low back pain (LBP) without sciatica, stenosis, or
severe deformity has been estimated at 33% and 73%.
Approximately 10% to 15% of physically active adults with LBP
may expect a serious episode of LBP each year. Most of these
individuals apparently experience significant resolution of their
symptoms within 1 month without formal medical intervention.
The utility (and wisdom) of ordering imaging early in the
course of LBP illness is not supported by clinical evidence.
Specific comorbidities are associated with risk of developing
persistent, disabling LBP, including preexisting ­psychological
distress, disputed compensation issues, chronic pain from
other (nonlumbar) anatomic sites, and job dissatisfaction.
The initial clinical determination involves two decision points:
(1) Does the patient have a significant risk of a serious under-
lying illness? (2) Is the back pain accompanied by a specific
pattern of pain radiation to the legs suggesting neurogenic
claudication or sciatic-type syndromes (radicular pain,
­numbness, or tingling down one or both lower extremities)?
A history of cancer, pain awakening the patient from sleep,
recent weight loss, lethargy, or other constitutional symptoms
suggested by the red flag survey warrant appropriate workup
for neoplasm, infection, or inflammatory conditions. Such a
history, along with associated neurologic findings, warrants
use of magnetic resonance imaging (MRI) early in the course
of an LBP episode.
Patients with persistent, debilitating LBP lasting more than 4
to 6 weeks should be assessed carefully and possibly referred
to the care of a spine specialist for secondary evaluation.
Common degenerative findings (e.g., Schmorl’s nodes, end
plate changes, disk desiccation) are expected on MRI and are
most commonly unrelated to symptoms.
Lumbar fusions performed on individuals for positive
­discographic findings are no more successful at reducing back
pain than fusions performed on patients selected for fusion
surgery without discography.
Treatment of LBP with narcotic pain medicines over the
long-term is usually discouraged.
The tricyclic and the tetracyclic classes of antidepressants
have been proved in randomized clinical trials to be effective
treatments for LBP in patients without preexisting clinical
depression.
Patients with high “fear avoidance” characteristics (a dispro-
portionate predilection to avoid activities or situations that
may produce discomfort) have poorer outcomes and are at
increased risk for developing chronic LBP illness than patients
who remain active despite their pain. Exercise, no matter what
type of regimen, seems to hasten patients’ return to usual
lifestyle activities.
Low Back Pain
Michael s. Wildstein  • 
EUGENE j. CARRAGEE
Concomitant medical and physical treatments seem to have
better outcomes with respect to pain and disability than
medical treatment alone.
Lumbar epidural steroid injections, facet blocks, trigger
point injections, and intradiscal anesthetics have not shown
­improvement in the outcomes of LBP patients who lack
­discrete radicular symptoms.
Spinal fusion or decompression in patients with back pain and
only common degenerative changes on imaging studies is not
clearly effective.
Short-term trials to date comparing disk replacement surgery
and spinal fusion have failed to show significant differences in
outcome.
For the internist or family physician, the problem of low
back pain (LBP) is one of the most commonly assessed
complaints. LBP is the most common cause of limitation of
activity in patients younger than 45 years old, is the second
most frequent reason for a visit to the physician’s office, is
the third most common surgical indication, and ranks fifth
among hospital admission diagnoses.1 Data from the 2002
National Health Interview Survey identified LBP as the
most prevalent pain syndrome, eclipsing migraine head-
aches, neck pain, and face/jaw pain.2 The financial impact
on society is even more staggering—$45 to $54 billion per
year in lost wages, loss in household services and tax rev-
enues, social security benefits, and lost productivity.3
In adult population studies, the point prevalence and
1-year prevalence of LBP without sciatica, stenosis, or severe
deformity has been estimated at 33% (point prevalence)4
and 73% (1-year prevalence).5 This prevalence may be even
higher in subgroups of adult laborers and military personnel
involved in training exercises.4 Despite the common preva-
lence, most individuals with LBP do not seek medical care
for this problem specifically.
Approximately 10% to 15% of physically active adults
with LBP may expect a serious episode of LBP each year.
Most of these individuals apparently experience significant
resolution of their symptoms within 1 month without formal
medical intervention.6 Recurrences of back symptoms are
common in this population, however, and can continue to
be intermittently bothersome for years after the first event
has passed. Despite the large proportion of adults who expe-
rience an episode or more of LBP, large population studies
prospectively examining LBP symptoms over 5 years show
that only a small proportion of respondents reported serious
or persistent disability. The diagnostic challenge is the early
identification and treatment of the few patients who either
have serious disease (e.g., tumor, infection, fracture) or, in
617
 618 Wildstein  |  Low Back Pain
Table 41-1  Magnetic Resonance Imaging Findings   be reserved (although they rarely are) as confirmatory, rather
in Asymptomatic Subjects
Radiographic Asymptomatic Patients
Abnormality with Abnormal Finding (%)
High intensity zone 14-33
Degenerative disk disease 25-70
Disk protrusion 25-50
the absence of clear radiographic or physical findings, might
benefit from interventions to alter the course of common
nonspecific LBP.
The utility (and wisdom) of ordering imaging studies
early in the course of LBP illness is not supported by clinical
evidence. Nonetheless, numerous patients who see a clini-
cian receive imaging modalities, such as radiographs or mag-
netic resonance imaging (MRI) scans, and are found to have
radiographically identifiable “abnormalities.” The findings
uncovered in such studies usually are nonspecific age-related
changes (disk desiccation, Modic end plate changes, annu-
lar fissures). Whether these common findings have much
to do with the patients’ chief complaints is unclear. Data
from cross-sectional population studies of subjects who are
asymptomatic for LBP show all of these findings can be seen
commonly in individuals with little or no back problems
(Table 41-1).7-9
Researchers also have examined patients who have had
baseline MRI scans before developing any serious LBP
problems and later went on to have a serious LBP episode.
When a subject in this study experienced a serious episode
of LBP and was reimaged in an attempt to identify new
­spinal pathology, the new MRI scan was compared with
baseline images. In less than 5% of cases, there was a dis-
crete new imaging finding compared with baseline studies.10
In prospective studies, investigators attempting to correlate
­baseline MRI findings in asymptomatic patients with future
LBP problems found these baseline common changes could
not predict with accuracy which patients would experience
disabling LBP symptoms at any point in the future.11-14
Studies have shown that certain specific comorbidities
are associated with risk of developing persistent, disabling
LBP. These conditions include preexisting psychological dis-
tress, disputed compensation issues, chronic pain from other
(nonlumbar) anatomic sites, and job dissatisfaction.13-16
Additionally, as noted earlier, common degenerative findings
on imaging studies have been identified in large proportions
of healthy, otherwise asymptomatic patients, complicating
­further the task of identifying appropriate treatments for
pain that may or may not be emanating from a specific ana-
tomic structure.7-9
DIAGNOSTIC METHODS
HISTORY
Paramount in the evaluation of patients with LBP is a thor-
ough history and physical examination. The small percent-
age of patients with serious disease causing back pain (e.g.,
tumor, infection, inflammatory disease) generally can be
suggested by the history and sometimes can be confirmed by
specific physical signs. Imaging modalities generally should
than diagnostic tests.
A primary care clinician typically performs the ini-
tial evaluation of a patient with LBP. The initial clinical
­determination involves two decision points: (1) Does the
patient have a significant risk of a serious underlying illness
(Fig. 41-1)? (2) Is the back pain accompanied by a specific
pattern of pain radiation to the legs suggesting neurogenic
claudication or sciatic-type syndromes (radicular pain,
numbness, or tingling down one or both lower extremities)?
In working up the subset of patients with LBP and radicu-
lar symptoms in the absence of identifiable trauma, the
­physician must keep in mind that young individuals gen-
erally experience disk herniations, and elderly patients are
more likely to have spinal stenosis. These two very differ-
ent sources of combined radicular and back pain, although
occasionally similar in presenting symptoms, require mark-
edly different treatment strategies. This chapter focuses pri-
marily on the evaluation of nonradicular LBP.
PHYSICAL EXAMINATION
Although patients with neurogenic claudication and radicu-
lopathy present with classic findings, typically LBP patients
without neurologic involvement exhibit few characteristic
findings on examination. The goal of the initial assessment
should always be to rule out the so-called red flags heralding
diagnoses, including tumor, infection, and fracture (Table
41-2). Generally, if after a thorough history and physical
examination, no serious abnormalities are identified (weak-
ness, ataxia, point tenderness over the spine, numbness in a
dermatomal pattern), patients should be reassured that the
situation is not likely dangerous, and that these types of pain
usually are self-limited. For persistent LBP lasting more than
4 to 6 weeks, imaging studies of the lumbar spine or blood
tests (erythrocyte sedimentation rate or C-reactive protein)
may be warranted to exclude serious structural disease. The
utility of specific laboratory tests and imaging studies, which
have low specificity, is to help rule out compression fractures
and other destructive bony pathology, including neoplasm,
and to allow a more aggressive rehabilitation plan. Patients
should understand before obtaining radiographs that com-
mon degenerative findings (e.g., spurring, disk height loss,
disk protrusion) are expected.
IMAGING STUDIES
A history of cancer, pain awakening the patient from
sleep, recent weight loss, lethargy, or other constitutional
symptoms suggested by the red flag survey (see Table 41-2)
warrant appropriate workup for neoplasm, infection, or
inflammatory conditions. Such a history, along with
­associated ­neurologic findings, warrants use of MRI early in
the course of an LBP episode. Robertson and coworkers17
addressed the debate over the use of MRI as a screening tool
in the evaluation of LBP. They showed that a rapid, two-
plane, single echo, fast spin-echo sequence protocol alone
is adequate to detect potentially significant degenerative
­disease of the lumbar spine. The significant time savings in
the scanner (2.5 ­minutes versus 28 minutes) for conventional
­protocols ­compared with rapid sequence and the attendant
cost savings may lead to re-evaluation of the role of MRI as
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN 619

Adults with low back pain (not for back pain associated
with major trauma, nonspinal back pain
or back pain due to systemic illness)

Perform a focused history and physical, evaluating:

Duration of symptoms
Risk factors for potentially serious conditions (e.g., tumor, infection,
cauda equina syndrome)
Symptoms suggesting radiculopathy or spinal stenosis
Presence and severity of neurologic deficits
Psychosocial risk factors

Cancer, infection, or cauda equina syndrome strongly Yes MRI or CT

suspected, or severe/progressive neurologic deficit? Specialist consultation

No
Consider imaging with initial plain
Cancer, infection, vertebral compression fracture, or radiography in most cases
Yes Consider ESR for evaluation of
other specific underlying condition not strongly
suspected, but one or more risk factors present? cancer or infection
If only weak risk factors for cancer,
No consider trial of therapy

Do not routinely obtain imaging or other diagnostic tests

Provide infomation and self-care advice to all patients Specific condition present?
Provide information on expected course and effective
No
self-care options Yes
Recommend continuing normal activities as much as possible
Review indications for reassessment and for diagnostic testing Evaluate and treat appropriately

Yes Continue self-care

Back pain mild and no significant
Review indications
functional deficits?
for reassessment
No

Consider pharmacologic or nonpharmacologic, noninvasive therapy for initial treatment

Pharmacologic options: acetaminophen, NSAIDs, opioids, tramadol, benzodiazepines,
skeletal muscle relaxants (for acute LBP), and tricyclic antidepressants (for chronic LBP)
Nonpharmacologic options for chronic LBP: acupuncture, exercise, massage, yoga,
behavorial therapy, spinal manipulation (also for acute LBP), and interdisciplinary
rehabilitation

Patient accepts risks and Yes Initiate time-limited trial of therapy

benefits of therapy? Assess response to treatment

No

Continue self-care
Reassess patient (within 1
month for acute low back pain)

Back pain resolved or improved Yes Continue self-care

with no significant functional deficits? Review indications for re-assessment

Figure 41-1  Algorithm for evaluation of a patient with low back pain. CT, computed tomography; ESR, erythrocyte sedimentation rate; LBP, low back
pain; MRI, magnetic resonance imaging; NSAIDs, nonsteroidal anti-inflammatory drugs.
 620 Wildstein  |  Low Back Pain
Table 41-2  Red Flags in the Evaluation of Low   disks can yield pain of a typical character and anatomic
Back Pain
Symptom Potential Causes
Night pain Tumor, infection
Recent weight loss Malignancy
Lytic lesion on imaging studies Malignancy/infection
Bowel/bladder incontinence Cauda equina syndrome
Progressive, unrelenting pain Malignancy/infection
Pain unrelieved by rest Malignancy
a screening tool for LBP. Typical laboratory tests consist of
C-reactive protein, erythrocyte sedimentation rate, and
serum or urine protein electrophoresis. Additional labora-
tory work may include complete blood count, serum creati-
nine, and serum calcium if myeloma is suspected.
Persistent, debilitating LBP lasting more than 4 to
6 weeks should be assessed carefully and possibly referred
to the care of a spine specialist for secondary evaluation.
Early in the evaluation, a consideration of psychosocial
impediments to recovery should be considered. As ­discussed
­previously, these psychosocial and other comorbid condi-
tions may be more important in predicting recovery than
imaging results. Neither MRI nor plain radiographs obtained
early in the course of LBP evaluation improves clinical out-
come, ­predicts recovery course, or reduces the overall cost of
care.18 If the patient has not already obtained further imag-
ing studies at the time of referral to a spine specialist and
his or her pain has persisted after 4 to 8 weeks of conserva-
tive care, a rapid-sequence MRI study may be warranted.
It should be reiterated to the patient that common degen-
erative ­ findings (e.g., Schmorl’s nodes, end plate changes,
disk desiccation) are fully expected and are most commonly
unrelated to symptoms.
PROVOCATIVE TESTING
Further attempts at identifying a single pain-causing struc-
ture, the so-called pain generator hypothesis, have focused
primarily on the intervertebral disks and facet joints. In
recent years, strategies aimed at sorting out specifically
the level of degenerative disk or facet joint most likely
­responsible for a patient’s discomfort have been presented.
Provocative discography is aimed at identifying primary
discogenic pain as the sole or major cause of LBP illness. In
a typical discography procedure, a patient is lightly sedated,
and radiopaque dye is injected under fluoroscopic guidance
into several intervertebral disks in sequence. The patient’s
pain responses are noted at each level. A concordant pain
response (i.e., one reproducing the patient’s usual pain)
at one level combined with a “negative” injection at an
­adjacent level is thought by proponents to be diagnostic
for “discogenic pain.” Advocates of the technique interpret
the data from such a “positive discogram” as defining the
disk itself as the primary or sole cause of LBP illness in that
patient, and suggest that eliminating the pain-generating
disk (via spinal fusion, disk arthroplasty, or percutaneous
intervention) would eliminate a patient’s symptoms.
Provocative discography has not been validated by stan-
dard methods of determining the accuracy of diagnostic
testing. Numerous reports have shown that injections into
location, although the actual cause of the patient’s LBP is
known not to originate from the disk in question.19 Addi-
tionally, currently asymptomatic subjects who previously
had undergone disk surgery, patients with psychological
distress, and individuals with disputed compensation claims
or remote chronic pain issues experienced pain with disk
injection 80% of the time.20,21 Many LBP patients who
are evaluated with discography fall into one or more of the
aforementioned categories, increasing the likelihood of a
false-positive test.
The ideal candidate for back surgery after positive discog-
raphy may be one with an annular disruption and advanced
disk degeneration on imaging studies, negative adjacent disk
pathology, low pressures on injection, no complicating com-
pensation claims, and normal psychometric testing. Studies
focusing on patients with these “ideal” parameters show low
rates of highly successful outcomes (<50% of subjects), even
in the presence of radiographically solid interbody fusion.
In the same group of model patients using high-grade relief
of LBP symptoms after fusion as the “gold standard” for con-
firmed diagnosis, the positive predictive value of an injec-
tion was at best only 50% to 60%.22
Whether using discography improves the outcomes of
individuals seeking surgery for LBP illness is unclear. In
the best evidence study to date, lumbar fusions performed
on individuals for positive discographic findings were no
more successful at reducing back pain than fusions per-
formed on patients selected for fusion surgery without
discography.23
Investigators also have experimented with anesthetic
blockade of the facet medial branch nerves and sacroiliac
joints. An injection’s success is gauged by the patient’s
­subjective perception of pain before and immediately after
the procedure. Although these modalities have shown anec-
dotal benefit, their validity remains questionable without a
histopathologic “gold standard” against which other treat-
ment modalities can be compared.24-26 Although using such
blocks to predict the outcome of specific neuroablative
­procedures of the lumbar facet joints has shown promise in
one study,27 the predominant interest to date of this inter-
vention is with disk arthroplasty candidates to rule out facet-
mediated pathology as the source of back pain. The degree
of success of facet and sacroiliac joint injections seems not
to correlate with the actual presence of or degree of local
pathology as seen on radiography or MRI.
NONSURGICAL INTERVENTIONS
PHARMACOLOGIC THERAPY
Treatment of LBP with narcotic pain medicines over the
long-term is usually discouraged.28,29 In addition to the
dependency issues that patients taking long-term narcotics
may develop, habituation to the analgesic effects of nar-
cotics can complicate postoperative pain control should
­surgery become necessary. Patients often are troubled by the
­gastrointestinal and sedative side effects of analgesic effects
of high-dose narcotics, and some patients require progres-
sively higher doses to achieve the same level of analgesia.
Classically, first-line pharmacologic treatment of LBP in
the absence of contraindications has been the ­nonsteroidal
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN 621

Table 41-3  Pharmacologic Treatment of Low Back Pain

Drug Name Mechanism of Action Anti-inflammatory Effects Side Effects
Acetaminophen Inhibits prostaglandin E synthesis +/− Hepatotoxic at high doses
NSAIDs Reversible inhibition of COX isoenzymes   +++ Gastrointestinal damage;  
blocking conversion of arachidonic   renal failure at high dose
acid to prostaglandin
COX-2 inhibitors Selective inhibition of COX-2 isoform;   +++ Increased risk of cardiovascular
blocks conversion of arachidonic   events
acid to prostaglandin
Muscle relaxants Inhibition of polysynaptic neuronal   − Sedation
events
Opioids Binds μ opioid receptors − Constipation, nausea,  
somnolence, dry mouth
Corticosteroids Inhibits phospholipase A2, preventing   ++++ Long-term use can lead to
leukotriene-mediated and   ­immunosuppression, aseptic
prostaglandin-mediated   ­necrosis of bone, impaired
inflammatory response wound healing, osteoporosis,
suppression of hypothalamic-
­pituitary-adrenal axis
Tricyclic   Unknown − Dry mouth, cardiac effects,
antidepressants ­sedation

COX, cyclooxygenase; NSAIDs, nonsteroidal anti-inflammatory drugs.

anti-inflammatory drug (NSAID) class of medications
(Table 41-3). A more recent Cochrane analysis has pointed
out the limited effect size of these medications in the treat-
ment of LBP. Studies suggest that the effects of NSAIDs
compared with placebo are quite small.28 In a month-long
trial with patients experiencing acute flare-up of previously
diagnosed LBP, pain scores improved an average of 40 points
on a 100-point scale with cyclooxygenase-2 inhibitors com-
pared with an average improvement of 30 points with pla-
cebo. Although not directly compared, acetaminophen
alone may have a comparable effect at a much lower cost
and risk of complications. Cyclooxygenase-2 inhibitors may
limit some of the gastrointestinal risks of NSAIDs, but are
considerably more expensive. Given their association with
an increased risk of cardiovascular events,30,31 long-term
control of chronic LBP with cyclooxygenase-2 inhibitors
should be carefully considered.
Adjuncts in the pharmacologic arsenal include mus-
cle relaxants, but the evidence for their use is limited as
well.28 Two randomized trials with a combined 222 patients
­compared treatment of LBP with 150 mg of the muscle
relaxant tetrazepam (divided dosing three times daily)
with placebo. The effect of tetrazepam, although statisti-
cally ­ significant, was clinically only of marginal benefit in
­alleviating symptoms.29
Only one other type of pharmacologic intervention has
consistently shown reproducible clinical benefits in the treat-
ment of LBP. The tricyclic and the tetracyclic classes of anti-
depressants have been proved in randomized clinical trials
to be effective treatments for LBP in patients ­without preex-
isting clinical depression. The selective serotonin reuptake
inhibitors and trazodone were no more effective at treating
LBP than placebo in the trials, whereas over 4 to 8 weeks,
patients taking the tricyclics or tetracyclics experienced a
20% to 40% greater reduction in pain than their placebo-
controlled counterparts.32 Although subjects’ pain scores
improved, their functional capabilities did not. In addition,
the use of antidepressant ­ pharmacologic ­ intervention is
not without risk. Greater than 20% of ­subjects ­experienced
some form of side effects while in the study.33
PHYSICAL MODALITIES
An important aspect in trying to prevent or reverse the
debilitating physical and mental effects of LBP is a patient’s
willingness to engage in physical and social activities despite
low back discomfort.34 Patients with high “fear avoidance”
characteristics (a disproportionate predilection to avoid
activities or situations that may produce discomfort) have
poorer outcomes and are at increased risk for developing
chronic LBP illness than patients who remain active despite
their pain.13,14,16,35 Exercise, no matter what type of regimen,
seems to hasten patients’ return to usual lifestyle activities.
In addition to the natural analgesic effects of endorphins
produced in the body with exercise, the preservation of
joint mobility and a more positive mental outlook seems to
be helpful in dealing with LBP.
A Cochrane review of randomized trials of numerous
exercise programs, including generalized stretching, the
McKenzie method of passive end-range stretching, conven-
tional physical therapy, and strengthening, showed equiva-
lence among interventions. All the programs seemed to offer
some benefit over the usual care given by primary care phy-
sicians.36 Of exercise programs, massage, physical therapy,
low-impact aerobics, osteopathic or chiropractic manipula-
tion, and classroom-style education (so-called back school)
on proper back mechanics, none have proven superior to
another.35,37-39 Concomitant medical and ­ physical treat-
ments seem to have better outcomes with respect to pain
and disability than medical treatment alone.35,40
Armed with this information, a more recent strategy for
the treatment of patients with severe LBP impairments or
psychosocial risk factors inhibiting recovery emphasizes a
functional restoration type of approach. This ­multifaceted
approach to LBP blends medical treatment strategies with
physical exercises and a cognitive behavioral scheme
 622 Wildstein  |  Low Back Pain
f­ocusing the patient’s efforts on achieving specific func-
tional goals (e.g., walking a set distance, ascending a certain
­number of stairs, lifting a set amount of weight a specific
number of repetitions). Compared with usual care scenar-
ios, these attempts at functional restoration also seem to
achieve superior results and minimize the amount of sick
leave taken.41-45 The long-term benefits of all back pain
rehabilitation programs are unproven, however.46
SURGICAL INTERVENTIONS
The surgical treatment of LBP in the absence of structural
findings is as varied as the clinicians advocating treatment.
Ranging from injections to neuroablation techniques to
fusion to disk arthroplasty, the procedures performed today
for the treatment of chronic LBP vary by surgical training,
familiarity with a specific treatment, and even geographic
region.
PERCUTANEOUS TECHNIQUES
Injections into facet joints, into sacroiliac joints, into the
lumbar epidural space, around nerve roots, and into trigger
points are frequently used in the treatment of LBP syndromes,
although there is no compelling evidence that injections are
effective in the absence of primary radicular pain. Frequent
corticosteroid injections (more than three or four a year)
may be associated with systemic or local side effects.
In randomized clinical trials, lumbar epidural ­steroid injec-
tions, facet blocks, trigger point injections, and intradiscal
anesthetics have not shown improvement in the outcomes
of LBP patients who lack discrete radicular ­symptoms.47-49
Sclerotherapy, sometimes called ­ prolotherapy, involves
injecting solutions of dextrose 12.5%, glycerin 12.5%, phe-
nol 1.25%, or lidocaine 0.25% into the spinal ligaments or
lumbodorsal fascia. The theory of prolotherapy stems from
the belief that LBP is generated from weakened or damaged
spinal ligaments. Repeatedly injecting these “damaged”
structures with irritant solutions is purported to strengthen
the ligaments, reducing pain and disability.50 There is con-
flicting evidence in the literature, however, with regard to
the ability of sclerosing agents to eliminate patients’ chronic
LBP and disability. Of some 200 articles authored on the
topic, only 4 met criteria for scientific validity, consisting of
a total of 344 subjects. Confounding variables exist in terms
of clinical heterogeneity among studies and by the pres-
ence of cointerventions that differed greatly among studies.
In the end, there was no clear evidence that ­prolotherapy
injections were more effective than control injections
alone.51-53
Radiofrequency ablation of the afferent medial branch
nerves to the facet joints was examined as a treatment for
facet-mediated pain. According to this hypothesis, facets
are sometimes thought to act as the pain generator in the
absence of obvious facet pathology. Neurotomy was found to
be marginally effective in the short-term (lasting 4 weeks)
in one study,54 whereas another randomized trial showed the
intervention to be ineffective.55 In a small subset of patients
with suspected facet-mediated pain that seemed to respond
well to placebo-controlled anesthetic blockade of the joint,
observational studies (without controls) have suggested rel-
atively good outcomes.56,57
Various percutaneous technologies aimed at the altera-
tion of intradiscal biomechanics and nociception have been
advocated by some clinicians. The two most commonly
employed treatments, intradiscal electrothermal treatment
and percutaneous neuromodulation therapy, met with skep-
ticism by some clinicians who believed that the use of radio-
frequency energy or heat around sensitive nerve structures
was an ill-conceived idea.
Intradiscal electrothermal treatment involves the percu-
taneous insertion of a navigable wire catheter into the disk
space. Thermal energy is delivered with the aim of decreas-
ing the nociceptive response to degenerative changes within
the intervertebral disk. The improvement in pain response
effected by intradiscal electrothermal treatment is thought
to occur through shrinkage of collagen fibers leading to
remodeling, cauterization of granulation tissue, and ther-
mocoagulation of nervous tissue resulting in denervation of
nociceptive nerve fibers within the disk.58 Randomized tri-
als have shown either no effect59,60 or a very small treatment
effect, but only in narrowly tailored cohorts.61 Additionally,
in a more recent study, the investigators failed to show any
correlation of the theoretically critical position of the abla-
tive catheter with patient outcomes.62
Percutaneous neuromodulation therapy is best likened
to acupuncture, wherein 10 needle probes are inserted to
various depths within the paraspinal musculature through
which an electric current is passed. By activating derma-
tomal sensory, myotomal sensorimotor, sclerotomal, and
sympathetic sensory nerves in the dermatomal distribution
of the pain, the postulated mechanism of action is a recal­
ibration of one or more of the neuronal feedback systems
involved in the regulation and maintenance of pain. There
are very few clinical studies and no long-term outcome stud-
ies on percutaneous neuromodulation therapy.
OPEN SURGICAL TECHNIQUES
Open surgical treatment of LBP is at the heart of a long and
continuing controversy. There is no doubt that open surgi-
cal management of some spinal fractures, infections, neo-
plasm, or progressive instability can be enormously helpful.
The usefulness of spinal fusion or decompression in patients
with back pain and only common degenerative changes on
imaging studies is unclear, however.
Shortly after the discovery that decompressive lami-
nectomy seemed to be a highly effective treatment for sci-
atica, many surgeons attempted to expand the ­indications
to LBP without radicular symptoms. Although many sur-
geons reported anecdotal successes with the application
of decompressive laminotomy for degenerative changes,
the more common observation was little, if any, symptom
­improvement. Today, laminectomy as a treatment for degen-
erative disk disease in the absence of neurologic ­symptoms
has been all but abandoned.63 Contemporary strategies
to address surgically degenerative spinal changes without
structural or neurologic compromise include lumbar spinal
fusion, nonfusion technologies such as disk arthroplasty and
dynamic stabilization, and the above-described percutane-
ous ­modalities.
Lumbar fusion as a treatment for LBP caused by a severe
structural deficiency is extremely effective. In the presence
of certain fracture types, spinal infections, ­spondylolisthesis,
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
and increasing coronal or sagittal imbalance, lumbar fusion
may have a dramatic effect in alleviating pain. Studies
place the success rate of lumbar fusion surgery in patients
with discrete and clear pathology at 70% to 90%,22,64 but
in patients who have had fusion for backache and common
­degenerative changes alone, outcomes lag far behind.65
There have been no randomized clinical trials of spinal
fusion versus nonsurgical care to date in the United States.
In two of three European randomized clinical trials compar-
ing fusion and nonoperative treatments, a cognitive behav-
ioral model seems to be more beneficial than usual treatment
as far as nonoperative modalities are concerned.66,67 The
cognitive behavioral therapy group also seemed to have
improved coping strategies, fewer complications, and pos-
sibly even improved occupational outcomes compared
with the fusion group (as measured by Oswestry disability
indices). The third study showed small, yet statistically sig-
nificant differences in the fusion group over unstructured
nonsurgical modes of therapy. It is important to realize,
however, that neither group did exceedingly well, with only
16% of the fusion patients and 6% of the nonoperated group
rating their overall result as “excellent.”68
Cohort studies in which lumbar fusion was undertaken
for a diagnosis of nonspecific degenerative disk disease or
internal disk derangement show small functional improve-
ments. In reviews of the literature on spinal fusion for non-
specific MRI changes along with presumed discogenic pain
based on discography, Carragee and colleagues showed that
ODI improvements were in the 6- to 15-point range (close
to the minimal detectable difference),68a and in another
similar study of workers’ compensation patients, satisfactory
outcomes were rarely observed.69
The role of artificial disk replacement (ADR) in the treat-
ment of LBP is unclear. The success of total joint arthro-
plasty in the knee and hip has led to the hope that similarly
positive outcomes could be realized with joint replacement
in the spine. In contrast to hips or knees, however, a spi-
nal motion segment comprises three discrete articular sur-
faces—two facet joints and the intervertebral disk space.
Arthroplasty of the spine addresses only one of the three
surfaces, leaving facet arthrosis untreated. The cause of the
severe impairment in hip and knee arthrosis is usually well
­correlated with the degree of arthritic change, whereas this
is not the case in LBP: Severe pain and disability are some-
times reported with minimal changes in the spine, or minimal
LBP can be reported with severe ­arthrosis. Hip arthrodesis
(which hip replacement supplanted) was an effective pain-
relieving operation for hip arthrosis, whereas spinal fusion,
as described previously, is only marginally effective. Short-
term trials to date have shown little ­­difference in outcome
between spinal fusion versus disk replacement surgery.
In theory, preserving motion segments in the spine may
decrease the rate at which adjacent segments degenerate.
Whether or not this benefit over fusion makes a clinical dif-
ference remains to be shown by carefully controlled clinical
assessment. The progressive disk degeneration is frequently
asymptomatic in patients with adjacent segment disease.
Current ADR instrumentation is widely inhomogeneous
with regard to mechanism of constraint, materials, and
mobility.
It has taken several decades of clinical research to
arrive at the current successful outcomes with total joint
623
a­ rthroplasty. In an elderly patient population, joint replace-
ments in the hip and knee now can be reliably predicted to
last 15 to 20 years from a purely materials standpoint. The
data on ADR, by comparison, are still in their infancy. Early
follow-up data show that potential problems of osteolysis
and aseptic loosening in ADR are infrequently encountered
in the first 5 years after implantation.
Clinical outcome comparisons between fusion and ADR
for nonspecific LBP have been equivocal. In terms of post-
operative functionality, narcotic use, pain scores, and occu-
pational disability, there do not seem to be large differences
between the two groups.70,71 Trials in the United States and
United Kingdom show clinical failure in half of the patients
who underwent ADR. This fact is especially surprising given
the narrowly tailored entry criteria for the initial ADR stud-
ies.70,72 Pioneering work on ADR was performed in Europe
with the Charité disk (Depuy, Warsaw, Ind). Long-term data
from the Charité experience show questionable preserva-
tion of motion at operated levels. The data also call into
question the clinical benefit of preserving motion segments
and the relationship to improving pain.73
PUBLIC HEALTH CAMPAIGNS
The immense public health cost of LBP to society has
prompted some governmental agencies to get involved.
Buchbinder and Jolley74 examined the long-term effects of
an Australian media campaign aimed at altering negative
beliefs about LBP, decreasing lost work time, and decreas-
ing medical use by individuals with LBP. Over the course
of 2 years, media advertisements encouraging Australians
to remain active and in the workforce despite having LBP
proved significantly effective at altering physicians’ and
patients’ perceptions regarding back pain. Workers’ com-
pensation claims for LBP were significantly reduced for the
2 years during which the ads ran, with an effect that per-
sisted for at least 3 years after the campaign. This lasting
effect suggests the potential effectiveness of primary preven-
tion strategies in altering the negative way in which LBP is
perceived in the medical and the lay communities.
CONCLUSION
Compared with many areas of our current fund of ortho-
paedic knowledge, the spine remains a comparative “black
box” in terms of understanding of certain specific disorders.
What is clear from the data is that an effective treatment
for chronic persistent and disabling LBP must focus on a
multifaceted approach. The answer at present seems to lie in
a combination of medical and physical modalities, although
current strategies for non-neurogenic back disorders remain
as varied as the physicians who provide them.
Current guidelines from numerous spine professional
groups agree that an early, gradual return to activities of
daily living represents a good strategy for coping with non-
specific LBP. Identification of psychological risk factors
may aid significantly in identifying patients who may not
respond well to surgical interventions. In the presence of
certain structural findings that are not well delineated in
their recommendations, the North American Spine Society
currently recommends a 2- to 4-month trial of nonoperative
modalities before attempting a surgical treatment for LBP.
 624 Wildstein  |  Low Back Pain
Surgical treatments have not been found to be highly effec-
tive. The assessment of LBP is best done on an individual
basis, taking into account the complex interconnectedness
of each patient’s specific physical demands, expectations
for recovery, psychosocial makeup, social and occupational
demands, and objective physical examination and radio-
graphic findings.
REFERENCES
  1. Anderson GB: Epidemiological features of low-back pain Lancet
354:581-585, 1999.
  2. U.S. Department of Health and Human Services: Summary Health
Statistics for U.S. Adults: National Health Interview Survey, 2002.
Vital and Health Statistics Series 10, No. 222 [DHHS Publication No.
(PHS) 2004-1550]. Hyattsville, Md, U.S. Department of Health and
Human Services, 2004. Available at: www.cdc.gov/nchs/data/series/
sr_10/sr10_222.pdf. Accessed January 8, 2007.
  3. National Academy of Sciences and Institute of Medicine: Musculo-
skeletal Disorders and the Workplace. Washington, DC, National
­Academy Press, 2001.
  4. Skovron ML, Szpalski M, Nordin M, et al: Sociocultural factors and
back pain: A population-based study in Belgian adults. Spine 19:
129-137, 1994.
  5. Cassidy JD, Carroll LJ, Cote P: The Saskatchewan Health and Back
Pain Survey: The prevalence of low back pain and related disability in
Saskatchewan adults. Spine 23:1860-1866, 1998.
  6. Carragee E, Cohen S: Reliability of LBP history in asymptomatic
­subjects? The prevalence and incidence of reported back pain corre-
lates with surveillance frequency. In Proceedings of the 19th Annual
Meeting of the North American Spine Society, Chicago, October
26-30, 2004, p 216 (abstract).
  7. Carragee EJ, Paragioudakis SJ, Khurana S: 2000 Volvo Award ­winner
in clinical studies: Lumbar high-intensity zone and discography in
­subjects without low back problems. Spine 25:2987-2992, 2000.
  8. Boden S, Davis D, Dina T, et al: Abnormal magnetic resonance scans
of the lumbar spine in asymptomatic subjects: A prospective investiga-
tion. J Bone Joint Surg Am 72:403-408, 1990.
  9. Jensen M, Brant-Zawadzki M, Obuchowski N, et al: Magnetic
­resonance imaging of the lumbar spine in people without back pain.
N Engl J Med 331:69-73, 1994.
10. Carragee E, Alamin T, Cheng I, et al: Are first-time episodes of serious
LBP associated with new MRI findings? Spine J 6:624-635, 2006.
11. Jarvik JJM, Hollingworth W, Heagerty P, et al: The ­ Longitudinal
Assessment of Imaging and Disability of the Back (LAIDBack)
study—baseline data. Spine 26:1158-1166, 2001.
12. Borenstein DG, O’Mara JW Jr, Boden SD, et al: The value of mag-
netic resonance imaging of the lumbar spine to predict low-back pain
in asymptomatic subjects: A seven-year follow-up study. J Bone Joint
Surg Am 83:1306-1311, 2001.
13. Carragee EJ, Alamin TF, Miller JL, et al: Discographic, MRI, and
­psychosocial determinants of low back pain disability and remission:
A prospective study in subjects with benign persistent back pain.
Spine J 5:24-35, 2005.
14. Boos N, Semmer N, Elfering A, et al: Natural history of individuals
with asymptomatic disc abnormalities in magnetic resonance ­imaging:
Predictors of low back pain-related medical consultation and work
incapacity. Spine 25:1484-1492, 2000.
15. Bigos SJ, Battie MC, Spengler DM, et al: A prospective study of work
perceptions and psychosocial factors affecting the report of back injury
[erratum in Spine 16:688, 1991]. Spine 16:1-6, 1991.
16. Burton AK, Tillotson KM, Main CJ, et al: Psychosocial predictors of
outcome in acute and subacute low back trouble. Spine 20:722-728,
1995.
17. Robertson WD, Jarvik JG, Tsuruda JS, et al: The comparison of a rapid
screening MR protocol with a conventional MR protocol for lumbar
spondylosis. AJR Am J Roentgenol 166:909-916, 1996.
18. Gilbert FJ, Grant AM, Gillan MG, et al: Low back pain: Influence
of early MR imaging or CT on treatment and outcome—multicenter
randomized trial. Radiology 231:343-351, 2004.
19. Carragee EJ, Tammer CM, Yang B, et al: False-positive findings on
lumbar discography: Reliability of subjective concordance assessment
during provocative disc injection. Spine 24:2542-2547, 1999.
20. Carragee EJ, Chen Y, Tanner CJ, et al: Provocative diskography in
patients after limited lumbar diskectomy: A controlled, randomized
study of pain response in symptomatic and asymptomatic subjects.
Spine 25:3065-3071, 2000.
21. Carragee EJ, Tanner CM, Khurana S, et al: The rates of false-­positive
lumbar discography in select patients without low back symptoms.
Spine 25:1373-1380, 2000.
22. Carragee EJ, Lincoln T, Parmar VS, et al: A gold standard evalua-
tion of the “discogenic pain” diagnosis as determined by provocative
­discography. Spine 31:2115-2123, 2006.
23. Madan S, Gundanna M, Harley JM, et al: Does provocative
­diskography screening of discogrnic back pain improve surgical out-
come? J Spinal Disord Tech 15:245-251, 2002.
24. Reid M, Lachs M, Feinstein A: Use of methodological standards
in diagnostic test research: Getting better but still not good. BMJ
274:645-651, 1995.
25. Sackett DL, Rosenberg WM, Gray JA, et al: Evidence based medicine:
What it is and what it isn’t. BMJ 312:71-72, 1996.
26. Greenhalgh T: Education and debate: Papers that report diagnostic or
screening tests. BMJ 315:1-12, 1997.
27. Dreyfuss P, Halbrook B, Pauza K, et al: Efficacy and validity of
­radiofrequency neurotomy for chronic lumbar zygoapophyseal joint
pain. Spine 25:1270-1277, 2000.
28. van Tulder MW, Scholten RJ, Koes BW, et al: Nonsteroidal anti-
inflammatory drugs for low back pain: A systematic review within
the framework of the Cochrane Collaboration Back Review Group.
Spine 25:2501-2513, 2000.
29. Van Tulder MW, Touray T, Furlan AD, et al: Muscle relaxants for non-
specific low back pain. Cochrane Database Syst Rev 2:CD004252, 2003.
30. Nussmeier NA, Whelton AA, Brown MT, et al: Complications of
the COX-2 inhibitors parecoxib and valdecoxib after cardiac surgery.
N Engl J Med 352:1081-1091, 2005.
31. Ostelo R, Tulder M, Vlaeyen J, et al: Behavioral treatment for chronic
low back pain. Cochrane Database Syst Rev 1:CD002014, 2005.
32. Staiger TO, Gaster B, Sullivan MD, et al: Systematic review of
­antidepressants in the treatment of chronic low back pain. Spine
28:2540-2545, 2003.
33. Salerno SM, Browning R, Jackson JL: The effect of antidepressant
treatment on chronic back pain: A meta analysis. Arch Intern Med
162:19-24, 2002.
34. Deyo RA, Diehl AK, Rosenthal M: How many days of bed rest
for acute low back pain? A randomized clinical trial. N Engl J Med
315:1064-1070, 1986.
35. Hurwitz EL, Morganstern H, Harber P, et al: A randomized trial of
medical care with and without physical therapy and chiropractic care
with and without physical modalities for patients with low back pain:
6 month follow-up outcomes from the UCLA low back pain study.
Spine 27:2193-2204, 2002.
36. Van Tulder MW, Malmivaara A, Esmail R, et al: Exercise therapy for
low back pain. Cochrane Database Syst Rev 2:CD000335, 2000.
37. Cherkin DC, Sherman KJ, Deyo RA, et al: A review of the evidence
for the effectiveness, safety, and cost of acupuncture, massage therapy,
and spinal manipulation for back pain. Ann Intern Med 138:898-906,
2003.
38. Assendelft WJ, Morton SC, Yu EI, et al: Spinal manipulative therapy
for low back pain: A meta-analysis of effectiveness relative to other
therapies. Ann Intern Med 138:871-881, 2003.
39. Mannion AF, Muntener M, Taimela S, et al: A randomized ­clinical
trial of three active therapies for chronic low back pain. Spine 24:
2435-2448, 1999.
40. Niemisto L, Lahtinen-Soupanki T, Rissanen P, et al: A randomized
trial of combined manipulation, stabilizing exercises and physician con-
sultation alone for chronic low back pain. Spine 28:2185-2191, 2003.
41. Schonstein E, Kenny DT, Keating J, et al: Work conditioning, work
hardening and functional restoration for workers with back and neck
pain. Cochrane Database Syst Rev 1:CD001822, 2003.
42. Guzman J, Esmail R, Karjalainen K, et al: Multidisciplinary bio-
­psycho-social rehabilitation for chronic low-back pain. Cochrane
Database Syst Rev 1:CD000963, 2002.
43. Fairbank J, Frost H, MacDonald J, et al: The MRC Spine Stabiliza-
tion Trial: A randomized controlled trial comparing surgical stabiliza-
tion of the lumbar spine with intensive rehabilitation in patients with
chronic low back pain. In Proceedings of the 31st Annual Meeting of
the International Society for the Study of the Lumbar Spine, Porto,
Portugal, May 31-June 5, 2004, p 38 (abstract).
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
44. Ivar Brox J, Sorensen R, Friis A, et al: Randomized clinical trial
of lumbar instrumentated fusion and cognitive intervention and
­exercises in patients with chronic low back pain and disc degenera-
tion. Spine 28:1913-1921, 2003.
45. Van Tulder MW, Ostelo R, Vlaeyen JW, et al: Behavioral treatment
for chronic low back pain: A systematic review within the frame-
work of the Cochrane Collaboration Back Review Group. Spine 26:
270-281, 2001.
46. Ostelo R, Tulder M, Vlaeyen J, et al: Behavioral treatments for chronic
low back pain. Cochrane Database Syst Rev 1:CD002014, 2005.
47. Carette S, Marcoux S, Truchon R, et al: A controlled trial of cortico-
steroid injections into facet joints for chronic low back pain. N Engl J
Med 325:1002-1007, 1991.
48. Nelemans PJ, deBie RA, deVet HC, et al: Injection therapy for
­subacute and chronic benign low back pain. Spine 26:501-515, 2001.
49. Khot A, Bowditch M, Powell J, et al: The use of intradiscal steroid
therapy for lumbar spinal discogenic pain: A randomized controlled
trial. Spine 29:833-836, 2004.
50. Dagenais S, Haldeman S, Wooley JR: Intraligamentous injection of
sclerosing solutions (prolotherapy) for spinal pain: A critical review of
the literature. Spine J 5:310-328, 2005.
51. Dechow E, Davies RK, Carr AJ, et al: A randomized, double-blind,
placebo-controlled trial of sclerosing injections in patients with
chronic low back pain. Rheumatology (Oxf) 38:1255-1259, 1999.
52. Yelland MJ, Glasziou PP, Bogduk N, et al: Prolotherapy injections,
saline injections, and exercises for chronic low-back pain: a random-
ized trial. Spine 29:9-16, 2004.
53. Yelland MJ, Mar C, Pirozzo S, et al: Prolotherapy injections for chronic
low-back pain. Cochrane Database Syst Rev 2:CD004059, 2004.
54. Van Kleef M, Barendse GA, Kessels A, et al: Randomized trial of
radiofrequency lumbar facet denervation for chronic low back pain.
Spine 24:1937-1942, 1999.
55. Leclaire R, Fortin L, Lambert R, et al: Radiofrequency facet joint
denervation in the treatment of low back pain: A placebo-controlled
clinical trial to assess efficacy. Spine 26:1411-1416, 2001.
56. Dreyfuss P, Halbrook B, Pauza K, et al: Efficacy and validity of radio-
frequency neurotomy for chronic lumbar zygoapophyseal joint pain.
Spine 25:1270-1277, 2000.
57. Van Kleef M, Weber WE, Kessels A, et al: Re: Efficacy and validity
of radiofrequency neurotomy for chronic lumbar zygoapophyseal joint
pain (Spine 25:1270-1277, 2000).
58. Saal JA, Saal JS: Management of chronic discogenic low back pain
with intradiscal thermal catheter Spine 25:382-388, 2000.
59. Barendse GA, van den Berg SG, Kessels AH, et al: Randomized
controlled trial of percutaneous intradiscal radiofrequency thermo-
coagulation for chronic discogenic back pain: Lack of effect from a
90-second 70 C lesion. Spine 26:287-292, 2001.
60. Freeman BJ, Fraser RD, Cain CM, et al: A randomized double-blind
controlled efficacy study: Intradiscal Electrothermal Therapy (IDET)
versus placebo. J Bone Joint Surg Br 86:484-485, 2004.
625
61. Pauza KJ, Howell S, Dreyfuss P, et al: A randomized, placebo-
­controlled trial of intradiscal electrothermal therapy for the treatment
of discogenic low back pain. Spine J 4:27-35, 2004.
62. Freeman BJ, Fraser RD, Cain CM, et al: A randomized, double-blind,
controlled trial: Intradiscal electrothermal therapy versus placebo
for the treatment of chronic discogenic low back pain Spine 30:
2369-2377, 2005.
63. Allan DB, Waddell G: An historical perspective on low back pain and
disability. Acta Orthop Scand 234S:1-23, 1989.
64. Swan J, Hurwitz E, Malek F, et al: Surgical treatment for unsta-
ble low grade isthmic spondylolisthesis in adults: A prospective
­controlled study of posterior instrumented fusion compared with
combined ­anterior-posterior fusion. Spine J 6:606-614, 2006.
65. Gibson JN, Waddell G: Surgery for degenerative lumbar spondylosis:
Updated Cochrane Review. Spine 30:2312-2320, 2005.
66. Ivar Brox J, Sorensen R, Friis A, et al: Randomized clinical trial of
lumbar instrumented fusion and cognitive intervention and exercises
in patients with chronic low back pain and disc degeneration. Spine
28:1913-1921, 2003.
67. Fairbank J, Frost H, Wilson-Macdonald J, et al: Randomised ­controlled
trial to compare surgical stabilization of the lumbar spine with an
intensive rehabilitation programme for patients with chronic low back
pain: The MRC spine stabilization trial. BMJ 330:1233, 2005.
68. Fritzell P, Hagg O, Wessberg P, et al: Group SLSS: 2001 Volvo
Award winner in clinical studies: Lumbar fusion versus nonsurgi-
cal treatment for chronic low back pain: A multicenter randomized
controlled trial from the Swedish Lumbar Spine Study Group. Spine
26:2521-2532, 2001.
68a. Don AS, Carragee E: A brief overview of evidence-informed manage-
ment of chronic low back pain with surgery. Spine J 8(1):258-265,
2008.
69. DeBerard MS, Masters KS, Colledge AL, et al: Outcomes of posterolat-
eral lumbar fusion in Utah patients receiving workers’ compensation:
A retrospective cohort study. Spine 26:738-746; discussion 747, 2001.
70. Freeman BJ, Davenport J: Total disc replacement in the lumbar spine:
A systematic review of the literature. Eur Spine J 15:439-447, 2006.
71. Delamarter RB, Bae HW, Pradhan BB: Clinical results of ProDisc-II
lumbar total disc replacement: Report from the United States clinical
trial. Orthop Clin North Am 36:301-313, 2005.
72. Blumenthal S, McAfee PC, Guyer RD, et al: A prospective, random-
ized, multicenter Food and Drug Administration investigational device
exemptions study of lumbar total disc replacement with the CHARITE
artificial disc versus lumbar fusion, part I: Evaluation of clinical out-
comes. Spine 30:1565-1575; discussion E387-E391, 2005.
73. Putzier M, Funk JF, Schneider SV, et al: Charite total disc replace-
ment—clinical and radiographical results after an average follow-up
of 17 years. Eur Spine J 15:183-195, 2006.
74. Buchbinder R, Jolley D: Population based intervention to change back
pain beliefs: Three year follow up population survey. BMJ 328:321,
2004.
 42
KEY POINTS
The clinician should be able to narrow the differential
diagnosis of hip and knee pain down to two to three
diagnoses after the history and physical examination.
Imaging studies should be used to confirm the diagnosis.
Conventional radiographs should be the initial imaging study
ordered.
Many vital structures in the knee can be palpated easily or
examined with provocative tests.
A knee effusion usually is associated with internal
derangement.
The clinician should suspect a torn meniscus if a patient has an
effusion, joint line tenderness, and pain with hyperextension
and with hyperflexion.
Patients with osteoarthritis often complain of stiffness and
pain with activity.
Inflammatory arthritis should be considered when a patient
continues to experience pain despite resting the joint.
Groin pain with internal rotation of the hip is due to hip
pathology until proved otherwise.
Concurrent hip and lumbosacral pathology are common.
It is estimated that musculoskeletal pain affects one third to
one half of the general population.1,2 A substantial increase
in the burden of musculoskeletal disease is expected in the
next decade as the “Baby Boomers” reach middle age and
beyond. Hip and knee replacement operations already have
increased in prevalence; operations increased by 16.2% to
884,400 procedures annually in the United States between
2002 and 2004.3 The prevalence of total knee and total hip
arthroplasty is expected to double by 2016 (for total knee
arthroplasty) and 2026 (for total hip arthroplasty).4 The
hip and knee joints are two of the most commonly affected
sites of musculoskeletal pain, with the prevalence of hip
pain ranging from 8% to 30% in individuals 60 years old
and older,5,6 and the prevalence of knee pain ranging from
20% to 52% in individuals 55 years old and older. Women
generally experience more musculoskeletal pain than men.7
Geographic and ethnic variations in the rates of hip and
knee pain also exist. There tends to be significantly less hip
and knee pain as latitude decreases and significantly less
hip pain and osteoarthritis in China than in the United
States.8-15
When evaluating complaints of knee or hip pain, knowl-
edge of the anatomy of these joints is necessary for formu-
lating a differential diagnosis. Given the thin soft tissue
envelope around the knee, and the fact that knee pain is
Hip and Knee Pain
James I. Huddleston  • 
Stuart B. Goodman
rarely referred, the pain generators around the knee often
can be elucidated with a complete history and thorough
physical examination. Diagnosis of hip pain may be more
challenging because the joint is deeper, and the region
is frequently the site of referred pain from the spine. An
understanding of the basic biomechanics of these joints also
is important in formulating a differential diagnosis because
certain activities are likely to cause specific injuries.
KNEE PAIN
HISTORY
A detailed history is perhaps the most important step in
accurately diagnosing the cause of knee pain. Knee com-
plaints generally fall into two broad categories—pain or
instability. Pain may arise from injury to the articular sur-
faces (e.g., osteoarthritis, inflammatory arthritis, osteochon-
dral defects, and osteochondritis dissecans), torn menisci,
quadriceps and patella tendon tears, bursitis, nerve dam-
age, fractures, neoplasia, or infection. Instability is usually
episodic and stems from injuries to the quadriceps–patellar
extensor mechanism, collateral ligaments, or cruciate liga-
ments. It is important to distinguish true instability from
the common complaint of “giving way” because the latter is
usually due to a robust pain response, rather than to specific
structural pathology.
Patients in certain age groups tend to experience simi-
lar injuries. In patients younger than 40 years, ligament
injuries, acute meniscus tears, and patellofemoral problems
are frequently encountered. In contrast, degenerative con-
ditions, such as osteoarthritis and degenerative meniscal
lesions, tend to occur more frequently in older patients.
The location and character of the pain are particularly
important when evaluating knee pain. It is useful to concep-
tualize the knee as three separate compartments—medial,
lateral, and patellofemoral. Each compartment should be
examined separately. The patient should be able to point to
the exact area where the pain is most severe. The onset of
the pain should be determined. Osteoarthritis and inflam-
matory arthritis tend to have an insidious onset, whereas
injuries to menisci and ligaments usually are associated with
a traumatic event. Knowing the details of a traumatic event
is helpful. A twisting injury, especially one sustained with
a flexed knee, suggests a meniscus tear, whereas a noncon-
tact knee injury associated with change of direction is more
likely to produce a tear of the anterior cruciate ligament.
Pain from degenerative arthritis tends to be associated with
stiffness; is generally worse with ongoing activity during the
day; and is exacerbated by exercise, stair climbing, getting
up from a chair, or getting in and out of a car.
627
 628 HUDDLESTON  |  Hip and Knee Pain
The presence or absence of knee swelling is an important
part of the history because knee effusions usually accom-
pany internal derangement. An effusion also may be pres-
ent with synovitis, osteoarthritis, inflammatory arthritis,
fractures, infection, and neoplasm. Distinguishing between
soft tissue swelling around the knee, synovial thickening,
and a true knee effusion is crucial (see later). The timing or
onset of the swelling also is important for determining the
diagnosis. An acute cruciate or collateral ligament injury
or osteochondral fracture usually manifests with an acute
hemarthrosis (occurring within 1 hour), whereas an effusion
associated with arthritis tends to be more insidious in its
development.
Complaints of “locking” are common. In a younger patient,
locking may be due to a displaced meniscal tear. In older
patients with degenerative arthritis, complaints of locking
are often due to loose bodies. It is important to distinguish
between true locking and diminished range of motion owing
to pain (so-called pseudolocking); this distinction determines
which imaging studies are most appropriate.
Timing of the pain with activity also is important for
making the correct diagnosis. Meniscus tears and ligament
injuries leading to instability are particularly troublesome
with activities such as walking on uneven surfaces, walking
up or down stairs, movements requiring knee flexion, and
pivoting. Osteoarthritis tends to be exacerbated by all load
bearing activities and is relieved by rest.
The clinician also should explore the patient’s exercise
tolerance and ability to perform activities of daily living.
Important details of the patient’s story include the use of
ambulatory assist devices (cane, crutches, walker, brace, and
wheelchair), walking tolerance, and capability for other
exercises (physical therapy).
A history of any previous treatment should be recorded.
The response to physical therapy, analgesics, nonsteroidal anti-
inflammatory drugs, nutritional supplements (e.g., glucosamine
and chondroitin), intra-articular injections of corticosteroids
Figure 42-1  Assessment of coronal alignment.
or hyaluronic acid derivatives, and any operative treatments
lends further insight into the accurate diagnosis and has impli-
cations for treatment when the diagnosis has been confirmed.
At the end of taking a detailed history, the clinician should
be able to formulate a differential diagnosis with a short list of
potential conditions.
PHYSICAL EXAMINATION
General
After a brief overall assessment of the patient, the physical
examination should begin with observation of the patient’s
lower extremity coronal alignment and leg lengths. It is
preferable to have the patient stand with legs slightly apart
while facing the examiner (Fig. 42-1). A goniometer is used
to measure the varus/valgus alignment of the knees. Evalu-
ation of leg lengths should be performed with step blocks of
known sizes. The total height of the blocks needed to make
the iliac crests level with the floor is equivalent to the leg-
length discrepancy (Fig. 42-2).
Gait is examined next. Although a comprehensive dis-
cussion of gait analysis is beyond the scope of this chapter,
there are a few basic observations that all clinicians should
make routinely when evaluating a patient with a knee prob-
lem. Antalgic gaits (shortened stance phase) and thrusts are
commonly seen. Any disorder that causes lower extremity
pain may cause an antalgic gait. Seen in the stance phase of
gait, thrusts may be due to a progressive angular deformity
secondary to degenerative changes or chronic ligamentous
instability. Medial thrusts result from medial collateral liga-
ment laxity, posteromedial capsular laxity or both. Lateral
thrusts arise from lateral collateral ligament or posterolat-
eral corner laxity (Fig. 42-3). Patients also may thrust into
Figure 42-2  The total height of the blocks needed to make the iliac
crests level is equal to the length discrepancy.
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
A B
Figure 42-3  A and B, The femur shifts medially during a medial thrust
(A) and laterally during a lateral thrust (B).
A B C of a goniometer.
Figure 42-4  A-C, Large effusions can be detected by “ballotting” the
patella with the knee in extension.
recurvatum (so-called back-knee deformity) secondary to
posterior capsular laxity or quadriceps weakness.
The patient should transfer to the examination table for
evaluation in a supine position. A pillow should be placed
under the knee if full extension is impossible because of
pain (e.g., fractures, displaced meniscus tears, or large effu-
sion). If it is not symptomatic, the contralateral knee can
serve as an adequate control. The lower extremity should be
inspected for any skin lesions, areas of ecchymosis, or surgi-
cal scars. Quadriceps atrophy should be noted, and a tape
measure should be used to record thigh circumference at the
same distance from the patella or joint line in each knee.
The presence of an effusion should be noted. Effusion is
seen as fullness or swelling in the suprapatellar pouch. The
effusion should be confirmed by ballottement of the patella
(Fig. 42-4). Small effusions require “milking” of the fluid
upward into the suprapatellar pouch and expressing it down
with the examiner’s fingers to appear as a “bulge” medial
629
Figure 42-5  Small effusions can be appreciated by the “milking” of
fluid into the suprapatellar pouch and then pressing it down, producing
a “bulge sign“ below the medial edge of the patella.
to the patella (Fig. 42-5). The active and passive range of
motion of both knees should be recorded, preferably by use
The examiner should proceed with palpation of all
structures of the knee. Palpation should be gentle, but firm
enough to detect subtle pathology. Structures to be pal-
pated include the quadriceps tendon, the patella (superior
and inferior poles), the pes anserinus bursa, the medial (Fig.
42-6A) and lateral (Fig. 42-6B) joint lines, the origins and
insertions of the collateral ligaments, the tibial tubercle,
and the popliteal fossa.
Ligaments
Injuries to the collateral or cruciate ligaments may lead to
knee instability. For each translational and rotational motion
of the knee, there are primary and secondary restraints. When
a primary restraint is disrupted, motion is limited by the sec-
ondary restraint. If a secondary restraint is injured, and the
primary restraint remains intact, motion is not abnormal.
The anterior cruciate ligament is the primary restraint to
anterior translation of the tibia, whereas the medial meniscus
is the secondary restraint. Anterior cruciate ligament disrup-
tion leads to a significant increase in anterior tibial transla-
tion. This translation is increased if the patient had a prior
medial menisectomy.16
The collateral ligaments can be examined with stress
applied in the coronal plane and should be examined in full
extension and in 30 degrees of flexion to remove the influ-
ence of the cruciate ligaments and the capsular restraints.
With the patient in a supine position, a varus force is applied
across the knee to test the lateral collateral ligament, and a
valgus force is applied across the knee to evaluate the medial
collateral ligament.
The anterior cruciate ligament is one of the most fre-
quently injured structures in the knee. Anterior cruciate
ligament insufficiency also is common in advanced osteo-
arthritis. Common mechanisms of injury include a direct
blow to the lateral side of the knee (the “clipping” injury in
football causing the triad of medial collateral ligament, ante-
rior cruciate ligament, and medial meniscus injuries17) and
 630 HUDDLESTON  |  Hip and Knee Pain
A
B
Figure 42-6  A and B, Palpation of the medial (A) and lateral (B) joint
lines.
noncontact injuries that occur during cutting, pivoting, and
jumping.18 Patients often report an audible “pop” accompa-
nied by the acute onset of knee swelling. Multiple tests have
been described to evaluate the anterior cruciate ligament.
The most sensitive tests for diagnosis of an anterior cruci-
ate ligament injury include the anterior drawer, Lachman,19
and pivot-shift tests.20,21 All three tests are performed with
the patient in the supine position. The anterior drawer test is
performed with the knee flexed to 90 degrees. The examiner
places his or her hands on the posterior surface of the proxi-
mal tibia and subluxates the tibia anteriorly (Fig. 42-7). Any
gross movement of the tibia that is different from the contra-
lateral side is considered abnormal. The Lachman test is per-
formed with the knee in 30 degrees of flexion (to remove the
contribution of secondary restraints). The examiner applies
an anterior force on the tibia while stabilizing the femur with
his or her contralateral hand. Any increase in anterior tibial
translation relative to the contralateral side is considered
abnormal (Fig. 42-8). The pivot-shift test is performed with
the knee in extension. The examiner holds the tibia in slight
internal rotation and applies a valgus stress while the knee
is slowly flexed. This combination of forces should cause
the tibia to subluxate anteriorly if the anterior cruciate liga-
ment is injured. The test is positive if the tibia reduces with a
“clunk” or a “glide” at 20 to 40 degrees of flexion.
Figure 42-7  The anterior drawer test is performed by subluxating the
tibia anteriorly with the knee in 90 degrees of flexion. The amount of an-
terior translation (mm) is noted. The end point is characterized as “soft”
or “hard.”
Figure 42-8  The Lachman test is performed by applying an anterior
force on the tibia while stabilizing the femur with the knee in 30 degrees
of flexion.
The posterior cruciate ligament is the strongest liga-
ment in the knee,22,23 and injuries to the posterior cruciate
ligament are usually a result of significant knee trauma. The
“dashboard” injury is a common mechanism for posterior
cruciate ligament injury and occurs during a motor vehicle
accident when the tibia with the knee flexed strikes the
dashboard (Fig. 42-9).
The posterior cruciate ligament can be evaluated with
the posterior drawer, posterior sag, and quadriceps active
tests. All tests are performed with the patient in the supine
position. The posterior drawer test is performed with the
knee in 90 degrees of flexion. The examiner applies a poste-
riorly directed force to the tibia. Placement of one’s thumb
tips at the anterior joint line allows for quantification of any
abnormal translation (Fig. 42-10). The posterior sag test is
positive when the tibia subluxates posteriorly with the knee
at 90 degrees of flexion. Loss of the medial tibial step-off
at the joint line should alert the examiner to a posterior
cruciate ligament injury (Fig. 42-11).22 This test is usually
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
Figure 42-9  An injury to the posterior cruciate ligament can occur
when the tibia strikes the dashboard, causing the tibia to subluxate pos-
teriorly on the femur.
Figure 42-10  The posterior drawer test is performed by subluxating
the tibia posteriorly with the knee in 90 degrees of flexion. The amount
of posterior translation (mm) is noted. The end point is characterized as
“soft” or “hard.”
positive in the setting of chronic pathology or under anes-
thesia in the setting of acute injury. The quadriceps active
test is performed with the knee in 60 degrees of flexion. The
patient is asked to extend the knee while keeping his or her
foot on the examination table. One sees reduction of the
tibia in a positive test.24
Injuries to the posterior cruciate ligament are often
accompanied by injuries to the “posterolateral corner,” a
complex structure that functions as a static and a dynamic
stabilizer of the knee.23 The posterolateral corner comprises
the lateral collateral ligament, the popliteofibular liga-
ment, the popliteomeniscal attachment, the arcuate liga-
ment, and the popliteus tendon and muscle.25 Injuries to
the posterolateral corner, the posterior cruciate ligament
or both can be examined with the “dial test” (Fig. 42-12).
631
Figure 42-11  The posterior sag test is positive when the tibia sublux-
ates posteriorly with the knee in 90 degrees of flexion.
The posterolateral corner structures restrain external rota-
tion at 30 degrees of flexion, whereas the posterior cruciate
ligament restrains external rotation at 90 degrees of flexion.
An increase of external rotation at 90 degrees of flexion
without an increase in external rotation at 30 degrees of
flexion suggests an isolated posterior cruciate ligament
injury. An increase of external rotation at 30 degrees of
flexion without an increase at 90 degrees of flexion suggests
an isolated injury to the posterolateral corner. Increased
external rotation at 30 degrees and 90 degrees of flexion
suggests combined posterior cruciate ligament and postero-
lateral corner injuries.
Menisci
Traumatic and degenerative meniscal injuries are among the
most common knee injuries. The menisci are considered the
“shock absorbers” in the knee. They also provide rotational
and translational restraint. The medial meniscus is bean-
shaped and is larger and less mobile than the lateral menis-
cus. The lateral meniscus is more C-shaped. Meniscal tears
usually occur with rotation of the flexed knee as it moves into
extension. Tears of the medial meniscus are more common
than tears of the lateral meniscus, likely owing to the relative
lack of mobility of the medial meniscus.26 Patients frequently
complain of “locking” and “clicking” or of something not
being “right” with the knee, and this usually results from
displacement of the torn meniscus during motion. Common
physical findings include pain with hyperflexion and with
hyperextension, joint line tenderness, and an effusion.
Many provocative tests have been described to diagnose
meniscal tears. The McMurray27 and Apley compression28
tests are performed frequently, although they do lack sen-
sitivity and specificity. The flexion McMurray test is per-
formed with the patient supine and the hip and knee flexed
to 90 degrees. A compressive and rotational force is applied
to the knee as it is moved from a flexed to an extended posi-
tion. The test is positive if the patient complains of pain
(Fig. 42-13). The Apley compression test is performed with
 632 HUDDLESTON  |  Hip and Knee Pain
Figure 42-12  A and B, The degree
of tibial external rotation is mea- A B
sured in the “dial” test.
Figure 42-13  A positive flexion McMurray test may indicate a torn 
meniscus.
the patient prone and the knee flexed to 90 degrees. In a
positive test, the patient complains of pain with rotation
of the tibia. The images in Figure 42-14 show a tear in the
posterior horn of the medial meniscus.
Quadriceps Tendon
Injuries to the quadriceps tendon are most common in the
sixth and seventh decades of life. Patients with systemic lupus
erythematosus, renal failure, endocrinopathies, diabetes, and
B
Figure 42-14  A and B, Images taken during knee arthroscopy reveal
a tear in the posterior horn of the medial meniscus before (A) and after 
(B) débridement.
various other systemic inflammatory and metabolic diseases
are at a higher risk for these injuries. Quadriceps tendon
rupture after total knee arthroplasty is a rare (0.1%) but
devastating complication.29 Patients usually present with
intense anterior knee pain after experiencing an eccentric
quadriceps contraction during a fall or twisting injury. Phys-
ical examination reveals a palpable defect in the tendon,
an effusion owing to hemarthrosis, and hypermobility of
the patella. Patients usually are unable to extend their knee
fully (Fig. 42-15).
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
Figure 42-15  An extensor lag resulting from a complete tear in the
quadriceps tendon.
Patella Tendon
Problems with the infrapatellar tendon include tendinitis
and rupture. Tendinitis is usually an overuse injury and is
often associated with jumping, changes in activity level,
and eccentric contractions during falls. Patients exhibit
tenderness at the tibial tubercle or at the inferior pole of
the patella. Rupture of the patella tendon usually occurs in
patients younger than 40 years old and is associated with
chronic patella tendinitis. Patients usually present with
anterior knee pain and the inability to extend the knee.
Patellofemoral Pain
Anterior knee pain is a common complaint and is more
common in women; it accounts for 25% of all sports-related
knee injuries.30 A variety of factors contribute to the biome-
chanics of the patellofemoral joint, including overuse, the
depth of the trochlea, the shape of the patella, quadriceps
strength, the line of pull of the quadriceps relative to the
patella tendon (the Q angle), the length of the patella ten-
don, the shape of the femoral condyles, and the articular
cartilage. Abnormalities of any of these factors may con-
tribute to this pain syndrome, and successful treatment is
possible only with correct identification of any contributing
factors.
Physical examination of the patellofemoral joint begins
with an analysis of coronal alignment of the knee because
any valgus deformity may contribute to lateral subluxation.
The height of the patella relative to the tibial tubercle should
be noted (patella alta or baja). The J sign is present when
the patella slides laterally at terminal extension, indicating
excessive pull of the vastus lateralis. The vastus medialis
obliquus is the primary stabilizer against lateral pull by the
vastus lateralis. With the knee extended and the quadriceps
relaxed, the examiner should make note of any patellar tilt.
Any audible or palpable crepitus should be noted as well.
Crepitus is common in osteoarthritis. A Q angle greater
than 15 degrees in women and greater than 8 to 10 degrees
in men is considered abnormal.30 Patellar mobility should be
assessed using a quadrant system for passive mediolateral dis-
placement of the patella relative to the trochlear groove. The
normal patella should not be displaced medially or laterally
633
Figure 42-16  The apprehension test is positive when subluxation of
the patella causes pain.
beyond the second quadrant. Any abnormality in mobility
may stem from changes in the tightness of the retinaculum.
The apprehension test is performed by attempting to sub-
luxate the patella with the knee in extension. The test is
positive when it elicits pain and an unwillingness to allow
the examiner to move the patella laterally (Fig. 42-16).
At the conclusion of the history and physical examina-
tion, the astute clinician should have formulated a short list
of possible diagnoses. With this list in mind, the imaging
studies, if appropriate, can now be obtained. The goal of
the initial imaging studies should be to confirm the diag-
nosis with the most appropriate and least expensive study.
Advanced imaging studies should not replace a thorough
history and physical examination.
IMAGING
Conventional Radiographs
Conventional radiographs are usually the first study obtained
after knee injury and should be read in a systematic manner.
Soft tissues should be evaluated before examining the bony
structures. Findings should be described in terms of radiolu-
cent and radiopaque lines. Only after the findings have been
described should the interpretation phase begin because it is
the natural tendency to bypass the description and proceed
directly to interpretation. If this is done, certain findings are
likely to be missed or dismissed prematurely.
The basic radiographic evaluation of the symptomatic
knee consists of standing anteroposterior weight-bearing,
lateral, and Merchant’s views. The anteroposterior view
allows for evaluation of coronal alignment and height of the
tibiofemoral joint spaces. The normal coronal alignment of
the knee should be 5 to 7 degrees of anatomic (tibiofemoral)
valgus. The lateral tibiofemoral joint space should be wider
than the medial tibiofemoral joint space in a normal knee.
Marginal osteophytes, joint space narrowing, subchondral
sclerosis, and cystic change are seen in the presence of
osteoarthritis (Fig. 42-17). Periarticular osteopenia, con-
centric joint space narrowing, and a paucity of osteophytes
are commonly seen in inflammatory arthritis (Fig. 42-18).
The lateral x-ray allows for evaluation of an effusion, patella
 634 HUDDLESTON  |  Hip and Knee Pain
C
Figure 42-17  A-C, Standing anteroposterior (A), lateral (B), and Merchant’s (C) views of an osteoarthritic knee.
tendon length, and the quadriceps tendon. Merchant’s view
is taken tangential to the patellofemoral joint.31 It allows
for detection of patellofemoral arthritis and malalignment
and should be obtained only when indicated for symptoms
or findings.
The posterior femoral condyles can be evaluated for
joint space narrowing with a posteroanterior standing view
with the knees flexed approximately 45 degrees, the tun-
nel or intercondylar notch view, and the 36-inch antero-
posterior standing view of bilateral lower extremities.32 The
flexed posteroanterior standing view is taken with the radio-
graphic beam directed 10 degrees caudad from anterior to
posterior. The tunnel view is obtained with the knee flexed
and the radiographic beam directed inferiorly at an angle
perpendicular to the tibial plateau. It is useful in detecting
posterior tibiofemoral joint space narrowing, tibial spine
fractures, loose bodies, and osteochondral lesions on the
medial aspect of the femoral condyles. The 36-inch stand-
ing view is used to determine the mechanical axis of the
lower extremity and to evaluate any deformity that may be
present. The normal mechanical axis is a straight line join-
ing the center of the hip, knee, and ankle joints. Surgeons
use it for preoperative planning and postoperative evalua-
tion in total knee arthroplasty and for the planning of distal
femoral and proximal tibia osteotomies in arthritis surgery.
Computed Tomography
Computed tomography (CT) has largely been replaced
by magnetic resonance imaging (MRI) in evaluation of
knee pathology. CT is now used primarily for detection of
bony tumors, in the trauma setting for detection of subtle
fractures that are not easily visualized with conventional
radiographs, and for a more thorough evaluation of intra-
articular fractures. In cases of distal femoral or proximal
tibia fractures, CT is used to help the surgeon plan operative
B
treatment and to assess axial alignment of the femoral and
tibial components in patients with a painful total knee
arthroplasty.33,34
Ultrasound
The use of ultrasound has become more common in the diag-
nosis of knee disorders as a result of more recent improve-
ments in transducer technology. Ultrasound is an attractive
imaging modality because of its low cost, real-time capa-
bilities, and portability. The ability to perform provocative
maneuvers during sonography is particularly appealing.
Ultrasound can easily and reliably detect joint effusions
and quadriceps and patella tendon disruptions. It has been
reported that ultrasound can detect a 1-mm increase in joint
fluid.35
Nuclear Scintigraphy
Nuclear scintigraphy is sensitive, but not specific, and it
is used to detect areas of increased osseous remodeling. It
requires clinical correlation and should be used in conjunc-
tion with other imaging modalities. Technetium phosphate
compounds are injected intravenously. Approximately 50%
of the tracer is excreted by the kidneys, and the remainder is
taken up in areas of increased osseous turnover. Imaging of
the skeleton is typically performed 2 to 3 hours after injec-
tion because this allows for maximal contrast between the
soft tissues and the skeletal structures, while still providing
for an adequate photon count.36
Three-phase bone scanning can yield additional informa-
tion. The three phases include an angiographic pool, a blood
pool, and bone imaging. The angiographic phase allows for
detection of regional hyperemia. This technique has been
reported to have greater specificity and can be used in cases
of suspected osteomyelitis, osteonecrosis, stress fracture, and
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
A B
C (B), and Merchant’s (C) views of the knee in a patient with
implant loosening.36 It has been reported that increased
radionuclide uptake can be seen for 12 to 18 months after
total knee arthroplasty. Asymmetric uptake in one area
around the prosthesis should raise the question of loosening
or periprosthetic fracture (Fig. 42-19).37,38 Addition of labeled
leukocytes to technetium-99m sulfur colloid yields 80% sen-
sitivity and 100% specificity for diagnosing infection.39
Magnetic Resonance Imaging
MRI has supplanted many imaging modalities because of its
direct multiplanar capabilities and superior soft tissue con-
trast. Although conventional radiographs remain the “gold
standard” for defining osseous structures, MRI provides
excellent visualization of articular cartilage, the cruciate
ligaments, the collateral ligaments, the patella tendon, the
quadriceps tendon, and the menisci (Fig. 42-20). It also is
highly sensitive for detecting bone marrow edema (contu-
sion), stress fractures, and mass lesions. Use of the “two-slice
touch” rule has improved the sensitivity and specificity of
MRI in accurately diagnosing meniscal tears. This rule clas-
sifies a meniscus as torn if there are two or more MR images
with abnormal findings and as possibly torn if there is only
one MR image with an abnormal finding. Using fast spin-
echo imaging, the sensitivity and specificity for diagnosing
medial and lateral meniscal tears were 95% and 85% (medial
635
Figure 42-18  A-C, Standing anteroposterior (A), lateral
rheumatoid arthritis.
tears) and 77% and 89% (lateral tears). This translates to a
positive predictive value of 91% to 94% for medial meniscus
tears and 83% to 96% for lateral meniscus tears.40
COMMON DISORDERS IN THE DIFFERENTIAL
DIAGNOSIS OF KNEE PAIN
General
Although there are many diseases that may involve the
knee, only a few are common. In evaluating the complaint
of knee pain, the clinician should be familiar with osteoar-
thritis; rheumatoid arthritis; inflammatory arthritis associ-
ated with the seronegative spondyloarthropathies; tears of
the menisci, ligaments, and tendons; osteochondritis disse-
cans; osteochondral fractures; fractures; referred pain from
the hip (e.g., with slipped capital femoral epiphysis in ado-
lescents); vascular claudication; neurogenic claudication;
complex regional pain syndrome; sarcoma; metastases; and
infection.
Bursitis
The prepatellar bursa lies between the retinaculum and
the subcutaneous fat and runs from the patella to the tibial
tubercle. The bursa may become inflamed and fill with fluid
 636 HUDDLESTON  |  Hip and Knee Pain

ANT POST

Figure 42-19  A bone scan reveals increased uptake of

radiotracer around the distal femur in a patient with an
infected total knee arthroplasty and septic loosening of
RT MED L LAT L MED RT LAT
the femoral component.

when exposed to a direct blow or repetitive microtrauma

(kneeling). Patients with prepatellar bursitis present with
anterior knee pain on flexion and a fluctuant mass over the
anterior knee. If the area becomes warm, tender to palpa-
tion, and erythematous, septic bursitis should be ruled out
by aspiration and culture. The pes anserinus bursa, located
over the insertions of the sartorius, gracilis, and semitendi-
nosus muscles on the proximal medial tibia, also can be a
source of knee pain when it is inflamed.

Neoplasia
Tumors around the knee are often diagnosed after trauma
prompts medical evaluation. Pain at night, pain at rest, and
constitutional symptoms should alert the clinician to con-
sider the appropriate workup. Some benign tumors seen
around the knee include enchondroma, pigmented villo­
nodular synovitis, osteochondromatosis, and giant cell tumor. Figure 42-20  Sagittal MRI shows linear signal change extending to the
Malignant tumors seen around the knee include, but are not meniscal surface consistent with a tear in the posterior horn of the me-
limited to, metastases, osteosarcoma, Ewing’s sarcoma, chon- dial meniscus.
drosarcoma, and malignant fibrous histiocytoma.
the underlying pathology (arthritis) with knee arthroplasty.
Popliteal pain and edema of the lower leg caused by a popli-
Popliteal Cysts
teal cyst can mimic thrombophlebitis, and anticoagulation
A popliteal cyst, originally called Baker’s cyst, is a synovial in such cases is not indicated.
fluid–filled mass located in the popliteal fossa. The most
common synovial popliteal cyst is considered to be a dis-
tention of the bursa located beneath the medial head of HIP PAIN
the gastrocnemius muscle. Usually, in an adult patient, an HISTORY
underlying intra-articular disorder (osteoarthritis or rheu-
matoid arthritis) is present. In children, the cyst can be iso- Generally, more conditions should be considered in the dif-
lated, and the knee joint can be normal. Patients usually ferential diagnosis for hip pain than for knee pain because
present with episodic posterior knee pain.41 The diagnosis is the hip is a common site for referred pain from lumbosacral
made by ultrasonography or MRI. Treatment options include and intrapelvic pathology. A detailed, comprehensive his-
benign neglect, aspiration, surgical excision, and removal of tory directs the clinician to a focused ­physical examination.
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
Most patients who present with hip pathology com-
plain of pain. It is important to define the exact location
of the pain because “hip” pain may refer to discomfort in
the groin, lateral thigh, or buttock. Pain in the groin or
medial thigh region is most often due to hip disease and is
believed to arise from irritation of the capsule or synovial
lining or both.42 Pain generated, the lumbosacral spine may
be referred to the buttocks, lateral thigh, or groin.43 Lateral
thigh pain may stem from so-called trochanteric bursitis
(usually abductor tendinitis). Activities or positions that
aggravate and relieve the pain and the severity, frequency,
and patterns of radiation should be explored. It is common
for knee pain to be referred from the hip joint. Metastatic
and primary tumors that occur in the pelvic and proximal
thigh regions and intrapelvic pathology from the prostate,
seminal vesicles, hernias, ovaries, gastrointestinal system,
and vasculature always should be included in the differen-
tial diagnosis.44,45
Knowledge of the patient’s general level of functioning
is important because this lends insight into the severity
of disease and may influence treatment. Patients with hip
pathology may have difficulty trimming their toenails, don-
ning shoes and socks, and using stairs. Walking tolerance
and use of assist devices also should be recorded. The Har-
ris Hip Score and WOMAC Osteoarthritis Index are two
rating scales that are widely used to assess function in this
patient population.46,47
The patient should be asked about any hip problems and
their treatment that he or she encountered in childhood.
Diseases such as developmental dysplasia, slipped capital
femoral epiphysis, Legg-Calvé-Perthes disease, polio, and
trauma may lead to osteoarthrosis later in life.48-50
Osteoarthritis and inflammatory arthritis are two com-
mon causes of hip pain. Generally, pain from osteoarthritis
is exacerbated by activity and relieved by rest. Mild arthri-
tis of the hip may not become symptomatic until a certain
activity level is reached. Stiffness (usually from synovitis)
also is a common complaint with degenerative and inflam-
matory arthritis. When the hip pain continues despite a trial
of rest, an underlying inflammatory or infectious process
should be considered. Any previous treatments for hip pain
should be discussed. The patient’s response to nonsteroi-
dal anti-inflammatory drugs, nutritional supplements (e.g.,
chondroitin and glucosamine), physical therapy, corticoste-
roid injections, local anesthetic injections, hyaluronic acid
injections, ultrasound, and operative interventions should
be recorded. Finally, a more general medical history should
be explored. The physician should always consider alcohol-
ism, fibromyalgia, neuromuscular disorders, and smoking
history as complicating factors in these patients.
PHYSICAL EXAMINATION
The physical examination of a patient with hip pain begins
as the clinician watches the patient for the first time. Ease
of chair rise, postures, and walking speed all provide insight
into the extent of a patient’s disability. A general evaluation
of the patient’s spine, lower extremity alignment, and leg
lengths comes next. With the examiner behind the patient,
the spine is examined for coronal and sagittal balance. The
patient is asked to touch his or her toes. A rib hump indi-
cates the presence of scoliosis. Any gross deformity of the
637
spine alerts the examiner to the potential of a pelvic obliq-
uity and resultant apparent leg-length discrepancy. The
overall coronal alignment of the lower extremities is evalu-
ated next. If a leg-length discrepancy is detected, blocks can
be used, as discussed previously, to determine the amount
of inequality. If the leg-length discrepancy is due to a fixed
pelvic obliquity from lumbosacral disease, blocks may not
be able to level the pelvis. Palpation of the bony landmarks
(iliac crest, anterior superior iliac spine, posterior superior
iliac spine, ischial tuberosity, coccyx, spinous processes, and
greater trochanter) should be performed (Fig. 42-21). The
femoral neck is located approximately three fingerbreadths
below the anterior superior iliac spine.
A basic evaluation of gait is useful. Although gait analy-
sis is a complex science, all clinicians should feel comfort-
able evaluating for common abnormalities. A patient with
hip pain may present with an antalgic gait. The severity of
the limp should be classified as mild, moderate, or severe.
Mild limps can be detected only by trained observers.
Moderate limps are noticed by the patient. A severe limp
is readily apparent and has a significant impact on speed
of ambulation. Common causes of limp include pain and
abductor (gluteus medius and gluteus minimus) muscle
weakness. Differentiating between these two etiologies is
an important part of the physical examination.
A patient with abductor dysfunction is likely to have an
abductor, or Trendelenburg, lurch.51 With a Trendelenburg
lurch, the patient compensates for abductor dysfunction
by leaning over the involved hip to shift the body’s cen-
ter of gravity in that direction (Fig. 42-22). If the patient
has a Trendelenburg lurch, we proceed to evaluate for a
Trendelenburg sign. A positive Trendelenburg sign occurs
when the pelvis tilts toward the unsupported side during
one-legged stance when viewed with the examiner behind
the patient. Causes of abductor weakness are numerous
and may include a contracted or shortened gluteus medius,
coxa vara, fracture, dysplasia, neurologic conditions (e.g.,
superior gluteal nerve injury, radiculopathy, poliomyelitis,
myelomeningocele, spinal cord lesions), and slipped capital
femoral epiphysis.
The patient is then asked to lay supine on the examination­
table. The range of motion of both hips should be evaluated
by recording flexion, extension, adduction, abduction, inter-
nal rotation in extension, and external rotation in exten-
sion. Hip extension is best evaluated with the patient in the
prone position. Normal range of motion values include 100
to 135 degrees for flexion (knee should be flexed to relax
the hamstrings), 15 to 30 degrees for extension, 15 to 30
degrees for adduction, 20 to 40 degrees for abduction, 20
to 40 degrees for internal rotation, and 15 to 60 degrees for
external rotation. Motion is often limited in cases of defor-
mity (e.g., limited internal rotation in slipped capital femo-
ral epiphysis) and advanced osteoarthritis. Internal rotation
and abduction are usually the first motions to be limited in
osteoarthritis. Motion also is painful in patients with syno-
vitis. Areas that are painful should be palpated.
A series of special tests can be performed to evaluate for
subtle muscle contractures and limitation of motion. The
presence of a hip flexion contracture is common in patients
with moderate-to-severe hip pathology and can be quanti-
fied with the Thomas test (Fig. 42-23).52 This test is per-
formed by having the patient bring his or her thighs to the
 638 HUDDLESTON  |  Hip and Knee Pain
Greater
Anterior trochanter
superior
iliac spine
Iliac
crest
Ischial
tuberosity
Spinous Posterior Coccyx
process superior
iliac spine
process
Figure 42-21  Diagram of the bony landmarks on the pelvis that can be
palpated during physical examination.
A B C reproduce snapping, patients may be able to demonstrate this
Figure 42-22  Physical examination of abductor function. A, Normal
single-legged stance. B, Positive Trendelenburg lurch and negative Tren-
delenburg sign. C, Positive Trendelenburg lurch with pelvic obliquity and
leaning over the involved hip to shift the body’s center of gravity.
chest while in the supine position. This allows for flatten-
ing of the spine, and the hip to be evaluated is allowed to
extend to neutral. If the patient is unable to reach neutral,
the amount of flexion contracture is recorded. The Ober test
measures tightness of the iliotibial band. The patient lies
on the unaffected side, and the examiner helps the patient
abduct the hip with the hip extended and the knee flexed
to 90 degrees. The leg is slowly released from abduction to
neutral, and the hip remains abducted if there is contracture
of the iliotibial band. Ely’s test detects a tight rectus femoris
muscle. The knee is passively flexed with the patient in the
prone position. If the rectus femoris is tight, the ipsilateral
hip spontaneously flexes. If the rectus femoris is normal, the
hip remains flush with the examination table.
Patients occasionally complain of a “snapping” sensation
in their hip. Although it may be difficult for the clinician to
Figure 42-23  In the Thomas test, a hip flexion contracture is measured
by flexing the contralateral hip to eliminate compensatory lumbar lor-
dosis. The ipsilateral hip is allowed to extend with gravity. The angle
between the examination table and the thigh is the degree of flexion
contracture.
by flexing and internally rotating their hip. Extra-articular
causes of hip snapping include a thickened iliotibial band
snapping over the greater trochanter, the iliopsoas tendon
gliding over the iliopectineal eminence, the long head of the
biceps tendon rubbing on the ischial tuberosity, and the ilio-
femoral ligament rubbing on the femoral head. Intra-articular
causes of snapping hip syndrome include loose bodies and
large labral tears.
In addition to using blocks with the patient standing,
leg lengths can be measured while the patient is in the
supine position (Fig. 42-24). The apparent leg length is the
distance from the umbilicus to the medial malleolus. The
true leg length is measured from the anterior superior iliac
spine to the medial malleolus. Pelvic obliquity and abduc-
tion/adduction of the hip create an apparent leg-length
discrepancy.
Sacroiliac disease should be included in the differential
diagnosis of hip pain. Although multiple provocative tests
have been described to elicit sacroiliac disease, the FABER
test (also known as Patrick’s test) can help distinguish
between hip and sacroiliac joint pathology. With the patient
supine, the clinician has the patient place his or her hip in
the flexion, abduction, and externally rotated positions. The
clinician presses the flexed knee and the contralateral ante-
rior superior iliac spine toward the floor. Pain in the buttocks
suggests sacroiliac joint disease, whereas pain in the groin
points to hip pathology. If the sacroiliac joint is implicated,
it is recommended that multiple other provocative tests be
performed. It has been shown that by using a combination
of the distraction, thigh thrust, compression, sacral thrust,
Gaenslen’s, and FABER tests, sacroiliac joint pathology is
the likely pain generator when three or more of the tests are
positive.53,54
The acetabular labrum is drawing attention as a previously
underappreciated cause of hip pain. Clinical presentation of
a labral tear of the acetabulum may vary, and the diagnosis is
often delayed. Patients usually see multiple providers before
the diagnosis is confirmed. In a series of 66 patients with
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN 639

A B C

Figure 42-24  Measurement of leg lengths. A, The apparent leg length is the distance from the umbilicus to the medial malleolus. B, Pelvic obliquity
causing an apparent leg-length discrepancy. C, The true leg length is the distance from the anterior superior iliac spine to the medial malleolus.

arthroscopically confirmed tears of the acetabular labrum,

92% of the patients complained of groin pain, 91% of the
patients had activity-related pain, 71% of the patients com-
plained of night pain, 86% of the patients described the
pain as moderate to severe, and 95% of the patients had a
positive impingement sign. The authors recommended that
a diagnosis of acetabular labral tear be suspected in young,
active patients complaining of groin pain with or without
trauma.55 The positive impingement test helps confirm the
diagnosis of labral tear. The test is positive if the patient
experiences groin pain with the hip flexed, adducted, and
internally rotated.
A thorough evaluation of the neurovascular system
should be completed after the musculoskeletal portion of
the physical examination for the hip or knee is completed.
This should include palpation or Doppler evaluation of the
femoral, popliteal, dorsalis pedis, and posterior tibial arter-
ies, as indicated. Strength testing with resisted isometric
movements for each muscle in the lower extremity is per- Figure 42-25  Anteroposterior radiograph of the pelvis shows the char-
formed, with a score of 5 being normal strength, 4 being full acteristic joint space narrowing, cystic changes, and osteophytes seen in
motion against gravity and against some resistance, 3 being osteoarthritis.
fair motion against gravity, 2 being movement only with
gravity eliminated, 1 being evidence of muscle contraction
but no joint motion, and 0 being no evidence of contractil- a low anteroposterior pelvis view (Fig. 42-25), an antero-
ity. Sensation in the lower extremity should be evaluated posterior hip view (Fig. 42-26), a frog-lateral view, and a
by assessing for light touch or appreciation of pin prick in a cross-table lateral view. The frog-lateral view provides a
dermatomal distribution. Patellar and ankle reflexes should lateral view of the proximal femur and is useful for detect-
be tested. The examiner should test for any abnormal clonus ing femoral head collapse (as seen in osteonecrosis) (Fig.
and Babinski’s reflexes as indicated. 42-27). There are numerous other special radiographs of the
hip; some of these include Judet 45-degree oblique views
and the false profile view. Judet views allow for easier visu-
IMAGING alization of the anterior (obturator oblique) and posterior
Conventional Radiographs (iliac oblique) columns. The false profile view allows for
evaluation of anterior bony coverage of the femoral head
Plain radiographs remain the primary diagnostic imaging in cases of acetabular dysplasia. Developmental dysplasia of
tool for the evaluation of hip pathology. All other imaging the hip is common, and we do not recommend the routine
modalities should be viewed as complementary to conven- use of any special views before referral to an orthopaedic
tional radiographs. Our standard screening series includes surgeon (Fig. 42-28).
 640 HUDDLESTON  |  Hip and Knee Pain
Figure 42-26  Anteroposterior radiograph of the hip shows the char-
acteristic concentric joint space narrowing, paucity of osteophytes, and
periarticular osteopenia seen in rheumatoid arthritis.
Figure 42-27  Frog-lateral radiograph shows femoral head collapse
from osteonecrosis.
Computed Tomography
CT is used for assessment of acetabular fractures, acetabu-
lar nonunions, femoral head fractures, subtle femoral neck
fractures, neoplasia, and bone stock in the setting of revi-
sion total hip arthroplasty. Owing to its limited soft tissue
contrast, CT has largely been replaced by MRI for detailed
evaluation of the soft tissues around the hip.
Nuclear Scintigraphy
The role of bone scanning in the evaluation of hip
pathology is similar to its role in the assessment of knee
pain. It should always be used in conjunction with other
Figure 42-28  Anteroposterior radiograph of the hip shows osteoar-
throsis from developmental dysplasia. The up-sloping lateral edge of the
acetabulum is characteristic for developmental dysplasia of the hip.
imaging modalities because of its limited specificity
(Fig. 42-29).
Magnetic Resonance Imaging
MRI provides unprecedented detail of the soft tissues around
the hip joint. Its use is now common for diagnosis of osteone-
crosis, labral pathology, neoplasia, effusion, synovitis, loose
bodies, tendinitis, transient osteoporosis of the hip, occult
femoral neck fractures, bone edema, gluteus medius tendon
avulsions, and nerve injury. Magnetic resonance arthrogra-
phy of the hip joint is useful for identifying gluteus medius
tendon avulsion after total hip arthroplasty (Fig. 42-30) and
for detecting labral tears. One study showed a 92% sensitiv-
ity for the detection of labral tears using magnetic resonance
arthrography.56 Delayed gadolinium-enhanced MRI of carti-
lage, a technique designed to measure early arthritis in the
hip joint, is now being used clinically in the management
of hip dysplasia.57 Despite the tremendous diagnostic capa-
bilities of MRI, its ability to detect bony pathology is lim-
ited. As such, conventional radiographs remain the imaging
modality of choice for screening.
Hip Arthrography
Hip arthrography is useful for detecting avulsions of the glu-
teus medius tendon from the greater trochanter and for dif-
ferentiating intra-articular hip pathology from lumbosacral
disease. In one study, intra-articular anesthetic injection
was 90% accurate in predicting intra-articular pathology
as confirmed by hip arthroscopy.58 Anesthetic arthrogram
of the hip has shown a 95% positive predictive value and
a 67% negative predictive value for pain relief after total
hip arthroplasty in patients with concurrent hip and lumbar
osteoarthritis.59
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN 641

ANT blood pool POS blood pool LAT RT hip

Figure 42-29  Bone scan shows

increased radiotracer uptake at the
proximal femur. The patient pre-
sented with activity-related thigh
pain 1-year status post primary
cementless total hip arthroplasty.
History, physical examination, and 
conventional radiographs suggested
failure of osseointegration. At the
ANT hips POS hips LAT LT hip time of surgery, the femoral compo-
nent was found to be grossly loose.

of the acetabular labrum, transient osteoporosis of the prox-

imal femur, infection, snapping hip syndrome, osteitis pubis,
neoplasia (osteosarcoma, chondrosarcoma, pigmented villo-
nodular synovitis, osteochondromatosis, malignant fibrous
histiocytoma, or metastases), inguinal hernia, or referred
pain (lumbosacral spine, sacroiliac joint, prostate, seminal
vesicles, uterus, ovaries, or lower gastrointestinal tract). This
list can be efficiently narrowed down by taking a detailed
history, performing a comprehensive examination of the
musculoskeletal and neurovascular systems, and obtaining
the appropriate imaging studies.

REFERENCES
Figure 42-30  Short tau inversion recovery coronal MRI shows complete
avulsion of the gluteus medius tendon from its insertion on the greater
trochanter. Note the signal change along the lateral aspect of the greater
trochanter, consistent with accumulation of intra-articular gadolinium at
the site where the gluteus medius tendon should be.
COMMON DISORDERS IN THE DIFFERENTIAL
DIAGNOSIS OF HIP PAIN
A detailed discussion of the numerous common causes of
hip pain is beyond the scope of this chapter. The differential
diagnosis of hip pain should include osteoarthrosis (most fre-
quently from developmental dysplasia, Legg-Calvé-Perthes
disease, or slipped capital femoral epiphysis), inflamma-
tory arthritis, osteonecrosis, fractures (acetabulum, femoral
head, femoral neck, intertrochanteric, or subtrochanteric),
trochanteric bursitis, femoroacetabular impingement, tears
  1. Mallen CD, Peat G, Thomas E, et al: Is chronic musculoskeletal pain in
adulthood related to factors at birth? A population-based case-control
study of young adults. Eur J Epidemiol 21:237-243, 2006.
  2. Peat G, McCarney R, Croft P: Knee pain and osteoarthritis in older
adults: A review of community burden and current use of primary
health care. Ann Rheum Dis 60:91-97, 2001.
  3. Mendenhall S: Mix shifts toward high-demand implants. OR Manager
21:13, 2005.
  4. Ong KL, Mowat FS, Chan N, et al: Economic burden of revision hip and
knee arthroplasty in Medicare enrollees. Clin Orthop 446:22-28, 2006.
  5. Aoyagi K, Ross PD, Huang C, et al: Prevalence of joint pain is higher
among women in rural Japan than urban Japanese-American women
in Hawaii. Ann Rheum Dis 58:315-319, 1999.
  6. Jacobsen S, Sonne-Holm S, Soballe K, et al: Radiographic case defini-
tions and prevalence of osteoarthrosis of the hip: A survey of 4 151
subjects in the Osteoarthritis Substudy of the Copenhagen City Heart
Study. Acta Orthop Scand 75:713-720, 2004.
  7. Helme RD, Gibson SJ: The epidemiology of pain in elderly people.
Clin Geriatr Med 17:417-431, 2001.
  8. Chen J, Devine A, Dick IM, et al: Prevalence of lower extremity pain
and its association with functionality and quality of life in elderly
women in Australia. J Rheumatol 30:2689-2693, 2003.
  9. Felson DT: Epidemiology of hip and knee osteoarthritis. Epidemiol
Rev 10:1-28, 1988.
10. Felson DT: An update on the pathogenesis and epidemiology of osteo-
arthritis. Radiol Clin North Am 42:1-9, 2004.
11. Felson DT, Nevitt MC: Epidemiologic studies for osteoarthritis: New
versus conventional study design approaches. Rheum Dis Clin North
Am 30:783-797, 2004.
 642 HUDDLESTON  |  Hip and Knee Pain
12. Gelber AC, Hochberg MC, Mead LA, et al: Joint injury in young
adults and risk for subsequent knee and hip osteoarthritis. Ann Intern
Med 133:321-328, 2000.
13. Horvath G, Than P, Bellyei A, et al: [Prevalence of musculoskeletal
symptoms in adulthood and adolescence (survey conducted in the
Southern Transdanubian region in a representative sample of 10.000
people]. Orv Hetil 147:351-356, 2006.
14. Leveille SG, Zhang Y, McMullen W, et al: Sex differences in muscu-
loskeletal pain in older adults. Pain 116:332-338, 2005.
15. Zeng QY, Westermark SQ, Rasmussen-Lestander A, et al: Low preva-
lence of knee and back pain in southeast China: The Shantou COP-
CORD study. J Rheumatol 31:2439-2443, 2004.
16. Butler DL, Noyes FR, Grood ES: Ligamentous restraints to ante-
rior-posterior drawer in the human knee: A biomechanical study.
J Bone Joint Surg Am 62:259-270, 1980.
17. O’Donoghue DH: Surgical treatment of fresh injuries to the major
ligaments of the knee. J Bone Joint Surg Am 32:721-738, 1950.
18. Griffin LY, Agel J, Albohm MJ, et al: Noncontact anterior cruciate
ligament injuries: Risk factors and prevention strategies. J Am Acad
Orthop Surg 8:141-150, 2000.
19. Torg JS, Conrad W, Kalen V: Clinical diagnosis of anterior cruci-
ate ligament instability in the athlete. Am J Sports Med 4:84-93,
1976.
20. Bach BR Jr, Warren RF, Wickiewicz TL: The pivot shift phenom-
enon: Results and description of a modified clinical test for ante-
rior cruciate ligament insufficiency. Am J Sports Med 16:571-576,
1988.
21. Noyes FR, Grood ES, Cummings JF, et al: An analysis of the pivot
shift phenomenon: The knee motions and subluxations induced by
different examiners. Am J Sports Med 19:148-155, 1991.
22. Harner CD, Hoher J: Evaluation and treatment of posterior cruci-
ate ligament injuries. Am J Sports Med 26:471-482, 1998.
23. Harner CD, Xerogeanes JW, Livesay GA, et al: The human posterior
cruciate ligament complex: An interdisciplinary study: Ligament mor-
phology and biomechanical evaluation. Am J Sports Med 23:736-745,
1995.
24. Fanelli GC: Posterior cruciate ligament injuries in trauma patients.
Arthroscopy 9:291-294, 1993.
25. Watanabe Y, Moriya H, Takahashi K, et al: Functional anatomy of the
posterolateral structures of the knee. Arthroscopy 9:57-62, 1993.
26. Andrews JR, Norwood LA Jr, Cross MJ: The double bucket handle
tear of the medial meniscus. J Sports Med 3:232-237, 1975.
27. McMurray T: The semilunar cartilages. Br J Surg 29:407, 1941.
28. Apley A: The diagnosis of meniscus injuries: Some new clinical meth-
ods. J Bone Joint Surg Br 29:78, 1929.
29. Dobbs RE, Hanssen AD, Lewallen DG, et al: Quadriceps tendon rup-
ture after total knee arthroplasty: Prevalence, complications, and out-
comes. J Bone Joint Surg Am 87:37-45, 2005.
30. Fredericson M, Yoon K: Physical examination and patellofemoral
pain syndrome. Am J Physical Med Rehabil 85:234-243, 2006.
31. Merchant AC: Classification of patellofemoral disorders. Arthroscopy
4:235-240, 1988.
32. Messieh SS, Fowler PJ, Munro T: Anteroposterior radiographs of the
osteoarthritic knee. J Bone Joint Surg Br 72:639-640, 1990.
33. Barrack RL, Schrader T, Bertot AJ, et al: Component rotation and
anterior knee pain after total knee arthroplasty. Clin Orthop 392:46-55,
2001.
34. Berger RA, Rubash HE: Rotational instability and malrotation after
total knee arthroplasty. Orthop Clin North Am 32:639-647, 2001.
35. van Holsbeeck M, Introcaso JH: Musculoskeletal ultrasonography.
Radiol Clin North Am 30:907-925, 1992.
36. Palmer EL, Scott JA, Strauss HW: Bone imaging. In Practical Nuclear
Medicine. Philadelphia, WB Saunders, 1992, pp 121-183.
37. Duus BR, Boeckstyns M, Kjaer L, et al: Radionuclide scanning after
total knee replacement: Correlation with pain and radiolucent lines:
A prospective study. Invest Radiol 22:891-894, 1987.
38. Kantor SG, Schneider R, Insall JN, et al: Radionuclide imaging of
asymptomatic versus symptomatic total knee arthroplasties. Clin Orthop
260:118-123, 1990.
39. Palestro CJ, Swyer AJ, Kim CK, et al: Infected knee prosthesis: Diag-
nosis with In-111 leukocyte, Tc-99m sulfur colloid, and Tc-99m MDP
imaging. Radiology 179:645-648, 1991.
40. De Smet AA, Tuite MJ: Use of the “two-slice-touch” rule for the
MRI diagnosis of meniscal tears. AJR Am J Roentgenol 187:911-914,
2006.
41. Fritschy D, Fasel J, Imbert JC, et al: The popliteal cyst. Knee Surg
Sports Traumatol Arthrosc 14:623- 628, 2006.
42. Kellgren JH: The sensitivity and innervation of the articular cap-
sule. J Bone Joint Surg Br 32:84, 1950.
43. Offierski CM, MacNab I: Hip-spine syndrome. Spine 8:316-321, 1983.
44. Dewolfe VG, Lefevre FA, Humphries AW, et al: Intermittent claudi-
cation of the hip and the syndrome of chronic aorto-iliac thrombosis.
Circulation 9:1-16, 1954.
45. Leriche R, Morel A: The syndrome of thrombotic obliteration of the
aortic bifurcation. Am Surg 127:193, 1948.
46. Bellamy N, Buchanan WW, Goldsmith CH, et al: Validation study
of WOMAC: A health status instrument for measuring clinically
important patient relevant outcomes to antirheumatic drug therapy
in patients with osteoarthritis of the hip or knee. J Rheumatol 15:
1833-1840, 1988.
47. Harris WH: Traumatic arthritis of the hip after dislocation and
acetabular fractures: Treatment by mold arthroplasty: An end-result
study using a new method of result evaluation. J Bone Joint Surg Am
51:737-755, 1969.
48. Harris WH: Etiology of osteoarthritis of the hip. Clin Orthop 213:
20-33, 1986.
49. Millis MB, Murphy SB, Poss R: Osteotomies about the hip for the
prevention and treatment of osteoarthrosis. Instr Course Lect 45:
209-226, 1996.
50. Millis MB, Poss R, Murphy SB: Osteotomies of the hip in the prevention
and treatment of osteoarthritis. Instr Course Lect 41:145-154, 1992.
51. Trendelenburg F: Dtsch Med Wschr (RSM translation) 21:21-24,
1895.
52. Thomas H: Hip, Knee and Ankle. Liverpool, Dobbs, 1976.
53. Laslett M: Pain provocation tests for diagnosis of sacroiliac joint pain.
Aust J Physiother 52:229, 2006.
54. Laslett M, Aprill CN, McDonald B: Provocation sacroiliac joint tests
have validity in the diagnosis of sacroiliac joint pain. Arch Phys Med
Rehabil 87:874; author reply 874-875, 2006.
55. Burnett RS, Della Rocca GJ, Prather H, et al: Clinical presentation
of patients with tears of the acetabular labrum. J Bone Joint Surg Am
88:1448-1457, 2006.
56. Toomayan GA, Holman WR, Major NM, et al: Sensitivity of MR
arthrography in the evaluation of acetabular labral tears. AJR Am J
Roentgenol 186:449-453, 2006.
57. Cunningham T, Jessel R, Zurakowski D, et al: Delayed gadolinium-
enhanced magnetic resonance imaging of cartilage to predict early
failure of Bernese periacetabular osteotomy for hip dysplasia. J Bone
Joint Surg Am 88:1540-1548, 2006.
58. Byrd JW, Jones KS: Diagnostic accuracy of clinical assessment, mag-
netic resonance imaging, magnetic resonance arthrography, and intra-
articular injection in hip arthroscopy patients. Am J Sports Med 2004;
32(7):1668-1674, 2004.
59. Illgen RL 2nd, Honkamp NJ, Weisman MH, et al: The diagnostic and
predictive value of hip anesthetic arthrograms in selected patients
before total hip arthroplasty. J Arthroplasty 21:724-730, 2006.
 43
KEY POINTS
The differential diagnosis for foot and ankle pain is vast.
Localizing symptoms by anatomic region helps to narrow this
differential.
On physical examination, most structures in the foot and
ankle are immediately subcutaneous and readily palpable.
Beyond medications, useful nonoperative treatments include
bracing, shoewear modification, orthoses, and physical
therapy.
Most surgical procedures in foot and ankle surgery fall into
one of the following categories: arthrodesis, arthroplasty,
corrective osteotomy, ostectomy, tendon débridement and
transfer, and synovectomy. Patient compliance and soft tissue
integrity are important factors when considering surgery.
Advances in medical management now make joint-sparing
procedures possible in many patients with inflammatory
arthritis who previously would have required arthrodesis.
Rheumatoid arthritis (RA) and other inflammatory arthri-
tides are potentially devastating systemic diseases that often
affect the foot and ankle. Foot and ankle pain is the pre-
senting complaint in approximately 15% to 20% of patients
newly diagnosed with RA.1 Of patients already diagnosed
with RA, the prevalence of foot and ankle involvement has
been estimated to be greater than 90%.2 Nevertheless, many
patients with RA have foot and ankle pain secondary to a
noninflammatory process, and many rheumatologists often
see patients without inflammatory arthritis. This chapter
provides a broad overview of foot and ankle pain, regardless
of etiology.
The evaluation of any patient with foot and ankle pain
begins with a thorough history and physical examination.
The location, timing, and duration of symptoms can help
establish a specific diagnosis and help guide the subsequent
course of treatment. Radiographs and advanced imaging
modalities provide useful adjuncts in the evaluation of spe-
cific foot and ankle pathologies.
The treatment of any disorder of the foot and ankle
is aimed at alleviating pain and preserving function (i.e.,
maintaining the ambulatory status of the patient). Initial
nonoperative treatment options include medical manage-
ment, physical therapy, shoewear modification, orthoses,
and bracing. These measures provide substantial relief for
many individuals. For recalcitrant symptoms, surgical inter-
vention may be necessary. Most surgical procedures fall into
one of the following general categories: arthrodesis (joint
fusion), arthroplasty (joint replacement), corrective oste-
otomy, ostectomy, tendon débridement and transfer, and
synovectomy (joint or tendon).
  Video available on the Expert Consult Premium Edition website.
Foot and Ankle Pain
Mark D. Price  • Christopher P. Chiodo
FUNCTIONAL ANATOMY
AND BIOMECHANICS
The ankle, or tibiotalar, joint comprises the articulation
between the foot (talus) and the lower leg (distal tibia and
fibula). Its primary motion is plantar flexion and dorsiflexion
in the sagittal plane. In addition, the articulation between
the distal tibia and fibula allows a lesser degree of internal
and external rotation to occur in the axial, or transverse,
plane.
The foot may be loosely divided into three anatomic
regions: the forefoot, the midfoot, and the hindfoot. The
forefoot consists of the toes and metatarsal bones, along
with the metatarsophalangeal (MTP) and interphalangeal
joints. The tarsometatarsal (TMT) joints connect the fore-
foot to the midfoot, which comprises the three cuneiform
bones, the navicular, and the cuboid. Finally, the hindfoot,
located below the ankle, consists of the talus and calcaneus.
The joints of the hindfoot include the talocalcaneal (subta-
lar), talonavicular, and calcaneocuboid articulations.
Forefoot and midfoot motion is primarily plantar flexion
and dorsiflexion in the sagittal plane, with some secondary
pronation and supination in the coronal plane and abduc-
tion/adduction in the axial plane. Motion in the hindfoot
consists primarily of inversion/eversion in the coronal plane,
with secondary internal/external rotation in the axial plane
and plantar flexion/dorsiflexion in the sagittal plane.
Rheumatoid arthritis affects the joints and the tendons of
the foot and ankle. Articular synovial disease results in loss
of cartilage and erosion of subchondral bone. In addition,
such articular disease also leads to attenuation of the joint
capsule and supporting collateral ligaments. As a result of
these combined structural insults, rheumatoid arthritis often
results in substantial deformity.
DIAGNOSTIC EVALUATION
PHYSICAL EXAMINATION
A thorough physical examination of the foot and ankle
begins with gait analysis, which is done by observing the
patient enter the examination room. Normal human gait is
classically divided into four segments and two phases. The
four segments are heel-strike, foot-flat, toe-off, and swing.
Stance phase is the weight-bearing portion of the gait
cycle. It extends from heel-strike to toe-off and constitutes
roughly 60% of the cycle. Meanwhile, the swing phase of
gait extends from toe-off to heel-strike and constitutes the
remaining 40% of the gait cycle.
Patients with an antalgic gait pattern have a short-
ened stance phase on the side of the affected limb, as they
643
 644 PRICE  |  Foot and Ankle Pain
attempt to transfer their weight quickly to the nonpainful
limb. In addition to an antalgic gait, foot and ankle pain
often results in the avoidance of ground contact with the
painful part of the foot. A further problem noted in stance
phase is dynamic collapse of the medial longitudinal arch,
most apparent at foot-flat and toe-off.
After gait analysis, the foot and ankle are inspected with
the patient sitting and standing. The location of swelling is
usually well correlated with the joints involved (e.g., ankle
versus talocalcaneal joint). Deformity also should be noted.
Common foot and ankle deformities include hallux valgus,
or bunion (Fig. 43-1), hammer toes, and flatfoot deformity
(characterized by hindfoot valgus/forefoot abduction). Cal-
losities develop over regions of increased pressure and are
associated with deformity and fat pad atrophy. Rheumatoid
nodules can appear anywhere on the foot, but are often
found in areas of repetitive trauma (i.e., at the site of irrita-
tion from a tight shoe counter). Similarly, ulcerations appear
in areas of repeated injury, such as those found in tight-fit-
ting shoes. Finally, wear patterns on shoes should be noted.
As Hoppenfeld3 observed, “A deformed foot can deform any
good shoe; in fact, in many cases the shoe is a literal show-
case for certain disorders.”
After inspection, the foot and ankle are thoroughly pal-
pated. The dorsum of the foot and ankle has little overlying
musculature. Many of the bones and tendons are immedi-
ately subcutaneous, and a great deal of information can be
gained from palpating these structures. It is helpful to pal-
pate the foot and ankle by anatomic location (i.e., forefoot,
midfoot, hindfoot, anterior and posterior ankle).
In the forefoot, the first metatarsal head and MTP joint
can be palpated at the base of the hallux (great toe), at the
medial aspect of the “ball” of the foot. Proceeding laterally,
the lesser metatarsal heads and MTP joints can be sequen-
tially palpated. In patients with RA or nonarthritic meta-
tarsalgia, such palpation often reveals tenderness, synovitis,
and swelling. In the second and third MTP joints, sagittal
Figure 43-1  Clinical photograph of hallux valgus deformity.
plane instability often results from attenuation of the plan-
tar joint capsule. This can be appreciated by gently translat-
ing the second and third toes dorsally. In the hindfoot, the
calcaneus is readily palpable, and its various parts can be
palpated individually. Tenderness over the posterior aspect
of this bone may indicate Achilles tendinitis, whereas pain
over the medial tubercle (palpable on the medial plantar
surface) may indicate plantar fasciitis. Tenderness over sinus
tarsi of the hindfoot (located laterally, just anterior and distal
to the tip of the fibula) indicates talocalcaneal joint pathol-
ogy. In the ankle joint proper, tenderness over the anterior
joint line usually correlates with ankle joint pathology.
After inspection and palpation, range of motion analysis is
performed. Passive range of motion of the ankle is normally
10 to 20 degrees of dorsiflexion and 40 to 50 degrees of plan-
tar flexion. Normal hindfoot inversion and eversion are each
approximately 5 degrees. The first MTP joint should have
approximately 45 degrees of plantar flexion (flexion) and 70
to 90 degrees of dorsiflexion (extension). Deviations from
these norms should be noted as part of the standard workup.
IMAGING
Despite the abundant availability of advanced imaging
modalities such as magnetic resonance imaging (MRI)
and computed tomography (CT), radiographs remain the
imaging mainstay in the evaluation of foot and ankle pain.
Weight-bearing images should be obtained whenever pos-
sible because joint space narrowing and deformity may
not be apparent in non–weight-bearing images. Standard
images consist of weight-bearing anteroposterior, lateral,
and oblique views of the foot and anteroposterior, mortise,
and lateral views of the ankle.
MRI provides reliable imaging of soft tissue structures
and is integral to the evaluation of foot and ankle pain. MRI
is especially crucial in the evaluation of synovitis, tendinitis,
tendinosis, and bursitis (Fig. 43-2).4 CT scan5 and nuclear
Figure 43-2  Axial MR image of ankle showing posterior tibial tendon
degeneration and synovitis.
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN 645

scintigraphy6 also are used in the evaluation of foot and Table 43-1  Differential Diagnosis of Foot  
ankle pain. Either method is helpful in delineating arthri- and Ankle Pain Based on Anatomic Location
tis and postoperatively evaluating the results of joint fusion Location Differential Diagnosis
surgery. Ultrasonography is gaining popularity in some
Hallux and first MTP joint Arthritis, hallux valgus, hallux
centers, especially when used to evaluate tendon integrity. rigidus, sesamoid disorders
The results are largely technique dependent, however, with
Lesser toes and MTP joints Lesser toe deformities, MTP insta-
minimization of artifacts being a concern. Ultrasound is bility, metatarsalgia, Morton’s
difficult to use in preoperative planning because important neuroma, bunionette
landmarks are often not well imaged.7 Midfoot Arthritis, accessory navicular,  
Anesthetic arthrograms can be extremely useful adjuncts osteonecrosis, peroneal tendi-
in diagnosing foot and ankle pain in patients with degen- nitis, posterior tibial tendinitis,
erative and inflammatory arthritis. Given the complex and fifth metatarsal stress fracture
crowded anatomy of the foot and ankle, it is sometimes diffi- Hindfoot Arthritis, posterior tibial tendinitis
cult to determine precisely which joint is symptomatic, or if and dysfunction
more than one joint is diseased. With an anesthetic arthro- Heel Plantar fasciitis, Achilles tendinitis,
gram, a mixture composed of a steroid, anesthetic, and con- nerve entrapment, calcaneal
trast material is injected under fluoroscopic guidance into a stress fracture
joint. This allows the clinician to determine more precisely Anterior ankle Arthritis, impingement,  
whether or not the injected joint is a significant pain gen- osteochondral defect
erator.8 Posterior ankle Achilles tendinitis/tendinosis,
bursitis
Posterolateral ankle Stress fracture, peroneal tendon
COMMON CAUSES OF ANKLE PAIN pathology (tendinitis, tear,
instability)
From a diagnostic standpoint, it is useful for the clinician to
conceptualize ankle pain based on anatomic location. This Posteromedial ankle Posterior tibial tendinitis/ 
dysfunction, FHL/FDL tendinitis,
approach applies to patients with and without arthritis and tarsal tunnel syndrome
helps to make a long differential list more manageable. Con-
FHL/FDL, flexor hallucis longus/flexor digitorum longus; MTP, metatar­
ditions affecting the foot and ankle and their correlation sophalangeal.
with anatomic location are summarized in Table 43-1.

patients ­without inflammatory arthritis. Although a history

ANTERIOR AND CENTRAL ANKLE PAIN
Anterior and central ankle pain is often the result of intra-
articular pathology. The reason for this is that anteriorly the
ankle joint is not shielded by the malleoli and is immediately
subcutaneous. With the exception of the anterior tibial ten-
don, the extensor tendons that cross the joint are not prone
to the development of tendinitis and tendinosis.
Spur and osteophyte formation are often seen in the
anterior ankle and localized to the superior talar neck and
anterior distal tibia. Nonarthritic spurs do occur in the
absence of degenerative and inflammatory arthritis, espe-
cially in active individuals who have a history of chronic
ankle instability. Clinically, anterior spurs and osteophytes
may result in symptoms of “impingement,” with anterior
joint line pain, swelling, and tenderness. Patients specifi-
cally note pain with ankle dorsiflexion, such as when they
walk up stairs or an incline. On physical examination, there
may be anterior tenderness or pain or both with terminal
passive dorsiflexion. Patients with more advanced global
ankle arthritis, whether degenerative or inflammatory, typi-
cally present with either anterior or central pain. “Start-up”
symptoms are common. Radiographs are essential to dif-
ferentiate between anterior impingement and more global
arthritis.
Two other bony causes of central ankle pain, stress frac-
ture and osteochondral defect, should always be consid-
ered. Stress fractures are commonly seen in patients with
inflammatory arthropathy secondary to periarticular and
generalized osteopenia. An osteochondral defect is a focal
defect in the articular cartilage and subchondral bone.
These lesions are encountered more commonly in younger
of trauma is often present, many osteochondral defects are
idiopathic or attributed to the ill-defined concept of “repeti-
tive microtrauma.”
Of the tendons that cross the front of the ankle joint, the
anterior tibial tendon is the most prone to developing ten-
dinitis or tendinosis. Nevertheless, the incidence of anterior
tibial tendon pathology is much lower compared with other
tendons such as the Achilles. With anterior tibial tendinitis
or tendinosis, a visible fullness is usually present. Tendon
dysfunction or rupture may result in a foot-slap or steppage
gait pattern.
POSTERIOR JOINT PAIN
Posterior ankle pain usually originates from the Achil-
les tendon, its insertion onto the calcaneal tuberosity, and
two associated bursae in this region. The Achilles tendon
is the largest tendon in the body. It lacks a true synovial
lining. Isolated Achilles tendinitis is uncommon. In most
instances, Achilles pain results from degenerative tendino-
sis, with or without an overlying tendinitis. Although asso-
ciated intratendinous spur formation is common, Achilles
spurs are a manifestation of associated tendon degeneration.
Spur excision also frequently entails tendon débridement,
reconstruction, and transfer.
The Achilles tendon is protected by two distinct bursae.
A more superficial bursa is immediately subcutaneous and
becomes inflamed primarily with irritation from ill-fitting
shoes with a tight counter (“pump bump”). The “retrocalca-
neal” bursa is a larger structure that lies deep to the Achilles
tendon. Inflammation of this structure often accompanies
 646 PRICE  |  Foot and Ankle Pain
Achilles tendinitis/tendinosis. It also may be irritated by an
enlarged posterior superior calcaneal tuberosity, sometimes
referred to as a Haglund’s deformity.
MEDIAL AND LATERAL ANKLE PAIN
As with anterior, central, and posterior ankle pain, the
origin of medial or lateral ankle pain also is anatomically
based. On the medial side, pain directly over the medial
malleolus should alert the clinician to the possibility of a
stress fracture. Pain anterior to the medial malleolus is usu-
ally articular in nature. Pain posterior to the medial mal-
leolus is often caused by inflammation or degeneration (or
both) of the posteromedial flexor tendons, including the
posterior tibial tendon and the flexor hallucis longus and
flexor digitorum longus tendons. The posterior tibial ten-
don is the largest and strongest of the posteromedial flexor
tendons. Its primary function is to invert the hindfoot and
support the medial longitudinal arch of the foot. Long-
standing synovitis and dysfunction of this tendon ultimately
may lead to collapse of the arch and the development of an
acquired flatfoot deformity. Finally, tarsal tunnel syndrome
is another cause of posteromedial ankle pain. This disorder
is believed to represent an entrapment neuropathy of the
tibial nerve in the tarsal tunnel. Patients typically complain
of pain that radiates into the plantar foot. On examination,
percussion of the tarsal tunnel reproduces these symptoms
(Tinel’s sign).
On the lateral side of the ankle, pain directly over the
lateral malleolus may be caused by a stress fracture. Simi-
lar to the medial side, pain anterior to the lateral malleolus
is usually articular in nature. Finally, pain posterior to this
lateral malleolus usually indicates peroneal tendon pathol-
ogy. In patients with and without inflammatory arthritis, the
peroneal tendons may be affected by tenosynovitis, longitu-
dinal “split” tears, and chronic tendon instability. With the
last-mentioned, the tendons sublux over the posterolateral
edge of the fibula, causing pain and attritional tearing.
COMMON CAUSES OF FOOT PAIN
FOREFOOT PAIN
The forefoot region is a common location of foot pain.
Patients with RA are particularly prone to symptoms9
because inflammation and progressive MTP synovitis even-
tually lead to capsular distention and destruction.10 Even-
tually, there may be loss of collateral ligament stability
and, finally, destruction of the articular cartilage and bone
(Fig. 43-3).
It is helpful to subdivide the forefoot further into two
regions: (1) the hallux and first MTP joint and (2) the
“lesser” (i.e., second through fifth) toes and MTP joints.
Several disorders frequently affect the hallux and first MTP
joint. The hallux valgus deformity, or bunion, is commonly
encountered in patients with and without inflammatory
arthritis (see Fig. 43-1). In RA, where the incidence of hal-
lux valgus has been estimated to be 70%,9 progression of this
deformity may be accelerated further by loss of support from
the adjacent lesser MTP joints. Hallux pain without defor-
mity may be caused by degenerative arthritis, commonly
referred to as hallux rigidus. Alternatively, it may be caused
by one of several sesamoid disorders, including sesamoiditis,
osteonecrosis, fracture, and arthritis.
In the lesser toes, three common deformities can cause
pain: claw toes, hammer toes, and mallet toes. Potential eti-
ologies of these deformities include arthritis, trauma, nerve/
muscle imbalance, and chronic use of shoes with inadequate
toe boxes. In the lesser MTP joints, instability may result
from mechanical causes or inflammatory disease. A particu-
larly common mechanical cause of MTP instability is a rela-
tively long second metatarsal, which increases the cyclical
loads of the MTP joint. With MTP instability, dorsiflexion
forces at toe-off can lead to progressive subluxation and dor-
sal dislocation. With this, the metatarsal head is prone to
forming keratotic skin lesions that can ulcerate. Lesser MTP
joint subluxation has been reported to be 70% with a con-
comitant incidence of pressure sores in approximately 30%
of those patients.10
Metatarsalgia is a general term used to denote pain
under the lesser metatarsal heads. There are several causes
of metatarsalgia. With a gastrocnemius contracture or tight
Achilles tendon, the forefoot is prematurely loaded during
the stance phase of gait. Alternatively, hammer toes and
mallet toes can result in downward pressure on the metatar-
sal heads, leading to metatarsalgia. In elderly patients and
patients with inflammatory arthritis, atrophy of the plantar
fat pad of the forefoot also can result in metatarsalgia.
In the lateral forefoot, two common causes of pain
include Morton’s neuroma and bunionette. Morton’s neu-
roma typically occurs in between the third and fourth meta-
tarsal heads and manifests with burning, aching, or shooting
pain in this region. Symptoms are especially exacerbated
with tight shoes. A bunionette is an angular deformity of
the fifth toe, typically causing pain over the lateral aspect of
the fifth metatarsal head.
MIDFOOT PAIN
In the midfoot, the most common cause of foot pain is
arthritis at the TMT joints, and most frequently the first
TMT joint on the medial side of the foot. If there is associ-
ated instability of the first TMT joint, repetitive stress can
lead to dorsiflexion of the first metatarsal. Alternatively,
midfoot arthritis can lead to an abduction deformity of the
foot, where the forefoot and metatarsals deviate outward.9
Arthritis of the lateral (fourth and fifth) TMT joints is
often asymptomatic. More commonly, lateral midfoot pain
results from insertional peroneal tendinitis or a stress frac-
ture of the fifth metatarsal. Simple palpation of these struc-
tures should allow one to distinguish readily between the
two diagnoses. On the medial side of the midfoot, pain not
due to arthritis may instead be secondary to an accessory
navicular bone, osteonecrosis of the native navicular bone,
and insertional posterior tibial tendinitis.
HINDFOOT PAIN
The three joints of the hindfoot (talonavicular, talocalca-
neal, and calcaneocuboid) all may be affected by degenera-
tive and inflammatory arthritis. In patients with RA, the
overall prevalence of hindfoot involvement is 21% to 29%.5
The talonavicular joint is most often affected, followed by
the talocalcaneal and calcaneocuboid joints. The incidence
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
A B
of hindfoot deformity in patients with RA of less than 5
years’ duration has been estimated to be 8% and increases
to 25% in patients with RA longer than 5 years.11 Clini-
cally, patients with talocalcaneal or calcaneocuboid arthri-
tis complain of lateral hindfoot pain. Patients with arthritis
and synovitis of the talonavicular joint complain of dorsal
or medial pain.
A common source of medial hindfoot pain in the gen-
eral population and patients with inflammatory arthritis is
posterior tibial tendinitis and dysfunction. The posterior
tibial tendon runs along the posteromedial ankle and hind-
foot and stabilizes the medial longitudinal arch. Inflamma-
tion, degeneration, and dysfunction of this tendon typically
result in medial ankle and hindfoot pain. An associated
flatfoot deformity, characterized by heel valgus and forefoot
­abduction, may be noted.
HEEL PAIN
Most heel pain can be attributed to one of four causes: plan-
tar fasciitis, Achilles tendinosis, nerve entrapment, or calca-
neal stress fracture. Plantar fasciitis typically causes inferior
heel pain, worse when first getting up in the morning or
getting up after sitting for a long time. Insertional Achilles
tendinosis usually results in posterior heel pain, worse dur-
ing or after exercise. Finally, entrapment of the first branch
of the lateral plantar nerve (Baxter’s nerve) can be a source
of medial heel pain and tenderness, whereas calcaneal stress
fracture can produce medial and lateral symptoms. Calca-
neal stress fracture usually can be distinguished by a positive
“squeeze test,” with compression of both sides of the heel.
NONOPERATIVE TREATMENT
Medical management is the cornerstone of treatment for
most foot and ankle disorders. With RA, many of the cur-
rent recommendations for operative treatment may soon
647
Figure 43-3  A and B, Preoperative (A) and
postoperative (B) anteroposterior radiographs
of hallux valgus deformity with lesser metatar-
sophalangeal joint erosions treated by fusion
and lesser metatarsal head resections.
be modified given the alteration of disease progression with
current medical regimens.12 The most common medical
management still consists of nonsteroidal anti-inflamma-
tory drugs, steroids, and disease-modifying antirheumatic
drugs. Although each of these drug classes has done much
to alleviate patient suffering, they have an impact on the
surgical management of rheumatic disease. There is concern
about lower fusion rates in the setting of nonsteroidal anti-
inflammatory drugs and increased infection rates in the set-
ting of steroids or disease-modifying antirheumatic drugs.13,14
Patients who have been on long-term steroids are at risk for
postoperative adrenal insufficiency and may require periop-
erative corticosteroids.15 Close communication and collabo-
ration between the rheumatologist and surgeon is essential
to good outcomes.
Beyond medications, shoewear modification should not
be overlooked because it often has profound benefits for
patients with foot and ankle pain. Shoes should be exam-
ined in the clinic to ensure that they can accommodate a
patient’s deformity. Patients often feel best in shoes with a
deep, wide toe box, a firm heel counter, and soft heel. Well-
constructed walking or jogging shoes usually provide suffi-
cient room for mild-to-moderate deformities. It is helpful to
provide patients with a list of suitable manufacturers when
making such recommendations.
Often it is necessary to prescribe a custom orthotic insert
for patients with more moderate deformities. It is typically
necessary to remove the insole of the shoe to make room for
the orthotic insert. Most walking or jogging shoes suffice.
Generally, custom orthoses can be divided into rigid, semi-
rigid, and softer accommodative devices. Rigid and semi-
rigid orthoses usually are used to correct supple deformities
and should be used with caution in patients with arthritis.16
More commonly, these patients, especially if they have RA,
benefit from accommodative orthoses (i.e., orthoses made
of softer material that can be molded to “accommodate”
a deformity).17 Accommodative orthoses can be modified
 648 PRICE  |  Foot and Ankle Pain
f­urther by incorporating a “relief” under a deformity, further
unloading it. When sending patients for orthoses, it is best
to provide the orthotist with a prescription that includes
the patient’s precise diagnosis (e.g., metatarsalgia) and the
type of orthosis and any modifications desired (e.g., a “cus-
tom accommodative orthosis with a relief under the lesser
metatarsal heads”).18
Injections of a mixture of anesthetic and corticosteroid to
areas of inflammation or bursitis are useful in the treatment of
inflammatory and noninflammatory conditions affecting the
foot and ankle. In the foot and ankle, such injections must
be judiciously employed, however. Most importantly, injec-
tions into and around tendons should be avoided. Because
of the forces associated with weight bearing and ambula-
tion, these tendons are under substantial load. The injec-
tion of a corticosteroid near or directly into a tendon can
adversely affect the biomechanical properties of the tendon,
ultimately leading to rupture.19 A further precaution is to
avoid corticosteroid injections into the lesser MTPs when
there is evidence of joint instability (manifested by devia-
tion on radiographs or instability on physical examination).
Such injections can lead to further attenuation of the joint
capsule and result in frank joint dislocation.
OPERATIVE TREATMENT
If symptoms persist despite nonoperative management, sur-
gical intervention should be considered. Two important fac-
tors must be taken into account when deciding whether or
not to proceed with surgery. First, the soft tissues and vas-
cular status must be assessed carefully. Both may be com-
promised and negatively affect outcome. Second, the ability
of patients to comply with the postoperative regimen (e.g.,
being able to use crutches and remain non–weight bearing if
necessary) must be considered. Even limited noncompliance
can lead to a poor outcome, especially in fusion surgery.
As noted earlier, most surgical procedures fall into one of
the following categories: arthrodesis (joint fusion), arthro-
plasty (joint replacement), corrective osteotomy, ostectomy,
tendon débridement and transfer, and synovectomy (joint
or tendon). Arthrodesis is a surgical cornerstone for arthritis
of the foot and ankle. With an arthrodesis procedure, the
two sides of the joint are roughened with a burr or small
chisel. Next, the two bones to be fused are compressed and
fixed together, usually with one or more screws (see Fig.
43-3B). In the weeks and months after surgery, the body is
“tricked” into thinking that there is a fracture present at the
fusion site and heals this with bone. The two bones become
one and are considered fused. Fusion surgery offers reliable
pain relief in most patients. One concern with fusion sur-
gery is the loss of motion. For the patient, however, this
usually results in only mild functional compromise. To the
untrained eye, there is remarkably little change in gait.
Commonly performed fusions include ankle arthro­
desis, isolated hindfoot fusions, triple arthrodesis, midfoot
arthrodesis, and arthrodesis of the first MTP joint. A triple
arthrodesis involves fusion of the talocalcaneal, talonavicu-
lar, and calcaneocuboid joints. Together, these joints allow
coronal plane motion and are most important when walking
on uneven ground.
Fusion remains the “gold standard” for patients with
degenerative and inflammatory ankle arthritis. If there is
minimal deformity and no loss of bone stock, ankle fusion
surgery may be performed arthroscopically or through a
mini-open approach. These techniques involve less soft tis-
sue dissection and stripping, minimizing loss of bony perfu-
sion. Nevertheless, the time period for which the patient
must remain non–weight bearing (6 to 12 weeks) remains
the same. The success rate of ankle fusion surgery in patients
with RA is generally 85% or greater. Although the osteope-
nia associated with the disease can compromise fixation, it
also theoretically can enhance fusion because there is less
sclerotic subchondral bone.
In the hindfoot, fusion surgery may be performed on one
or more of the three joints of this part of the foot (i.e., the
talocalcaneal, talonavicular, and calcaneocuboid joints). If
only one of these joints is diseased, an isolated fusion of this
joint is acceptable.20 This procedure reduces surgical mor-
bidity and the extent of the procedure. Nevertheless, with
fusion of just one of the joints of the hindfoot, motion in the
other joints is reduced.21 If more than one joint is diseased,
a double or triple arthrodesis is necessary.
In the midfoot, fusion surgery results in negligible loss of
motion because the joints of the midfoot normally have less
than 10 degrees of motion. With osteoarthritis and inflam-
matory arthritis, symptoms most often are limited to the
medial (first through third) TMT joints. The lateral (fourth
and fifth) TMT joints are infrequently symptomatic, even
in the setting of advanced radiographic changes.
In the forefoot, fusion surgery is indicated only for the
first MTP joint. This procedure is used for arthrosis and
advanced hallux valgus (bunion) deformities. When the first
MTP joint is fused, it is positioned in a slightly dorsiflexed
position to facilitate ambulation. With MTP fusion in 47
feet, Coughlin22 reported 96% good-to-excellent results and
100% fusion at an average 6.2-year follow-up.
Fusion surgery generally provides reliable pain relief and
a stable, plantigrade foot. Nevertheless, the loss of motion
of the fused joint can lead to increased motion and altered
biomechanics at adjacent joints. This alteration ultimately
may lead to arthritic changes in these joints.23 Fusion surgery
may lead to subtle, albeit real changes in gait.24 Finally, the
minimal ramifications of fusing just one joint may become
much greater in the setting of a subsequent fusion in either
the ipsilateral or the contralateral limb.
Such concerns drive many researchers to work toward
improving joint replacement surgery (arthroplasty) in the
foot and ankle. Most notably, total ankle replacement sur-
gery continues to evolve and remains a controversial topic
among orthopaedic surgeons. Although many orthopaedic
surgeons perform ankle replacement surgery, many do not or
do so on a limited basis. To this end, the U.S. Food and Drug
Administration currently approves only two ankle prosthe-
ses for implantation. Long-term survival data as published
for hip and knee arthroplasty are not yet available.
The main advantage of ankle arthroplasty is preserva-
tion of motion. Its main two disadvantages are technical
complexity and the difficulty with subsequent fusion if the
procedure fails. Generally, ankle replacement surgery is
indicated for late middle-age and elderly individuals with
low functional demands and minimal deformity. Bilateral
disease and concomitant ipsilateral hindfoot fusion increase
the benefits afforded by arthroplasty. The paradox of ankle
replacement surgery remains as follows: Ankle replacement
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
is contraindicated in young patients for whom preservation
of motion is most important. Arthroplasty is more com-
monly performed in older patients for whom preservation
of motion is less important, and who would do well with a
fusion. Nevertheless, total ankle replacement surgery con-
tinues to evolve, and reported success rates with modern
designs continue to improve (Fig. 43-4).25
In the foot, arthroplasty is performed by some surgeons
for the first MTP joint. The relevant literature is still con-
flicted, however. Although there were some encouraging
early results with arthroplasty, other studies have shown high
rates of implant failure and loosening secondary to synovitis
from Silastic particle wear.25-27 Advanced deformity, often
present in patients with RA and inflammatory arthritis, is
considered to be a relative contraindication to first MTP
joint arthroplasty. Nevertheless, new implant designs may
hold increased promise. Generally, these implants are lower
profile and resect less bone, which also makes it easier to
perform a subsequent fusion, if necessary.
Corrective osteotomies are used primarily for two rea-
sons in the treatment of arthritis: to correct deformity and
to redistribute forces on a joint or the terminal aspect of a
bone. Examples of osteotomies to correct deformity include
calcaneal osteotomies for pes planovalgus and metatarsal
osteotomies for hallux valgus. Previously, patients with RA
and concomitant pes planovalgus or hallux valgus under-
went fusion surgery. With advances in medical management
of the disease, however, it is reasonable to attempt joint
preservation surgery in patients who have mild-to-moderate
disease, good soft tissues, and flexible deformities.
Examples of osteotomies to redistribute forces include
tibial osteotomies in the setting of eccentric ankle arthritis
and metatarsal osteotomies in the setting of metatarsalgia.
Patients requiring surgery for ankle RA previously under-
went fusion surgery only, and patients requiring surgery
Figure 43-4  Anteroposterior ankle radiograph of a total ankle arthro-
plasty.
649
for metatarsalgia underwent metatarsal head resection.
Advances in medical management of the disease allow joint
preservation osteotomies to be considered, however. This
is especially the case for metatarsalgia, which is common
in patients with RA, yet increasingly does not entail frank
dislocation or articular erosion.
Some patients with early arthritis or chronic ankle insta-
bility may present with symptoms of mechanical impinge-
ment from anterior ankle spurs or osteophytes. In cases
without global joint destruction, surgical resection of the
spurs, or cheilectomy, is a reasonable treatment. Although
no studies have examined cheilectomy in RA specifically,
patients with less severe erosive changes tend to be more
satisfied with the results of cheilectomy.28
Some clinicians believe that early synovectomy of either
the affected joint or the tendon may help halt the progress
of joint destruction in RA. For patients whose symptoms
persist despite nonoperative treatment, synovectomy can
provide substantial pain relief.29 When considering synovec-
tomy of an arthritic joint, there should be preservation of
the articular cartilage. Similarly, tenosynovectomy is most
reliable when performed on tendons with little or no under-
lying tendinosis.
SUMMARY
Foot and ankle pain is a prevalent and potentially debili-
tating problem for many patients with and without
inflammatory arthritis. A proper history and physical
examination, performed in conjunction with appropri-
ate imaging modalities, are essential for establishing an
­accurate diagnosis. Conceptualizing the various causes of
foot and ankle pain according to anatomic location nar-
rows the differential diagnosis and allows the clinician
to take a categorical approach to the diagnostic process.
Nonoperative modalities, such as medications, bracing,
­physical therapy, orthoses, and shoewear modification,
relieve pain and maintain function for many individu-
als. For ­ recalcitrant symptoms, substantial relief may be
afforded by ­surgical intervention.
REFERENCES
  1. Vanio E: Rheumatoid foot: Clinical study with pathological and
roentgenological comments. Ann Chir Gynaecol Fenniae 45(S):
1-107, 1956.
  2. Flemming A, Crown JM, Corbett M: Early rheumatoid disease, I:
Onset. Ann Rheum Dis 35:357-360, 1976.
  3. Hoppenfeld S: Physical Examination of the Spine and Extremities.
Norwalk, Conn, Appleton & Lange, 1976.
  4. Boutry N, Flipo RM, Cotten A: MR imaging appearance of rheu-
matoid arthritis in the foot. Semin Musculoskelet Radiol 9:199-209,
2005.
  5. Seltzer SE, Weismann BN, Braunstein EM, et al: Computed tomog-
raphy of the hindfoot with rheumatoid arthritis. Arthritis Rheum
28:1234-1242, 1985.
  6. Groshar D, Gorenberg M, Ben-Haim S, et al: Lower extremity scintig-
raphy: The foot and ankle. Semin Nucl Med 28:62-77, 1998.
  7. Riente L, Delle Sedie A, Iagnocco A, et al: Ultrasound imaging for the
rheumatologist, V: Ultrasonography of the ankle and foot. Clin Exp
Rheumatol 24:493-498, 2006.
  8. Khoury NK, el-Khoury GY, Saltzman CL, et al: Intraarticular foot and
ankle injections to identify source of pain before arthrodesis. AJR Am
J Roentgenol 167:669-673, 1996.
  9. Vidigal E, Jacoby RK, Dixon AS, et al: The foot in chronic rheuma-
toid arthritis. Ann Rheum Dis 34:292-297, 1975.
 650 PRICE  |  Foot and Ankle Pain
10. Jaakkola JI, Mann RA: A review of rheumatoid arthritis affecting
the foot and ankle. Foot Ankle Int 25:866-874, 2004.
11. Spiegel TM, Spiegel JS: Rheumatoid arthritis in the foot and ankle—
diagnosis, pathology and treatment. Foot Ankle 2:318-324, 1982.
12. Matteson EL: Current treatment strategies for rheumatoid arthritis.
Mayo Clin Proc 75:69-74, 2000.
13. Conn DL, Lim SS: New role for an old friend: Prednisone is a disease-
modifying agent in early rheumatoid arthritis. Curr Opin Rheumatol
15:192-196, 2003.
14. Mohan AK, Cote TR, Siegel JN, et al: Infectious complications of
biologic treatment of rheumatoid arthritis. Curr Opin Rheumatol 15:
179-184, 2003.
15. Coursin DB, Wood KE: Corticosteroid supplementation for adrenal
insufficiency. JAMA 287:236-240, 2002.
16. Clark H, Rome K, Plant M, et al: A critical review of foot orthoses in
the rheumatoid arthritic foot. Rheumatology 45:239-245, 2006.
17. Woodburn J, Barker S, Helliwell PS: A randomised controlled trial
of foot orthoses in rheumatoid arthritis. J Rheumatol 29:1377-
1383, 2002.
18. Magalhaes E, Davitt M, Filho DJ, et al: The effect of foot orthoses in
rheumatoid arthritis. Rheumatology 45:449-453, 2006.
19. Hugate R, Pennypacker J, Saunders M, et al: The effects of intratendi-
nous and retrocalcaneal intrabursal injections of corticosteroid on the
biomechanical properties of rabbit Achilles tendons. J Bone Joint Surg
Am 86:794-801, 2004.
20. Chiodo CP, Martin T, Wilson MG: A technique for isolated arthro­
desis for inflammatory arthritis of the talonavicular joint. Foot Ankle
Int 21:307-310, 2000.
21. Astion DJ, Deland JT, Otis JC, et al: Motion of the hindfoot after
simulated arthrodesis. J Bone Joint Surg Am 79:241-246, 1997.
22. Coughlin M: Rheumatoid forefoot reconstruction: A long term fol-
lowup study. J Bone Joint Surg Am 82:322-341, 2000.
23. Coester LM, Saltzman CL, Leupold J, et al: Long-term results fol-
lowing ankle arthrodesis for post-traumatic arthritis. J Bone Joint
Surg Am 83:219-228, 2001.
24. Thomas R, Daniels TR, Parker K: Gait analysis and functional out-
comes following ankle arthrodesis for isolated ankle arthritis. J Bone
Joint Surg Am 88:526-535, 2006.
25. Deheer PA: The case against first metatarsal phalangeal joint implant
arthroplasty. Clin Podiatr Med Surg 23:709-723, 2006.
26. Bommireddy R, Singh SK, Sharma P, et al: Long term followup of
Silastic joint replacement of the first metatarsophalangeal joint. Foot
12:151-155, 2003.
27. Shankar NS: Silastic single-stem implants in the treatment of hallus
rigidus. Foot Ankle Int 16:487-491, 1995.
28. Hattrup SJ, Johnson KA: Subjective results of hallux rigidus treatment
with cheilectomy. Clin Orthop 226:182-191, 1988.
29. Aho H, Halonen P: Synovectomy of the MTP joints in rheumatoid
arthritis. Acta Orthop Scand Suppl 243:1, 1991.
 44
KEY POINTS
Patients with carpal tunnel syndrome typically present with
nocturnal paresthesias associated with intermittent pain or
paresthesia during the day.
Ganglia are mucin-filled cysts arising from joint capsules
or tendon sheaths. If they are particularly symptomatic,
­corticosteroid injections may be tried, but surgical excision
may be necessary to effect a cure.
Tendinitis of the extensor pollicis longus tendon can be
­particularly dangerous because of the risk of tendon rupture.
DeQuervain’s disease, inflammation of the extensor pollicis
brevis and abductor pollicis longus tendons in the first dorsal
extensor compartment, is common in women and is associated
with repetitive hand activities, such as caring for an infant.
Painful osteoarthritis involving the carpometacarpal joint of
the thumb usually can be treated successfully by splinting.
Trigger fingers, caused by thickening of the A1 retinacular
pulley in the palm, usually can be treated by corticosteroid
injections and splinting.
The multiple functions that the hand performs in daily life
are usually taken for granted until they become affected by
disease or injury. Depending on the nature of the disorder,
patients have different capacities to adapt. Patients present-
ing with pain and dysfunction of the hand or wrist or both
represent a wide spectrum, diverse in age, occupations, and
avocations. These patients have a broad range of medical
conditions that may or may not be related to their current
problem. Each patient has a different story to tell about his
or her hand and wrist and why he or she is seeking treat-
ment. It is up to the clinician to sort out these various fac-
tors, some of which may seem confounding, and determine
the most appropriate diagnosis and course of treatment.
This chapter presents guidelines that are useful in the
evaluation of patients presenting with hand and wrist pain.
Complete coverage of all of the various conditions that can
affect the hand and wrist is beyond the scope of this chapter.
Instead, the conditions discussed include the most common
pathologies seen by general practitioners and hand sur-
geons. The conditions are grouped by their anatomic area
to include pain localized to the volar, dorsal, radial, or ulnar
wrist; the base of the thumb; and the palm and digits.
PATIENT EVALUATION
ANATOMY
The complex anatomy of the hand and wrist involves
many structures interacting in close proximity to one
another. ­ Several different diagnoses can manifest with
Hand and Wrist Pain
CARRIE R. SWIGART
similar symptom patterns despite varying pathologies. A
precise knowledge of the anatomy of the hand and wrist
often eliminates several diagnostic considerations on the
basis of the physical examination alone. The history of the
illness and the examination also help to narrow further
investigation by enabling the physician to choose appro-
priate additional diagnostic tests better. Several common
sites of pain in the hand and wrist and their corresponding
leading diagnoses are illustrated in Figure 44-1. Pain in one
location can have multiple etiologies depending on the
patient profile and the history of the problem. A thorough
review of the pertinent regional ­anatomy is important to
help differentiate successfully the many possible causes of
hand and wrist pain.
HISTORY
Important patient factors include age, sex, hand dominance,
occupation, and hobbies or sports. When determining the
history of the problem, a history of recent or distant trauma
should be sought, and an estimation of the severity of the
trauma should be noted. Next, questions about the dura-
tion and frequency of the pain and the intensity and quality
should be addressed. The pain of degenerative arthritis is
often described as a localized “toothache”-type pain, which
is always present at a low level and increases with activity,
whereas the pain of tendinitis may be sharp, poorly localized,
and present only with activity. Rheumatoid arthritis mani-
fests initially with hand and wrist involvement in 25% of
patients and is characterized by joint effusion, with bilateral
hand and wrist involvement and morning stiffness. Night-
time symptoms of a burning-type pain in the hand and wrist
that are exacerbated by arm position are often associated
with nerve entrapment syndromes. Specific activities that
either cause pain or alleviate it also should be noted. Arthri-
tis at the base of the thumb, or first carpometacarpal joint, is
often aggravated by such activities as opening jars, turning
doorknobs, and doing needlework or other hobbies.
PHYSICAL EXAMINATION
A thorough examination of the involved extremity and com-
parison with the uninvolved extremity are essential. Atten-
tion should be paid to abnormalities of the more proximal
joints of the elbow and shoulder and the cervical spine. As
the differential diagnosis narrows, the examination should
be tailored as needed to include or eliminate any possible
systemic etiologies. As with other musculoskeletal exami-
nations, a complete record of the range of motion of the
involved joints and comparison measurements of the oppo-
site side should be made. Any difference between active and
651
 652 swigart  |  Hand and Wrist Pain
Carpal tunnel syndrome
Ulnar nerve entrapment
FCR/FCU tendinitis
Hamate fracture
TFCC injury/ulnar impaction syndrome
ECU tendinopathy
Lunotriquetral ligament injury
Pisotriquetral arthritis
A this chapter.
B Advances in ultrasound and MRI technology have
Ganglion
Carpal boss
Extensor tendinopathies
..
Kienbock’s disease
Scapholunate interosseous ligament injury
Gout and inflammatory arthritis
DeQuervain’s disease and intersection syndrome
Basal joint pathology
Volar ganglia
Scaphoid fracture/nonunion
Figure 44-1  A, Palmar and ulnar view of the hand and wrist with areas
of pain and tenderness marked with their corresponding leading differ-
ential diagnoses. B, Dorsal and radial view of the hand and wrist with
areas of pain and tenderness marked with their corresponding leading
differential diagnoses. ECU, extensor carpi ulnaris; FCR/FCU, flexor carpi
radialis/flexor carpi ulnaris; TFCC, triangular fibrocartilage complex.
passive motion should be noted. Careful palpation for the
site of maximal tenderness is important in differentiating
the source of pain and is particularly important when try-
ing to exclude possible factors of secondary gain. Measure-
ments of grip and pinch strength also are helpful in many
situations as a diagnostic aid and a baseline measurement to
follow for improvement. Many provocative maneuvers are
useful in differentiating etiologies; these are discussed with
the specific pathology with which they are associated.
IMAGING STUDIES
Technologic advances have increased the availability of
imaging studies for the hand and wrist. Improvements in
magnetic resonance imaging (MRI) resolution using small
joint coils allow more precise imaging of small structures in
the hand and wrist. Advancements in ultrasound technol-
ogy have allowed this tool to be increasingly used in the
diagnosis of musculoskeletal complaints. With the multitude
of ancillary studies available, it is important to be selective
in using these to establish or refute diagnostic possibilities.
In this era of cost containment, imaging studies should be
used most often to confirm a diagnosis rather than to find
one. An understanding of the advantages and limitations of
each study is necessary to enable using them to their fullest
potential.
Plain radiographs are the easiest and most readily avail-
able study that can be obtained in most offices. A routine
hand or wrist series, including anteroposterior, lateral, and
oblique views, is a useful screening tool, but often lacks the
specificity required. Depending on the suspected diagnosis,
there are many available special views. These are discussed
with the specific diagnoses to which they pertain later in
If further detail of the bony anatomy is required, com-
puted tomography (CT) is the best available tool today. The
most common uses for CT in the hand and wrist include
evaluation of intra-articular fractures of the distal radius and
metacarpals, scaphoid fractures and nonunions, and intraos-
seous cysts or tumors.1,2
enhanced the ability to evaluate the soft tissue structures of
the hand and wrist. Smaller ultrasound probes with higher
resolution have made it possible to visualize and differen-
tiate structures such as flexor tendons, ganglion cysts, and
ligaments. Doppler ultrasound can help to differentiate vas-
cular disorders of the hand. MRI technology is constantly
improving and allowing for new uses in the hand and wrist.
By altering the parameters of this test, information about
anatomy and physiology can be obtained.3 Specific uses of
these tests and others such as arthrography and bone scans
are addressed with the diagnoses for which they are most
useful.
ADDITIONAL DIAGNOSTIC TESTS
Neurodiagnostic Tests
Neurodiagnostic tests, including nerve conduction studies
and electromyography, are useful in the diagnosis of sus-
pected neurologic disorders of the upper extremity. Specify-
ing the type and nature of examination required enhances
the information gained by these studies. If a nerve compres-
sion syndrome such as carpal or cubital tunnel syndrome is
suspected, nerve conduction studies may be sufficient with-
out the added cost and patient discomfort of formal electro-
myography testing. Nerve conduction studies evaluate the
speed of conduction of motor and sensory nerves across a set
distance at a specific location and compare this with estab-
lished normal values. A decrease in the speed of nerve con-
duction, as evidenced by an increase in the latency, is seen
with localized nerve compression and is shown in several
different nerves concomitantly in demyelinating diseases,
such as multiple sclerosis. When more severe nerve inju-
ries are suspected, or if there is clinical evidence of muscle
weakness or atrophy, an electromyogram can be useful to
delineate better the extent of the process or rule out a myo-
pathic process.4
Injections and Aspirations
The use of injections and aspirations can be therapeutic
and diagnostic. A so-called lidocaine challenge can be used
to discriminate between different diagnoses when placed
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
precisely in one joint or painful area. Corticosteroids can be
given selectively in conjunction with the local anesthetic
for more lasting relief and in some cases can be curative.5-11
Some of the most common sites for injection are the A1
pulley region of the finger for trigger finger, the carpal canal
for carpal tunnel syndrome, and the first dorsal compart-
ment of the wrist for deQuervain’s disease.
Aspiration of joints or other fluid collections such as
ganglia can yield vital diagnostic information and can be
therapeutic. If infection is suspected, aspiration should be
used to obtain a sample of joint fluid for Gram stain, cell
count, and culture. Diagnoses such as gout and pseudogout
can be confirmed by crystal analysis under polarized light.
Many ganglia and retinacular cysts can be treated temporar-
ily or permanently with simple aspiration.12,13
Arthroscopy
Direct visualization of a joint via arthroscopy can be an
invaluable diagnostic tool. Despite the increasing sensitiv-
ity of imaging techniques such as MRI, arthroscopy provides
a dynamic evaluation that static imaging cannot provide.14
Since the first published report of a series of cases by Roth
and colleagues in 1988,15 it has become the “gold standard”
for evaluation of chronic wrist pain.16-18 With new surgi-
cal techniques being developed, surgeons often can pro-
ceed directly to the definitive treatment using arthroscopy
entirely or in part.19-24
COMMON ETIOLOGIES FOR HAND
AND WRIST PAIN
WRIST PAIN—PALMAR
Carpal Tunnel Syndrome
Carpal tunnel syndrome is the most commonly diagnosed
compression neuropathy in the upper extremity. It usually
occurs as an isolated phenomenon, but symptoms of carpal
tunnel syndrome can accompany many systemic diseases,
such as congestive heart failure, multiple myeloma, and
tuberculosis.25-28 More commonly, carpal tunnel syndrome
is associated with conditions such as pregnancy, diabetes,
obesity, rheumatoid arthritis, and gout.29-39
The classic constellation of symptoms consists of weak-
ness or clumsiness of the hand; paresthesias or hypesthesias
in the thumb, index, and long fingers; and nocturnal pares-
thesias in the affected digits. Patients often may complain of
forearm and elbow pain that is aggravated by activities, but
is poorly localized and aching in nature. Occasionally, more
proximal symptoms, such as shoulder pain, are the main pre-
senting complaint.40 Past reports have indicated a 3:1 preva-
lence of carpal tunnel syndrome in women. Approximately
half of patients are 40 to 60 years old, although carpal tunnel
syndrome occasionally has been diagnosed in children.41,42
The diagnosis of carpal tunnel syndrome is usually clini-
cal. Tinel’s sign, shown by radiating paresthesias in the
median nerve distribution with gentle percussion over the
volar wrist, indicates nerve irritation. Reproduction of
symptoms with wrist flexion, as described by Phalen,43 and
with the carpal compression test, as described by Durkan,44
has been shown to be more specific.45 Decreased sensibility
and thenar atrophy are late signs seen in advanced median
653
nerve entrapment. Bilateral electrodiagnostic tests, spe-
cifically nerve conduction velocity testing, should be used
to confirm the diagnosis, particularly in patients claiming
a compensable injury or in patients with atypical signs or
symptoms. Prolonged motor and sensory latencies across the
carpal canal confirm pathologic compression of the median
nerve.46-48 In patients with classic clinical findings, a study
found that carpal tunnel syndrome could be diagnosed with
a high degree of accuracy on clinical grounds alone, and
that the addition of electrodiagnostic tests did not increase
the accuracy.49 When attempting to differentiate carpal tun-
nel syndrome from more proximal nerve entrapments such
as cervical root compression or thoracic outlet syndrome,
the addition of electromyography of the cervical paraspinal
muscles and proximal conduction tests (H reflex, f waves)
can be useful.50
Conservative treatment for carpal tunnel syndrome
consists of splinting of the wrist in neutral position and
consideration of oral nonsteroidal anti-inflammatory drugs
(NSAIDs) for pain control. Splinting should be used spar-
ingly during the workday to prevent secondary muscle
weakness and fatigue, but is best prescribed to prevent pro-
vocative wrist positioning at night. The splint should not
hold the wrist in extension beyond 10 degrees. Although
splinting may be beneficial for relief of symptoms in cases
of mild compression, its long-term effectiveness is limited.51
The use of vitamin B6 (100 to 200 mg/day) has been helpful
in some cases, but its efficacy has not been confirmed in a
randomized trial. The popularity of injections of corticoste-
roid in the treatment of carpal tunnel syndrome has waxed
and waned over the last half century. Although it has been
shown to be quite effective in the short-term, the long-term
efficacy is mixed.52-54 Also, injections have been associated
with exacerbation of the condition and permanent median
nerve injury if performed incorrectly.55,56 For these reasons,
injections are most often indicated in cases when the condi-
tion is thought to be temporary, such as with pregnancy, or
if surgery has to be deferred because of a medical condition
or major life event.
Surgical release is indicated for patients with confirmed
carpal tunnel syndrome who have failed a course of conser-
vative treatment. In patients who exhibit late findings of
objective sensory loss or thenar atrophy, early surgery should
be recommended.
Ulnar Nerve Entrapment—Cubital Tunnel Syndrome
Entrapment of the ulnar nerve as it passes through the cubital
tunnel just posterior to the medial epicondyle of the elbow
can manifest with symptoms localized to the ulnar border
of the hand. There also may be associated medial forearm
pain and irritability of the ulnar nerve at the elbow. Present-
ing symptoms usually consist of paresthesias or numbness or
both in the small and ring fingers. Percussion of the nerve in
the cubital tunnel elicits Tinel’s sign. Prolonged elbow flex-
ion reproduces the symptoms. In contrast to carpal tunnel
syndrome, it is not unusual for patients to present with early
atrophy of the intrinsics, most easily appreciated in the first
dorsal interosseous muscle.
Electrodiagnostic studies can help to confirm the diag-
nosis and differentiate cubital tunnel syndrome from more
distal compression of the ulnar nerve in Guyon’s canal
 654 swigart  |  Hand and Wrist Pain

(see later). If malalignment of the elbow is present, or the calcific tendinitis and is most commonly seen around the
patient relates a history of childhood trauma, radiographs flexor carpi ulnaris tendon.70,71
should be obtained to rule out a supracondylar or epicondy-
lar malunion. So-called tardy ulnar nerve palsy can develop Hamate Fracture
years after a supracondylar fracture of the elbow.57
Conservative treatment includes strategies to help the An uncommon and underdiagnosed etiology of palmar pain
patient avoid having the elbow flexed for prolonged periods, in young, active individuals is a fracture of the hook of the
particularly at night. Soft, or semirigid, elbow splints prevent hamate. These fractures can occur from a fall on an extended
elbow flexion beyond 50 to 70 degrees. Medial elbow pads wrist, a “dubbed” golf shot, or from forcefully striking a ball
can be used if the patient’s job or hobbies requires resting with a club or bat. Plain radiographs of the wrist are usually
the medial elbow on a hard surface. NSAIDs can be benefi- read as normal. The condition should be established and
cial in acute or traumatic cases. Surgical decompression of treated expeditiously because it may lead to ulnar nerve
the nerve is indicated if a patient fails to obtain relief from entrapment, ulnar artery thrombosis, or rupture of flexor
splinting and activity modification, or if there is clinical or tendons.72 Pain in the base of the palm overlying the hamate
electrodiagnostic evidence of muscle denervation. is the most common presenting symptom. Often, the pain
is present only with the activity that caused the fracture,
such as driving a golf ball or swinging a bat. Because of the
Ulnar Nerve Entrapment—Guyon’s Canal
proximity of the ulnar nerve, patients also can have sensory
In 1861, Guyon58 published a description of the contents of and motor symptoms of distal ulnar neuropathy. Occasion-
an anatomic canal at the wrist. The distal branches of the ally, in the acute setting, vascular complaints, such as cold
ulnar nerve and the ulnar artery pass through this space. As intolerance or frank ischemia, from ulnar artery thrombosis
it exits the canal, the ulnar nerve divides into its sensory can be the presenting condition.
and motor branches. Compression of the nerve within or A carpal tunnel view, obtained with the wrist in a hyper-
proximal to the canal usually manifests with a combination extended position, may show the fracture (Fig. 44-2A).
of sensory and motor symptoms in the ulnar nerve distribu- Alternatively, a selective CT scan through the hamate is a
tion. Patients complain of numbness and paresthesias of the more accurate way to confirm the diagnosis (Fig. 44-2B).73
palmar aspect of the ring and small fingers. Motor symptoms If diagnosed within 2 to 3 weeks of injury, casting should
usually are described as a cramping weakness with grasping be attempted to allow the fracture to heal.74 If this fails, or
and pinching. As with median neuropathy, atrophy of the if the fracture is diagnosed late, surgical treatment is indi-
intrinsics and objective sensory loss are late findings. cated, and most authors favor excision of the hook followed
In contrast to carpal tunnel syndrome, in which patients by a gradual return to activities.75-78
usually have an ill-defined onset of symptoms, ulnar nerve
compression in the canal of Guyon is often of more acute
onset. It can be associated with repeated blunt trauma,59,61
a fracture of the hamate or the metacarpal bases, or occa-
sionally a fracture of the distal radius.62,63 Space-occupying
lesions, such as a ganglion, lipoma, or anomalous muscle,
also can cause compression.64-68 Because of the difference
in etiology, this nerve entrapment syndrome is often not
amenable to conservative treatment. If there is an anatomic
lesion, such as a fracture or a mass, this must be addressed.
If repetitive blunt trauma is the cause, without associated
fracture or arterial thrombosis, splinting and activity modi-
fication can alleviate the symptoms.
A
Flexor Carpi Radialis and Flexor Carpi Ulnaris
Tendinitis
Similar to other tendinopathies around the wrist, irritation
of the wrist flexors occurs with stress of the wrist in a par-
ticular position. Activities that require forced wrist flexion
for prolonged periods or with repetition put patients at risk
for inflammation around the flexor carpi radialis tendon69
or the flexor carpi ulnaris tendon or both. The condition
manifests with tenderness along the course of the tendon,
especially near its insertion. Wrist flexion against resistance
with radial or ulnar deviation reproduces the symptoms.
Treatment consists of splinting and rest, elimination of
activities that cause pain, and oral NSAIDs. Injection of B
corticosteroid into the flexor carpi radialis or flexor carpi Figure 44-2  A, Carpal tunnel view radiograph showing a hamate hook
ulnaris sheath may be curative. Sharp pain, associated with fracture (arrow). B, Coronal CT scan showing the same hamate hook
an intense inflammatory localized reaction, is suggestive of ­fracture.
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
WRIST PAIN—DORSAL
Ganglion
Ganglia account for 50% to 70% of all soft tissue tumors of
the hand and wrist. Of these, 60% to 70% occur around the
dorsal wrist. These mucin-filled cysts usually arise from an
adjacent joint capsule or tendon sheath. The most common
site of origin is the scapholunate ligament, and the main
body of the cyst may be located elsewhere on the dorsum
of the wrist and attached to this ligament by a long pedicle.
Although most ganglia occur as a well-circumscribed and
obvious soft mass, some are subtler and are evident only with
the wrist in marked volar flexion. As a result of their char-
acteristic appearance, ganglia are not often misdiagnosed,
but should be differentiated from the less well-demarcated
swelling of extensor tenosynovitis, lipomas, and other hand
tumors. Plain radiographs are usually normal, but occasion-
ally show an intraosseous cyst or an osteoarthritic joint.
Some ganglia may not be clinically apparent and are known
as “occult” ganglia. Ultrasound and MRI have been shown
to be useful in the diagnosis of these ganglia.79,80
Not all ganglia are painful, and the tendency is for smaller
ganglia to be more painful. Patients may present with com-
plaints of wrist weakness or simply because of the cosmetic
appearance of the cyst. In approximately 10% of cases, there
is evidence of associated trauma to the wrist. The ganglia
may appear suddenly or develop over many months. Inter-
mittent complete resorption followed by reappearance
months or years later is common.
Most conservative measures, such as splinting and rest,
have only a temporary effect on ganglia. They tend to
diminish in size with rest and enlarge with increased activity.
Spontaneous rupture is common, and at one time attempt-
ing to rupture the cyst with a heavy object, such as a large
book, was recommended as treatment. Aspiration can be
performed, but has mixed results because of the thick gelati-
nous nature of the fluid within the cyst. Even if adequate
decompression of the cyst can be achieved, reaccumulation
of the fluid usually occurs. Aspiration in conjunction with
irrigation or injection of corticosteroids can be effective in
alleviating the symptoms for varying periods of time.12,13,81
Occasionally, a ganglion can become so large that it
can interfere with the function of the wrist by limiting the
motion, especially in extension. Pressure of the mass on the
terminal branches of the posterior interosseous nerve may
be painful. Excision is generally curative, but may result in
short-term stiffness and some loss of terminal flexion sec-
ondary to surgical scarring. Occasionally, a patient desires
excision of the cyst for cosmetic reasons. With proper exci-
sion, recurrence is less than 10%,82-84 but if the dissection is
incomplete and fails to identify the origin of the cyst, recur-
rence rates can be 50%. Arthroscopic resection has been
shown to be a safe and effective method of treating dorsal
wrist ganglia.23,24
Carpal Boss
Often confused with a dorsal ganglion, the carpal boss is a
bony, nonmobile prominence on the dorsum of the wrist.
It is an osteoarthritic spur that forms at the second or third
carpometacarpal joints.85 The boss is most evident with the
wrist in volar flexion. Patients usually present with pain and
655
localized tenderness over the prominence. The condition is
twice as common in women as in men, and most patients are
in their 20s to 30s. It is not unusual for a small ganglion to
be associated with the boss. Radiographs are best taken with
the hand and wrist in 30 to 40 degrees of supination and 20
to 30 degrees of ulnar deviation to put the bony prominence
on profile (the “carpal boss view”).86 Conservative treatment
consists of rest, immobilization, NSAIDs, and occasionally
injection with corticosteroids. If persistently painful despite
these measures, surgical excision of the boss may be neces-
sary, but is associated with a prolonged recovery and contin-
ued symptoms in a high percentage of patients.
Extensor Tendinopathies
The extensor pollicis longus (EPL) tendon can be irritated
as it passes around Lister’s tubercle. This condition, in con-
trast to other tendinopathies around the wrist, carries a
significant risk of tendon rupture. Early diagnosis and some-
times urgent operative treatment are necessary to prevent
this complication. Localized pain, swelling, and tenderness
are the hallmarks of this condition, and similar to other ten-
dinopathies, initial treatment consists of decreased activ-
ity and splinting. A short course of oral anti-inflammatory
medication can be useful in decreasing symptoms. Diagnos-
tic injections with lidocaine can help to differentiate the
condition from other causes of wrist pain, but corticosteroid
injections are not routinely used in this condition because
of a propensity for the EPL to rupture in chronic cases.
A patient commonly may present with a rupture of the
EPL without antecedent pain or swelling. There is a well-
known association of EPL rupture with fractures of the dis-
tal radius that likely occurs owing to a relative “watershed
zone” of vascular supply within its tight retinacular sheath.
Tendon rupture most often occurs with minimally displaced
or nondisplaced fractures and can occur several weeks or
months after the original injury.87-90 Individuals with rheu-
matoid arthritis and systemic lupus erythematosus are espe-
cially prone to rupture of the EPL and other tendons.
Kienböck’s Disease
Kienböck’s disease is so named for Kienböck,91 who first
described in 1910 what he postulated were avascular changes
in the lunate. Nearly a century later, the cause of this disease
remains unclear; it is likely multifactorial. Kienböck’s dis-
ease should be suspected when a young adult presents with
pain and stiffness of the wrist and swelling and tenderness
around the region of the dorsal lunate. There is an increased
propensity of the disease among patients with an ulna that is
anatomically shorter than the radius (so-called ulnar nega-
tive variance). Radiographs are needed to confirm and stage
the process. Kienböck’s disease is staged by the degree of
fragmentation and collapse of the lunate, associated osteoar-
thritis, and carpal collapse in a system originally proposed by
Stahl.92 In this system, the earliest sign of the disease is a lin-
ear or compression fracture in the lunate. Later stages show
sclerosis of the lunate, followed by lunate collapse and a loss
of carpal height. In the final stage, the carpus shows signs of
diffuse osteoarthritis with complete collapse and fragmenta-
tion of the lunate (Fig. 44-3). With the increased sensitivity
of MRI, it is possible to identify avascular changes within
 656 swigart  |  Hand and Wrist Pain
Figure 44-3  Advanced Kienböck’s disease,
showing carpal collapse, intercarpal and ra-
diocarpal arthrosis, and fragmentation of the A B
lunate. A, Posteroanterior view. B, Lateral view.
the lunate before they become evident on plain radiographs.
This is referred to as “stage zero” Kienböck’s disease.
The treatment for Kienböck’s disease is largely surgical.
Depending on the stage of the disease and the postulated
etiology, several surgical procedures have been described. In
early stages of the disease, when there is little lunate collapse
and no osteoarthritis, the goal of surgery is to “unload” the
lunate by redistributing articular contact forces and allow it
to revascularize.93-96 The most common procedure is a radial
shortening osteotomy, performed to neutralize ulnar variance.
In later stages, various intercarpal arthrodeses have been used
to readjust and maintain carpal height and alignment.97-99
Microsurgical techniques have been used more recently to
revascularize the lunate with promising early results.100
Scapholunate Interosseous Ligament Injury
The interosseous ligament between the scaphoid and the
lunate is a stout structure, especially dorsally, and usually
requires a significant force to cause disruption. The typical
mechanism of injury is a fall onto the outstretched hand
with the wrist extended. Early diagnosis is essential to pre-
vent the late sequelae of carpal collapse. The key radio-
graphic features of scapholunate dissociation (scapholunate
interval widening) are shown in Figure 44-4. The antero-
posterior view shows the scapholunate interval better than
the posteroanterior view.101 Early surgical intervention is
recommended with the goal of maintaining carpal align-
ment and prevention of an otherwise inevitable progression
to carpal collapse and degenerative arthritis.
Gout and Inflammatory Arthritis
All of the inflammatory arthritides, including the crystalline
arthropathies, can manifest as dorsal wrist pain. Approxi-
mately 25% of patients with a diagnosis of rheumatoid
arthritis present initially with hand and wrist symptoms. The
reader is referred to Chapters 87 and 88 for further details.
WRIST PAIN—ULNAR
Triangular Fibrocartilage Complex Injury
and Ulnocarpal Impaction Syndrome
One of the most complex and confusing areas of the wrist
from a diagnostic standpoint is the articulation of the ulna
with the carpus. The triangular fibrocartilage complex
(TFCC), so named by Palmer and Werner,102 comprises the
articular disk itself and the immediately surrounding ulno-
carpal ligaments. It can be injured by a variety of acute and
chronic mechanisms. Hyperpronation and hypersupination
of the carpus during forceful activities are the usual causes of
acute injuries, whereas repetitive pronation and supination
more often cause attritional changes in the TFCC. Careful
physical examination is important to determine the origin
of the pain and to try to discover the maneuver or wrist posi-
tion that most closely reproduces the symptoms.
The radius and ulna must remain congruent through
a 190-degree arc.103 Limitation of motion and pain with
pronation and supination are consistent with a tear of the
supporting ligaments and resultant distal radioulnar joint
(DRUJ) instability. If a sufficient portion of the stability has
been lost, the ulna appears clinically dislocated or sublux-
ated, and there is severe limitation of forearm rotation. Lat-
eral radiographs of the wrist in neutral and full pronation
and supination are not generally specific enough to confirm
ulnar subluxation. To evaluate better the congruency of the
DRUJ through its range of motion, and to assess for subtle
subluxations, CT can be performed on both wrists simulta-
neously in positions of neutral, full pronation, and full supi-
nation.104-107
Tears of the TFCC may manifest with painful clicking
during wrist rotation. Patients generally have localized ten-
derness on the midaxial border of the wrist and directly
beneath the extensor carpi ulnaris tendon. If forced ulnar
deviation of the wrist or gripping or both reproduce the
patient’s symptoms, a degenerative tear of the central por-
tion of the TFCC is more likely. The degenerative tear is
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
Figure 44-4  Anteroposterior radiograph of the wrist showing scaph-
olunate interosseous space widening, scaphoid foreshortening, and the
cortical ring sign associated with scapholunate interosseous ligament
disruption.
frequently a component of the ulnocarpal impaction syn-
drome, a condition associated with higher than normal
loads on the ulnar carpus secondary to a congenitally posi-
tive ulnar variance.
Plain radiographs are most useful in determining ulnar
variance and for ruling out fractures or arthritis as a cause
of ulnar wrist pain. Because of the variable relationship of
the radius and ulna depending on forearm rotation, it is
important to take standardized films when measuring ulnar
variance.108,109 A posteroanterior view of the wrist with the
shoulder abducted to 90 degrees and the elbow flexed to 90
degrees shows the DRUJ in neutral forearm rotation and is
easily reproducible (Fig. 44-5). Because the ulna lengthens
relative to the radius during power grip, a radiograph in the
same position during maximal grip best shows impaction of
the ulna on the carpus.
Ancillary studies for TFCC tears include three-compart-
mental arthrography and MRI. In arthrography, sequential
injections of radiopaque dye are performed into the carpal
joint, midcarpal joint, and DRUJ. The test is considered
positive when the dye is seen leaking from one compartment
to another. The site of the leak determines the location of
the torn structure.110 Several studies have shown, however,
that there are age-related attritional tears, which occur in
the TFCC and other ligamentous structures of the wrist.111-113
Technologic advancements in MRI have improved the abil-
ity to visualize and diagnose abnormalities in the TFCC.
MRI can be combined with arthrography in an MRI arthro-
gram to visualize better the TFCC and the intrinsic wrist
ligaments. Peripheral detachments and central degenerative
tears of the TFCC can be visualized. MRI remains highly
operator-dependent and technique-dependent, and the
studies should be interpreted in the context of the findings
on physical examination.114
Patients presenting with pain localized to the ulnar side
of the wrist often respond to simple splinting and rest. This
conservative treatment and NSAIDs can be used effectively
657
Figure 44-5  Posteroanterior radiograph of the wrist in neutral forearm
rotation showing the method of measuring ulnar variance by drawing
tangential lines to the distal ulna and distal radius. The space between
these lines in millimeters is the ulnar variance. A positive value indicates
the ulnar length is greater than the radial length.
while a workup is in progress. A course of rest and splinting,
followed by a gradual return to activities, may completely
alleviate ulnar-sided symptoms.
Despite the advancements in imaging techniques, there
is often no substitute for direct visualization of the ulno-
carpal joint or DRUJ or both. Arthroscopy has become an
invaluable diagnostic and surgical tool. Tears of the TFCC
can be visualized and their clinical significance better deter-
mined. Arthroscopy, done in conjunction with fluoroscopy,
can assess for instability of the DRUJ or intercarpal joints
or both. Several surgical procedures can now be performed
entirely or in part through the arthroscope.115,116
Extensor Carpi Ulnaris Tendinitis and Subluxation
The extensor carpi ulnaris tendon can become irritated with
forced pronation/supination activities, such as putting top-
spin on a tennis ball. In severe cases, the tendon can begin
to sublux around the ulnar head as its restraining dorsal
retinaculum becomes increasingly lax. Patients complain
of pain with forceful rotation of the forearm, and some-
times there is an associated snapping of the extensor carpi
ulnaris tendon. Early treatment consists of immobilization
of the wrist and forearm to prevent rotation. Anti-inflam-
matory medication can help to decrease the inflammation
more quickly. After an adequate period of rest, if the acute
inflammation resolves, but the extensor carpi ulnaris tendon
continues to be unstable, surgery may be indicated to recon-
struct or release the sheath at the wrist.
Lunotriquetral Ligament Injury
Tears in the short, stout intraosseous ligament connecting
the lunate and the triquetrum are uncommon and often dif-
ficult to diagnose. As with the aforementioned diagnoses,
patients present with ulnar-sided wrist pain usually wors-
ened by either pronation or supination. Forceful translation
 658 swigart  |  Hand and Wrist Pain
of the triquetrum against the lunate causes pain in affected
individuals. If diagnosed within 3 to 4 weeks of injury, a
short arm cast allows healing and eliminates symptoms.
Chronic tears may lead to advanced carpal instability and
collapse. MRI or wrist arthroscopy or both may be necessary
to make the diagnosis. Treatment is predicated on the stag-
ing of instability and ranges from simple casting for acute
instability to ligament reconstruction or intercarpal fusion
for more advanced cases.
Pisotriquetral Arthritis
Degenerative changes in the pisotriquetral articulation usu-
ally are post-traumatic in nature. Patients may recall a fall
onto the extended wrist with direct trauma to the ulnar
side of the palm. Affected patients present with pain during
passive wrist hyperextension and exacerbation with flex-
ion against resistance. With palpation of the pisotriquetral
joint, there is tenderness and often crepitus. As with many
joints, splinting, NSAIDs, and occasionally injection with
corticosteroid and lidocaine are the mainstays of conserva-
tive treatment. If this is inadequate to control the symp-
toms, surgical resection of the pisiform is indicated.
WRIST PAIN—RADIAL AND BASE OF THUMB
de Quervain’s Disease and Intersection Syndrome
One of the most common sites of tendon irritation around
the wrist is in the first dorsal extensor compartment, a phe-
nomenon known as de Quervain’s disease. The tendons
involved are the extensor pollicis brevis and the abductor
pollicis longus. At the level of the radial styloid, these two
tendons pass through an osteoligamentous tunnel composed
of a shallow groove in the radius and an overlying ligament.
Anatomic studies have shown that a high percentage of
patients have a divided first dorsal compartment, and this
can account for failure of conservative treatment and injec-
tions.117-119
Patients with de Quervain’s disease are typically women
in their 30s and 40s, although men and women can develop
the condition at any age. This is the most common ten-
dinopathy to develop in women in the postpartum period
because of the specific hand and wrist position requirements
in the care of an infant. Any activity requiring repeated
thumb abduction and extension in combination with wrist
radial and ulnar deviation can aggravate this problem.
Patients complain of pain along the course of these tendons
with grasping activities. Clinically, there is tenderness along
the affected compartment, and there may be swelling over
the radial styloid. In severe cases, a creaking sound can be
elicited with movement of the involved tendons. Finkel-
stein’s test of forced ulnar deviation of the wrist with the
thumb clasped in the fisted palm is pathognomonic of the
condition120,121
A less common condition that may occur in the same
general location in the wrist is intersection syndrome.
Although initially attributed to friction between the first
and second dorsal compartment tendons, Grundberg and
Reagan122 subsequently showed that the condition repre-
sented a tendinopathy of the radial wrist extensors within
the second dorsal compartment.
The primary treatment for de Quervain’s disease and inter­
section syndrome is rest with splinting. For de Quervain’s
disease, the wrist should be held in slight extension and the
thumb abducted in a thumb spica splint to the level of the
interphalangeal joint. Immobilization of the wrist alone, in
approximately 15 degrees of extension, is usually adequate
for intersection syndrome. The addition of a 2- to 4-week
course of anti-inflammatory medication also can be helpful.
Phonophoresis with a cortisone cream and injection of the
compartment with cortisone are second-line treatments if
immobilization alone fails to give adequate relief. Injection
of corticosteroid into the affected first dorsal compartment
is curative for de Quervain’s disease in approximately 75%
of patients.123 Surgery may be indicated for patients who
do not respond to a course of conservative treatment. For
de Quervain’s disease and intersection syndrome, surgery
consists of releasing the stenotic retinacular sheath of the
involved compartment.
Basal Joint Arthropathy
Inflammation and pain related to the carpometacarpal joint
of the thumb are common and can occur at any age. In
younger patients, instability secondary to ligamentous laxity
is associated with joint subluxation and abnormal cartilage
wear and may lead to pain with mechanical activities. In
women older than 45 years, studies show 25% have radio-
graphic evidence of degeneration of the basal joint.124,125
Patients generally present with pain at the base of the
thumb, worsened by pinch and highly dexterous activities.
They often report difficulty with tasks such as opening jars
and bottles, turning doorknobs and keys, and other activi-
ties of daily living. The thumb carpometacarpal joint may be
swollen and subluxed and is generally tender to palpation.
The joint should be assessed for the presence of increased
laxity by manual subluxation of the base of the metacar-
pal out of the trapezial “saddle” with radial and volar force.
With advanced degenerative disease, crepitus is sometimes
appreciated.
Radiographs should be obtained to determine the stage
of the disease. The addition of a basal joint posteroanterior
stress film, in which the patient presses the tips of the thumbs
together firmly with the nail plates facing up, is helpful in
assessing joint subluxation (Fig. 44-6). The most commonly
Figure 44-6  “Basal joint stress” radiograph showing stage three 
degeneration of the left thumb and stage four degeneration of the right
thumb.
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
used staging system was developed by Eaton and Glickel126
and is based on the degree of involvement of the trape-
ziometacarpal joint and whether or not the scaphotrape-
zial joint is involved.126 Advancing stages show increased
subluxation of the basal joint, with development of joint
space narrowing, osteophytes, and subchondral cysts.
Stage 4 implies the presence of pantrapezial ­degenerative
­disease.
Regardless of the stage of the disease, the first line of
treatment is immobilization of the thumb metacarpal,
leaving the interphalangeal joint free. Splinting has been
shown to alleviate the symptoms of carpometacarpal joint
inflammation in more than 50% of patients.127 NSAIDs can
be a useful adjunct. Injections of corticosteroid and local
anesthetic mixture are quite effective, but usually for a lim-
ited time. Although therapy for thenar muscle strength-
ening has been advocated, especially in early stages, its
benefits are minimal, and it can occasionally aggravate the
­problem.
Many patients are able to manage their symptoms with
a combination of splinting, medications, corticosteroid
injections, and activity modification. If this combination is
insufficient, surgery may be indicated in young patients to
reconstruct the ligaments that stabilize the metacarpal base.
In patients with advanced degenerative changes and whose
symptoms continue to interfere sufficiently with their daily
activities, surgery is indicated to replace the joint with a
prosthetic device or to excise the trapezium and reconstruct
the soft tissue supports.
Volar Ganglion
Another common location for ganglia is the radial side
of the volar wrist. Ganglia typically originate from the
scaphotrapezial joint, but become superficial and are
clinically evident at or near the distal wrist crease over
the flexor carpi radialis tendon. Volar ganglia can occur
in close proximity to the radial artery and should be dif-
ferentiated from a radial artery aneursym. Aspiration, if
attempted, should be performed carefully to avoid vascu-
lar injury, and surgery should be preceded by performance
of an Allen test to document patent ulnar arterial flow.
Volar ganglia are associated with a higher recurrence
rate and a higher complication rate than their dorsal
­counterparts.128
Scaphoid Fracture and Nonunion
Occasionally, a young or middle-aged patient presents
with a nonunited scaphoid fracture without recollection
of a traumatic incident. When evaluating a relatively
young patient with pain at the base of the thumb, wrist
swelling in the region of the anatomic snuffbox, and a
decreased range of motion of the wrist, plain radiographs
and a specialized ulnar-deviation “navicular” x-ray should
be obtained to rule out scaphoid pathology. In patients
in whom a scaphoid nonunion has been present for a sig-
nificant period, secondary changes in carpal alignment
and joint degeneration have usually occurred. Although
splint or cast immobilization can be tried, surgical repair
of the scaphoid or other wrist salvage procedure is usually
required.
659
PALM
Trigger Finger
Painful clicking and locking of the digits in flexion is one of
the most common causes of pain in the hand. This condi-
tion, caused by a thickening of the A1 retinacular pulley in
the palm, is commonly known as trigger finger. The thumb is
the most commonly affected digit, followed by the ring and
long fingers.129 Patients may present with isolated activity-
related pain in the proximal interphalangeal joint without
frank clicking or locking. Early clicking is felt as a snapping
sensation during digital motion and is frequently worst on
awakening. As the condition progresses, the digital range
of motion can be reduced and secondary proximal inter-
phalangeal joint contractures develop. The final stage is a
locked trigger finger that cannot be straightened actively.
Primary trigger finger is the most common type, found
most often in middle-aged individuals. Triggering of the
thumb is four times more frequent in women than in men.5
Secondary triggering is seen in association with such dis-
eases as rheumatoid arthritis, diabetes, and gout. In this
type, trigger fingers are often multiple and can coexist with
other stenosing tendinopathies, such as de Quervain’s dis-
ease or carpal tunnel syndrome. Congenital or developmen-
tal triggering can be identified in children and is much less
common. Similar to in adults, the thumb is most commonly
affected, but in contrast to adults, these often present with
the interphalangeal joint locked in flexion.
Nonoperative treatment of this condition consists pri-
marily of splinting and local steroid injections. In adults,
injection of steroid into the tendon sheath has been shown
to be quite effective.5,6,130 Injection is used infrequently in
infants or children. When nonoperative treatments fail to
give lasting relief, surgical treatment consists of longitudi-
nal division of the A1 pulley at the level of the metacarpal
head. It is a simple procedure that yields reliable and perma-
nent results with few complications.
Retinacular Cysts
Retinacular ganglion cysts can occur in conjunction with a
triggering digit or in isolation. They are located at the base
of the digit over the A1 pulley as a discrete, firm, pea-sized
nodule. They originate from the flexor tendon sheath or
annular pulleys and contain synovial fluid. Patients usually
complain of pain with gripping objects or with direct pres-
sure over the cyst. A retinacular cyst is most easily treated
initially by needle decompression, with care to avoid injury
to the sensory nerves that lie immediately adjacent to the
flexor tendon and associated cyst. Approximately 50% recur
after aspiration, and surgical resection may be required.
DIGITS
Mallet Finger
Mallet finger refers to a loss of terminal extension of the dis-
tal interphalangeal joint of the digit and can be classified as
bony or soft tissue depending on where the disruption in the
extensor mechanism occurred. Mallet fingers can occur with
minimal trauma, such as tucking in bed sheets, and may not
be recalled by the patient. This sometimes leads to a delay
 660 swigart  |  Hand and Wrist Pain

in diagnosis and treatment. When a patient presents with

a digit that droops at the distal interphalangeal joint and
cannot be actively extended, but has full passive motion,
a radiograph should be obtained to determine if there is
an associated fracture of the distal phalanx. An extension
splint is the treatment of choice for bony and soft tissue mal-
let fingers. The distal interphalangeal joint should be held
in full extension, and care should be taken not to force the
distal interphalangeal joint into hyperextension to prevent
dorsal skin ischemia and necrosis. Splinting is employed
full time for 6 weeks. The patient should not remove the
splint for showering or any other activity, but may change
the splint carefully for skin care, provided that the joint is
maintained in extension throughout. Proximal interphalan-
geal flexion exercises are initiated from the outset and are
important to help reset the tension in the extensor mecha-
nism. Gentle distal interphalangeal flexion exercises are
begun at 6 weeks, and splinting is decreased to nighttime
between 6 and 8 weeks. Patients usually can expect a small
extension lag, on the order of 5 degrees, and a return of most
of their flexion.
Osteoarthritis of the Digits
Osteoarthritis of the interphalangeal joints is extremely
common in older patients and is most often manifested as
Heberden’s nodes of the distal interphalangeal joint. Despite
gross deformities, pain and dysfunction may be minimal. A
mucous cyst may appear in association with degenerative
arthritis. Mucous cysts appear on the dorsum of the joint
and can cause nail growth deformities owing to pressure
on the germinal matrix (Fig. 44-7). The changes in nail
growth may precede clinical detection of the cyst. These
cysts should not be aspirated with a needle because of the
close proximity of the distal interphalangeal joint and the
risk of secondary joint infection. Treatment consists of distal
interphalangeal joint immobilization to control symptoms
or surgical excision of the cyst and in particular the underly-
ing osteophytic spurs.
Tumors
Benign bone tumors, such as simple bone cysts and enchon-
dromas, are common in the phalanges. These usually cause
no symptoms and frequently are diagnosed as incidental
findings on routine hand radiographs. Enchondromas are
most commonly located in the metaphysis of the proximal
phalanx and may lead to fracture with minimal trauma
as a result of weakening of the bone structure. If a patho-
logic fracture occurs, nonoperative treatment is indicated
until the fracture heals. The bone tumor subsequently can
be addressed with curettage and bone grafting. Occasion-
ally, because of malalignment, earlier surgical intervention
becomes necessary.
Many soft tissue tumors can occur in the hand and dig-
its. Some common benign tumors are giant cell tumors of
the tendon sheath, lipomas, and glomus tumors. Lipomas
and giant cell tumors of the tendon sheath manifest clini-
cally as painless, slow-growing masses in the palm and digits.
Surgical excision is necessary for diagnosis. Glomus tumors
arise from the pericytes in the fingertip or subungual area
and typically present with intermittent sharp pain in the
Figure 44-7  Dorsal view of a digit with an as yet clinically inapparent
mucous cyst and the corresponding groove deformity of the nail plate.
fingertip. These vascular tumors become intensely symptom-
atic when the hand is exposed to cold temperatures, owing
to abnormal arteriovenous shunting through the hypertro-
phic glomus system. Surgical excision is generally curative
and should be preceded by MRI to rule out multifocal sites.
Infection
The most common infection in the hand is the paronychia.
It involves the fold of tissue surrounding the fingernail.
Staphylococcus aureus is the usual pathogen, introduced by
a hangnail, a manicure instrument, or nail biting. Patients
present with an exquisitely painful and erythematous swell-
ing involving a part of the nail fold. Occasionally, the infec-
tion can progress to surround the nail in a horseshoe fashion
and undermine the nail plate. If seen early, within the first 24
to 48 hours, oral antibiotics and local treatment of the finger
with warm soaks can be effective. Superficial abscesses can
be drained with a sharp blade through the thin skin without
requiring local anesthesia. Larger or more chronic infections
require surgical drainage.
An infection of the distal pulp of the fingertip, known
as a felon, is a particular problem in diabetic patients. This
infection differs from other subcutaneous infections because
of the vertical fibrous septa that divide and stabilize the pulp
of the fingertip. Often patients have had some recent pene-
trating injury in the area. Because of the tightly constrained
area of the infection, patients present with an intensely
painful fingertip. There may be an area of “pointing” over
the abscess. Surgical drainage is required followed by soaks
and oral antibiotics, and intravenous antibiotics are gener-
ally recommended in diabetic patients.
Although similar in appearance to a paronychia, herpetic
whitlow is caused by herpes simplex virus and must be differ-
entiated from other fingertip infections because of a radically
different treatment protocol.131,132 Whitlow was common
among dental hygienists before the widespread use of gloves
for all health care workers. As with bacterial ­infections, the
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
area becomes painful and erythematous; local tenderness
is much less severe, however. Diagnosis is by clinical pre-
sentation and history. If seen early, vesicles can be ruptured
for fluid analysis and viral culture. Nonoperative treatment
with oral antiviral agents is recommended.
Other hand and digit infections, such as suppurative
flexor tenosynovitis, deep space infections of the palm,
pyogenic arthritis, infections from bite wounds, and osteo-
myelitis, should be evaluated initially with radiographs of
the hand and appropriate blood work. If possible, antibiot-
ics should be withheld until definitive cultures are obtained
from the affected area. Antibiotics should be administered
intravenously, and the hand and wrist should be immobi-
lized. Most infections require surgical drainage for definitive
treatment.
REFERENCES
  1. Metz VM, Gilula LA: Imaging techniques for distal radius fractures
and related injuries. Orthop Clin North Am 24:217-228, 1993.
  2. Larsen CF, Brondum V, Wienholtz G, et al: An algorithm for acute
wrist trauma: A systematic approach to diagnosis. J Hand Surg (Am)
18:207-212, 1993.
  3. Schreibman KL, Freeland A, Gilula LA, et al: Imaging of the hand
and wrist. Orthop Clin North Am 28:537-582, 1997.
  4. Kaufman MA: Differential diagnosis and pitfalls in electrodiagnos-
tic studies and special tests for diagnosing compressive neuropathies.
Orthop Clin North Am 27:245-252, 1996.
  5. Marks MR, Gunther SF: Efficacy of cortisone injection in treatment
of trigger fingers and thumbs. J Hand Surg (Am) 14:722-727, 1989.
  6. Newport ML, Lane LB, Stuchin SA: Treatment of trigger finger by
steroid injection. J Hand Surg 15:748-750, 1990.
  7. Freiberg A, Mulholland RS, Levine R: Nonoperative treatment of
trigger fingers and thumbs. J Hand Surg (Am) 14:553-558, 1989.
  8. Gelberman RH, Aronson D, Weisman MH: Carpal tunnel syn-
drome: Results of a prospective trial of steroid injection and splint-
ing. J Bone Joint Surg 62:1181-1184, 1980.
  9. Avci S, Yilmaz C, Sayli U: Comparison of nonsurgical treatment
measures for de Quervain’s disease of pregnancy and lactation.
J Hand Surg (Am) 27:322-324, 2002.
10. Lane LB, Boretz RS, Stuchin SA: Treatment of de Quervain’s disease:
Role of conservative management. J Hand Surg (Am) 26:258-260,
2001.
11. Taras JS, Raphael JS, Pan WT, et al: Corticosteroid injections for
trigger digits: Is intrasheath injection necessary? J Hand Surg (Am)
23:717-722, 1998.
12. Esteban JM, Oertel YC, Mendoza M, et al: Fine needle aspiration in
the treatment of ganglion cysts. South Med J 79:691-693, 1986.
13. Richman JA, Gelberman RH, Engber WD, et al: Ganglions of the
wrist and digits: Results of treatment by aspiration and cyst wall
puncture. J Hand Surg (Am) 12:1041-1043, 1987.
14. Easterling KJ, Wolfe SW: Wrist arthroscopy: An overview. Contemp
Orthop 24:21-30, 1992.
15. Roth JH, Poehling GG, Whipple TL: Arthroscopic surgery of the
wrist. Instr Course Lect 37:183-194, 1988.
16. Adolfsson L: Arthroscopy for the diagnosis of post-traumatic wrist
pain. J Hand Surg (Am) 17:46-50, 1992.
17. Koman LA, Poehling GG, Toby EB, et al: Chronic wrist pain: Indica-
tions for wrist arthroscopy. Arthroscopy 6:116-119, 1990.
18. Terrill RQ: Use of arthroscopy in the evaluation and treatment of
chronic wrist pain. Hand Clin 10:593-603, 1994.
19. DeSmet L, Dauwe D, Fortems Y, et al: The value of wrist arthroscopy:
An evaluation of 129 cases. J Hand Surg (Am) 21:210-212, 1996.
20. Kelly EP, Stanley JK: Arthroscopy of the wrist. J Hand Surg
15:236-242, 1990.
21. Poehling GP, Chabon SJ, Siegel DB: Diagnostic and operative
arthroscopy. In Gelberman RH (ed): The Wrist: Master Techniques
in Orthopedic Surgery. New York, Raven Press, 1994 pp 21-45.
22. Bienz T, Raphael JS: Arthroscopic resection of the dorsal ganglia of
the wrist. Hand Clin 15:429-434, 1999.
23. Luchetti R, Badia A, Alfarano M, et al: Arthroscopic resection of
dorsal wrist ganglia and treatment of recurrences. J Hand Surg (Am)
25B:38-40, 2000.
661
24. Ho PC, Griffiths J, Lo WN, et al: Current treatment of ganglion of
the wrist. Hand Surg 6:49-58, 2001.
25. Arnold AG: The carpal tunnel syndrome in congestive cardiac fail-
ure. Postgrad Med J 53:623, 1977.
26. Doll DC, Weiss RB: Unusual presentations of multiple myeloma.
Postgrad Med 61:116-121, 1977.
27. Klofkorn RW, Steigerwald JC: Carpal tunnel syndrome as the initial
manifestation of tuberculosis. Am J Med 60:583, 1976.
28. Mayers LB: Carpal tunnel syndrome secondary to tuberculosis. Arch
Neurol 10:426, 1964.
29. Champion D: Gouty tenosynovitis and the carpal tunnel syndrome.
Med J Aust 1:1030, 1969.
30. Gould JS, Wissinger HA: Carpal tunnel syndrome in pregnancy.
South Med J 71:144-145, 1978.
31. Green EJ, Dilworth JH, Levitin PM: Tophacceous gout: An unusual
cause of bilateral carpal tunnel syndrome. JAMA 237:2747-2748,
1977.
32. Leach RE, Odom JA: Systemic causes of the carpal tunnel syndrome.
Postgrad Med 44:127-131, 1968.
33. Massey EW: Carpal tunnel syndrome in pregnancy. Obstet Gynecol
Surg 33:145, 1978.
34. Michaelis LS: Stenosis of carpal tunnel, compression of median
nerve, and flexor tendon sheaths, combines with rheumatoid arthritis
elsewhere. Proc R Soc Med 43:414, 1950.
35. O’Hara LJ, Levin M: Carpal tunnel syndrome and gout. Arch Intern
Med 120:180, 1967.
36. Phillips RS: Carpal tunnel syndrome as manifestation of systemic dis-
ease. Ann Rheum Dis 26:59, 1967.
37. Stallings SP, Kasdan ML, Soergel TM, et al: A case-control study
of obesity as a risk factor for carpal tunnel syndrome in a population
of 600 patients presenting for independent medical examination.
J Hand Surg (Am) 22:211-215, 1997.
38. Karpitskaya Y, Novak CB, Mackinnon SE: Prevalence of smoking,
obesity, diabetes mellitus, and thyroid disease in patients with carpal
tunnel syndrome. Ann Plast Surg 48:269-273, 2002.
39. Mondelli M, Giannini F, Giacchi M: Carpal tunnel syndrome inci-
dence in a general population. Neurology 58:289-294, 2002.
40. Kummel BM, Zazanis GA: Shoulder pain as the presenting complaint
in carpal tunnel syndrome. Clin Orthop 83:41-47, 1972.
41. Lettin AWF: Carpal tunnel syndrome in childhood. J Bone Joint
Surg 47:556-559, 1965.
42. al-Qattan MM, Thomson HG, Clarke HM: Carpal tunnel syndrome
in children and adolescents with no history of trauma. J Hand Surg
(Am) 21B:108-111, 1996.
43. Phalen GS: Spontaneous compression of the median nerve at the
wrist. JAMA 145:1128, 1951.
44. Durkan JA: A new diagnostic test for carpal tunnel syndrome.
J Bone Joint Surg 73:535-538, 1991.
45. Gonzalez del Pino J, Delgado-Martinez AD, Gonzalez Gonzalez I,
et al: Value of the carpal compression test in the diagnosis of carpal
tunnel syndrome. J Hand Surg (Am) 22:38-41, 1997.
46. Kemble F: Electrodiagnosis of the carpal tunnel syndrome. J Neurol
Neurosurg Psychiatry 31:23, 1968.
47. Ludin HP, Lutschg J, Valsangiacomo F: Comparison of orthodromic
and antidromic sensory nerve conduction, 1: Normals and patients
with carpal tunnel syndrome. EEG EMG 8:173, 1977.
48. Richier HP, Thoden U: Early electroneurographic diagnosis of carpal
tunnel syndrome. EEG EMG 8:187, 1977.
49. Szabo RM, Slater RR Jr, Farver TB, et al: The value of diagnostic
testing in carpal tunnel syndrome. J Hand Surg (Am) 24:704-714,
1999.
50. Melvin JL, Schuckmann JA, Lanese RR: Diagnostic specificity of
motor and sensory nerve conduction variables in the carpal tunnel
syndrome. Arch Phys Med Rehabil 54:69, 1973.
51. Gerritsen AAM, deVet HCW, Scholten RJPM, et al: Splinting vs
surgery in the treatment of carpal tunnel syndrome: A randomized
controlled trial. JAMA 288:1245-1251, 2002.
52. Gonzalez MH, Bylak J: Steroid injection and splinting in the
treatment of carpal tunnel syndrome. Orthopedics 24:479-481,
2001.
53. Irwin LR, Beckett R, Suman RK: Steroid injection for carpal tunnel
syndrome. J Hand Surg (Am) 21B:355-357, 1996.
54. Irwin LR, Beckett R, Suman RK: Steroid injection for carpal tunnel
syndrome. J Hand Surg (Am) 21:355-357, 1996.
55. Linskey ME, Segal R: Median nerve injury from local steroid
injection for carpal tunnel syndrome. Neurosurgery 26:512-515,
1990.
 662 swigart  |  Hand and Wrist Pain
56. Tavares SP, Giddins GE: Nerve injury following steroid injection for  
carpal tunnel syndrome: A report of two cases. J Hand Surg (Am)
21:208-209, 1996.
57. Ogino T, Minami A, Fukada K: Tardy ulnar nerve palsy caused by  
cubitus varus deformity. J Hand Surg (Am) 11:352-356, 1986.
58. Guyon F: Note sur une disposition anatomique proper a la face anter-  
ieure do la region du poignet et non encores decritie par la docteur.
Bull Soc Anat Paris 36:184-186, 1861.  
59. Blunden R: Neuritis of deep branch of the ulnar nerve. J Bone Joint
Surg 40:354, 1958.  
60. Eckman PB, Perlstein G, Altrocchi PH: Ulnar neuropathy in bicycle
riders. Arch Neurol 32:130-131, 1975.
61. Uriburu IJF, Morchio FJ, Marin JC: Compression syndrome of the  
deep branch of the ulnar nerve (piso-hamate hiatus syndrome). J
Bone Joint Surg 58:145-147, 1976.
62. Poppi M, Padovani R, Martinelli P, et al: Fractures of the distal radius  
with ulnar nerve palsy. J Trauma 18:278-279, 1978.
63. Vance RM, Gelberman RH: Acute ulnar neuropathy with fractures at
the wrist. J Bone Joint Surg 60:962-965, 1978.  
64. Jeffery AK: Compression of the deep palmar branch of the ulnar
nerve by an anomalous muscle. J Bone Joint Surg 53:718-723, 1971.  
65. Kalisman M, Laborde K, Wolff TW: Ulnar nerve compression sec-
ondary to ulnar artery false aneurysm at the Guyon’s canal. J Hand
Surg (Am) 7:137-139, 1982.  
66. McFarland GB, Hoffer MM: Paralysis of the intrinsic muscles of
the hand secondary to lipoma in Guyon’s canal. J Bone Joint Surg
53:375-376, 1971.  
67. Richmond DA: Carpal ganglion with ulnar nerve compression. J
Bone Joint Surg 45:513-515, 1963.
68. Toshima Y, Kimata Y: A case of ganglion causing paralysis of intrin-  
sic muscles innervated by the ulnar nerve. J Bone Joint Surg 43:153,
1961.
69. Bishop AT, Gabel G, Carmichael SW: Flexor carpi radialis ten-
donitis, part I: Operative anatomy. J Bone Joint Surg 76:1009-1014,
1994.
70. Carroll RE, Sinton W, Garcia A: Acute calcium deposits in the hand.
JAMA 157:422-426, 1955.
71. Moyer RA, Bush DC, Harrington TM: Acute calcific tendonitis of
the hand and wrist: A report of 12 cases and a review of the literature.
J Rheum 16:198-202, 1989.
72. Yang SS, Kalainov DM, Weiland AJ: Fracture of the hook of hamate
with rupture of the flexor tendons of the small finger in a rheumatoid
patient: A case report. J Hand Surg (Am) 21:916-917, 1996.
73. Kato H, Nakamura R, Horii E, et al: Diagnostic imaging for fracture
of the hook of the hamate. Hand Surg 5:19-24, 2000.
74. Whalen JL, Bishop AT, Linscheid RL: Nonoperative treatment of
acute hamate hook fractures. J Hand Surg (Am) 17:507-511, 1992.
75. Bishop AT, Bechenbaugh RD: Fracture of the hamate hook. J Hand
Surg (Am) 13:863-868, 1988.
76. Carter PR, Eaton RG, Littler JW: Ununited fracture of the hook of
the hamate. J Bone Joint Surg (Am) 59:583-588, 1977.
77. Stark HH, Chao EK, Zemel NP, et al: Fracture of the hook of the
hamate. J Bone Joint Surg 71:1202-1207, 1989.
78. Stark HH, Jobe FW, Boyes JH, et al: Fracture of the hook of the
hamate in athletes. J Bone Joint Surg 59:575-582, 1977.
79. Cardinal E, Buckwalter KA, Braunstein EM, et al: Occult dorsal
carpal ganglion: Comparison of US and MR imaging. Radiology
193:259-262, 1994.
80. Vo P, Wright T, Hayden F, et al: Evaluating dorsal wrist pain: MRI
diagnosis of occult dorsal wrist ganglion. J Hand Surg (Am) 20:
667-670, 1995.
81. Zubowicz VN, Ishii CH: Management of ganglion cysts of the hand
by simple aspiration. J Hand Surg (Am) 12:618-620, 1987.
82. Angelides AC, Wallace PF: The dorsal ganglion of the wrist: Its
pathogenesis, gross and microscopic anatomy, and surgical treatment.
J Hand Surg (Am) 1:228-235, 1976.
83. Clay NR, Clement DA: The treatment of dorsal wrist ganglia by radi-
cal excision. J Hand Surg (Am) 13:187-191, 1988.
84. Janzon L, Niechajev IA: Wrist ganglia: Incidence and recurrence rate
after operation. Scand J Plast Reconstr Surg 15:53-56, 1981.
85. Angelides AC: Ganglions of the hand and wrist. In Green DP (ed):
Operative Hand Surgery. New York, Churchill Livingstone, 1993.
86. Cuono CB, Watson HK: The carpal boss: Surgical treatment and
etiological considerations. Plast Reconstr Surg 63:88-93, 1979.
87. Bonatz E, Dramer TD, Masear VR: Rupture of the extensor pollicis
longus tendon. Am J Orthop 25:118-122, 1996.
88. Stahl S, Wolff TW: Delayed rupture of the extensor pollicis longus
tendon after nonunion of a fracture of the dorsal radial tubercle.
J Hand Surg (Am) 13:338-341, 1988.
89. Hove LM: Delayed rupture of the thumb extensor tendon: A 5-year
study of 18 consecutive cases. Acta Orthop Scand 65:199-203, 1994.
90. Dawson WJ: Sports-induced spontaneous rupture of the extensor pol-
licis longus tendon. J Hand Surg (Am) 17:457-458, 1992.
91. Kienbock R: Uber Traumatische Malazie des Mondbeins und Kom-
pression Fracturen. Fortschr Roentgenstrahlen 16:77-103, 1910.
92. Stahl F: On lunatomalacia (Keinbock’s disease): Clinical and roent-
genological study, especially on its pathogenesis and late results of
immobilization treatment. Acta Chir Scand 126(suppl):1-133, 1947.
93. Wada A, Miura H, Kubota H, et al: Radial closing wedge osteotomy
for Kienbock’s disease: An over 10 year clinical and radiographic fol-
low-up. J Hand Surg (Am) 27B:175-179, 2002.
94. Wintman BI, Imbriglia JE, Buterbaugh GA, et al: Operative treat-
ment with radial shortening in Kienbock’s disease. Orthopedics
24:365-371, 2001.
95. Quenzer DE, Dobyns JH, Linscheid RL, et al: Radial recession oste-
otomy for Kienbock’s disease. J Hand Surg (Am) 22:386-395, 1997.
96. Nakamura R, Imaeda T, Miura T: Radial shortening for Kienbock’s
disease: Factors affecting the operative result. J Hand Surg (Am)
15:40-45, 1990.
97. Oishi SN, Muzaffar AR, Carter PR: Treatment of Kienbock’s disease
with capitohamate arthrodesis: Pain relief with minimal morbidity.
Plast Reconstr Surg 109:1293-1300, 2002.
98. Watson HK, Monacelli DM, Milford RS, et al: Treatment of Kien-
bock’s disease with scaphotrapezio-trapezoid arthrodesis. J Hand Surg
(Am) 21:9-15, 1996.
99. Chuinard RG, Zeman SC: Kienbock’s disease: An analysis and ratio-
nale for treatment by capitate-hamate fusion. Orthop Trans 4:18,
1980.
100. Kakinoki R, Matsumoto T, Suzuki T, et al: Lunate plasty for
Kienbock’s disease: Use of a pedicled vascularised radial bone graft
combined with shortening of the capitate and radius. Hand Surg
6:145-156, 2001.
101. Thompson TC, Campbell RD Jr, Arnold WD: Primary and secondary
dislocation of the scaphoid bone. J Bone Joint Surg 46:73-82, 1964.
102. Palmer AK, Werner FW: The triangular fibrocartilage complex of
the wrist: Anatomy and function. J Hand Surg (Am) 6:153-161,
1981.
103. King GJ, McMurtry RY, Rubenstein JD, et al: Kinematics of the dis-
tal radioulnar joint. J Hand Surg (Am) 11:798-804, 1986.
104. Burk DL Jr, Karasick D, Wechsler RJ: Imaging of the distal radioul-
nar joint. Hand Clin 7:263-275, 1991.
105. King GJ, McMurtry RY, Rubenstein JD, et al: Computerized tomogra-
phy of the distal radioulnar joint: Correlation with ligamentous pathol-
ogy in a cadaveric model. J Hand Surg (Am) 11:711-717, 1986.
106. Mino DE, Palmer AK, Levinsohn EM: The role of radiography and
computerized tomography in the diagnosis of subluxation and disloca-
tion of the distal radioulnar joint. J Hand Surg (Am) 8:23-31, 1983.
107. Mino DE, Palmer AK, Levinsohn EM: Radiography and computerized
tomography in the diagnosis of incongruity of the distal radio-ulnar
joint: A prospective study. J Bone Joint Surg 67:247-252, 1985.
108. Steyers CM, Blair WF: Measuring ulnar variance: A comparison of
techniques. J Hand Surg (Am) 14:607-612, 1989.
109. Epner RA, Bowers WH, Guilford WB: Ulna variance: The effect of
wrist positioning and roentgen filming technique. J Hand Surg (Am)
7:298-305, 1982.
110. Gilula LA, Hardy DC, Totty WG: Distal radioulnar joint arthrogra-
phy. AJR Am J Roentgeol 150:180-189, 1988.
111. Mikic ZD: Age changes in the triangular fibrocartilage of the wrist
joint. J Anat 126:367-384, 1978.
112. Mikic ZD: Arthrography of the wrist joint: An experimental study.
J Bone Joint Surg 66:371-378, 1984.
113. Palmer AK, Levinsohn EM, Kuzma GR: Arthrography of the wrist.
J Hand Surg (Am) 8:18-23, 1983.
114. Potter HG, Asnis-Ernberg L, Weiland AJ, et al: The utility of high-
resolution magnetic resonance imaging in the evaluation of the
triangular fibrocartilage complex of the wrist. J Bone Joint Surg
79:1675-1684, 1997.
115. Feldon P, Terronon AL, Belsky MR: The wafer procedure: Partial
distal ulnar resection. Clin Orthop 275:124-129, 1992.
116. de Araujo W, Poehling GG, Kuzma GR: New Tuohy needle tech-
nique for triangular fibrocartilage complex repair: Preliminary stud-
ies. Arthroscopy 12:699-703, 1996.
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
117. Lacey T II, Goldstein LA, Tobin CE: Anatomical and clinical study
of the variations in the insertions of the abductor pollicis longus
tendon, associated with stenosing tendovaginitis. J Bone Joint Surg
33:347-350, 1951.
118. Leao L: De Quervain’s disease: A clinical and anatomical study.
J Bone Joint Surg 40:1063-1070, 1958.
119. Strandell G: Variations of the anatomy in stenosing tenosynovitis at
the radial styloid process. Acta Chir Scand 113:234-240, 1957.
120. F inkelstein H: Stenosing tendovaginitis at the radial styloid pro-
cess. J Bone Joint Surg 12:509-540, 1930.
121. Pick RY: De Quervain’s disease: A clinical triad. Clin Orthop
143:165-166, 1979.
122. Grundberg AB, Reagan DS: Pathologic anatomy of the forearm:
Intersection syndrome. J Hand Surg (Am) 10:299-302, 1985.
123. Weiss AP, Akelman E, Tabatabai M: Treatment of de Quervain’s dis-
ease. J Hand Surg (Am) 19:595-598, 1994.
124. Kelsey JL, Pastides H, Kreiger N, et al: Arthritic Disorders, Upper
Extremity Disorders: A Survey of Their Frequency and Cost in the
United States. St. Louis, CV Mosby, 1980.
663
125. Armstrong AL, Hunter JB, Davis TRC: The prevalence of degenera-
tive arthritis of the base of the thumb in post-menopausal women.
J Hand Surg (Am)19:340-341, 1994.
126. Eaton RG, Glickel SZ: Trapeziometacarpal osteoarthritis: Staging
as a rationale for treatment. Hand Clin 3:455-469, 1987.
127. Swigart CR, Eaton RG, Glickel SZ, et al: Splinting in the treatment
of trapeziometacarpal joint arthritis. J Hand Surg (Am) 24:86-91,
1999.
128. Gundes H, Cirpici Y, Sarlak A, et al: Prognosis of wrist ganglion
operations. Acta Orthop Belg 66:363-367, 2000.
129. Bonnici AV, Spencer JD: A survey of ‘trigger finger’ in adults. J Hand
Surg (Am) 13:202-203, 1988.
130. Murphy D, Failla JM, Koniuch MP: Steroid versus placebo injec-
tion for trigger finger. J Hand Surg (Am) 20:628-631, 1995.
131. Fowler JR: Viral infections. Hand Clin 5:533-552, 1989.
132. LaRossa D, Hamilton R: Herpes simplex infections of the digits.
Arch Surg 102:600-603, 1971.
 45
Key Points
Temporomandibular joint (TMJ) pain must be distinguished
from the pain that more commonly arises from the muscles of
mastication (myofascial pain), which can produce similar signs
and symptoms.
TMJ pain also must be distinguished from pain coming from
the ear or parotid gland.
TMJ and masticatory muscle pain generally are accompanied
by limitation of mouth opening, but not pain arising from the
ear or parotid gland.
All the forms of arthritis that involve other joints in the body
also involve the TMJ and may give rise to pain and limited jaw
movement.
Displacement of the intra-articular disk in the TMJ produces
pain that is accompanied by a clicking or popping sound or
sudden onset of jaw locking.
Pain in the temporomandibular joint (TMJ) region, a com-
monly encountered symptom, affects more than 10 million
Americans. Because of its diverse etiology, however, there is
often considerable difficulty in proper diagnosis and treat-
ment. Owing to the proximity of the ear and parotid gland
and the similar nature of pain in these areas, pathologic
conditions involving these structures are often confused
with conditions arising in the TMJ. Pain occurring in the
adjacent muscles of mastication, also a frequently encoun-
tered situation, not only is similar to TMJ pain in character
and location, but also is associated with jaw dysfunction,
a common finding with painful conditions directly involv-
ing the TMJ. For these reasons, a knowledge of the various
painful conditions occurring in the TMJ region is essential
in establishing a correct diagnosis.
Because patients with primary TMJ disease often have
secondary myofascial pain in the muscles of mastication,
and patients with primary myofascial pain problems in the
masticatory muscles can develop secondary TMJ disease,
the generally accepted term used to describe this overlap-
ping group of conditions is temporomandibular disorders.
These conditions are subdivided for purposes of diagnosis
and treatment into conditions that primarily involve the
TMJ (TMJ problems) and conditions that primarily involve
the muscles of mastication (myofascial pain and dysfunction
[MPD], masticatory myalgia). From a diagnostic standpoint,
it also is important to consider the numerous conditions that
mimic the temporomandibular disorders or MPD by produc-
ing similar signs and symptoms (Tables 45-1 and 45-2).
Table 45-3 lists the various pathologic entities that com-
monly involve the TMJ. Although there are a variety of
Temporomandibular
Joint Pain
Daniel J. Laskin
conditions, only three types commonly are considered to
produce pain: the various arthritides, derangements of the
intra-articular disk, and certain neoplasms.
ARTHRITIS OF THE
TEMPOROMANDIBULAR JOINT
Arthritis is the most common condition affecting the TMJ,
just as it is in other joints. Although degenerative arthritis
and rheumatoid arthritis are encountered most frequently,
cases of infectious arthritis, metabolic arthritis, and the
spondyloarthropathies also have been reported. Traumatic
arthritis is another common occurrence.
Degenerative Arthritis (Osteoarthritis)
Degenerative arthritis is the most common type of arthritis
involving the TMJ and the most frequent cause of pain in
that region. Clinical symptoms of the disease have been
reported in 16% of the general population,1 but radiographic
evidence has been found in 44% of asymptomatic individu-
als.2 Although the TMJ is not a weight-bearing joint in
the same sense as the joints of the long bones, the stresses
associated with such parafunctional habits as clench-
ing and grinding of the teeth are sufficient to contribute
to similar degenerative changes in some patients.3 Acute
and chronic trauma and derangements of the intra-articu-
lar disk also are common causes of secondary degenerative
arthritis.
Clinical Findings
Primary degenerative arthritis, which usually is seen in older
individuals, is insidious in its onset; it generally produces
only mild discomfort, and individuals rarely complain about
the condition. Secondary degenerative arthritis usually occ­
urs in younger patients (20 to 40 years old) and tends to
be painful. In contrast to primary degenerative joint disease
and rheumatoid arthritis, it often is limited to only one
TMJ, although it may become bilateral in the late stages,
and involvement of other joints is uncommon. The condi-
tion is characterized by TMJ pain that is increased by func-
tion, joint tenderness, limitation of mouth opening, and
occasional clicking and popping sounds. In the late stages,
there may be crepitation in the joint.
Imaging Findings
The earliest radiologic feature of degenerative arthritis of
the TMJ, whether primary or secondary, is subchondral scle-
rosis in the mandibular condyle. If the condition progresses,
665
 666 LASKIN  |  Temporomandibular Joint Pain

Table 45-1  Differential Diagnosis of Nonarticular Conditions Mimicking Temporomandibular Joint Pain  
or Myofascial Pain in the Masticatory Muscles
Disorder Jaw Limitation Muscle Tenderness Diagnostic Features
Pulpitis No No Mild-to-severe ache or throbbing; intermittent or constant; aggravated by
thermal change; eliminated by dental anesthesia; positive radiographic
findings
Pericoronitis Yes Possible Persistent mild-to-severe ache; difficulty swallowing; possible fever; local
inflammation; relieved with dental anesthesia
Otitis media No No Moderate-to-severe earache; pain constant; fever; usually history of upper
respiratory infection; no temporomandibular joint tenderness
Parotitis Yes No Constant aching pain, worse when eating; pressure feeling; absent salivary
flow; ear lobe elevated; suppuration from duct
Sinusitis No No Constant aching or throbbing; worse with change of head position; nasal
discharge; often maxillary molar pain not relieved by dental anesthesia
Trigeminal neuralgia No No Sharp stabbing pain of short duration; trigger zone; pain follows nerve
pathway; older age group; often relieved by dental anesthesia
Atypical (vascular) No No Diffuse throbbing or burning pain of long duration; often associated auto-
­neuralgia nomic symptoms; no relief with dental anesthesia
Temporal arteritis No No Constant throbbing preauricular pain; artery prominent and tender;  
low-grade fever; may have visual problems; elevated erythrocyte  
sedimentation rate
Trotter’s syndrome Yes No Aching pain in ear, side of face, and lower jaw; deafness; nasal obstruction;
cervical lymphadenopathy
Eagle’s syndrome No No Mild-to-sharp stabbing pain in ear, throat, and retromandible; provoked by
swallowing, turning head, carotid compression; usually post-tonsillec-
tomy; styloid process >2.5 cm
Modified from Laskin DM, Block S: Diagnosis and treatment of myofascial pain dysfunction (MPD) syndrome. J Prosthet Dent 56:75-84, 1986.

Table 45-2  Differential Diagnosis of Nonarticular Conditions Producing Limitation of Mandibular Movement
Disorder Pain Muscle Tenderness Diagnostic Features
Odontogenic infection Yes Yes Fever; swelling; positive radiographic findings; tooth tender to
percussion; pain relieved and movement improved with dental
anesthesia
Nonodontogenic   Yes Yes Fever; swelling; negative dental findings on radiograph; dental anes-
infection thesia may not relieve pain or improve jaw movement
Myositis Yes Yes Sudden onset; jaw movement associated with pain; areas of muscle
tenderness; usually no fever
Myositis ossificans No No Palpable nodules seen as radiopaque areas on radiograph; involve-
ment of nonmasticatory muscles
Neoplasia Possible Possible Palpable mass; regional nodes may be enlarged; may have paresthe-
sia; radiograph may show bone involvement
Scleroderma No No Skin hard and atrophic; masklike facies; paresthesias; arthritic joint
pain; widening of periodontal ligament
Hysteria No No Sudden onset after psychological trauma; no physical findings; jaw
opens easily under general anesthesia
Tetanus Yes No Recent wound; stiffness of neck; difficulty swallowing; spasm of facial
muscles; headache
Extrapyramidal   No No Patient on antipsychotic drug or phenothiazine tranquilizer; hyper-
reaction tonic movement; lip smacking; spontaneous chewing motions
Depressed zygomatic   Possible No History of trauma; facial depression; positive radiographic findings
arch
Osteochondroma   No No Gradual limitation; jaw may deviate to unaffected side; possible
coronoid ­clicking sound on jaw movement; positive radiograph findings
From Laskin DM, Block S: Diagnosis and treatment of myofascial pain dysfunction (MPD) syndrome. J Prosthet Dent 56:75-84, 1986.

condylar flattening and marginal lipping may be noted.
In the later stages, erosion of the cortical plate, osteophyte
­formation, or both may occur. There also occasionally may
be breakdown of the subcortical bone resulting in the for-
mation of bone cysts. Although the changes in the ­articular
fossa generally are not as severe as the changes in the
condyle, cortical erosion sometimes can be seen. Narrowing
of the joint space also occurs in the late stages, and this is
indicative of concomitant degenerative changes in the intra-
articular disk. Although the changes in the TMJ ­usually can
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN 667

Table 45-3  Differential Diagnosis of Temporomandibular Joint Diseases

Disorder
Agenesis
Condylar hypoplasia
Condylar hyperplasia
Neoplasia
Infectious arthritis
Rheumatoid arthritis
Spondyloarthropathies
Psoriatic arthritis
Ankylosing spondylitis
Metabolic arthritis
Gout
Pseudogout
Traumatic arthritis
Degenerative arthritis
Ankylosis
Internal disk degeneration
TMJ, temporomandibular joint.
Modified from Laskin DM, Block S: Diagnosis and treatment of myofascial pain dysfunction (MPD) syndrome. J Prosthet Dent 56:75-84, 1986.
Pain Jaw Limitation Diagnostic Features
No Yes Congenital; usually unilateral; mandible deviates to affected side; unaffected side
long and flat; severe malocclusion; often ear abnormalities; radiograph shows
condylar deficiency
No No Congenital or acquired; affected side has short mandibular body and ramus, fullness
of face, deviation of chin; body of mandible elongated and face flat on unaffected
side; malocclusion; radiograph shows condylar deformity, antegonial notching
No No Facial asymmetry with deviation of chin to unaffected side; cross-bite malocclusion;
prognathic appearance; lower border of mandible often convex on affected side;
radiograph shows symmetric enlargement of condyle
Possible Yes Mandible may deviate to affected side; radiographs show enlarged, irregularly
shaped condyle or bone destruction, depending on type of tumor; unilateral
condition
Yes No Signs of infection; may be part of systemic disease; radiograph may be normal
early, later can show bone destruction; fluctuance may be present; pus may be
obtained on aspiration; usually unilateral
Yes Yes Signs of inflammation; findings in other joints (hands, wrists, feet, elbows, ankles);
positive laboratory test results; retarded mandibular growth in children; anterior
open bite; radiograph shows bone destruction; usually bilateral
Yes Yes Presence of cutaneous psoriasis; nail dystrophy; involvement of distal interphalan-
geal joints; radiograph shows condylar erosion; negative for rheumatoid factor
Yes Yes Frequent involvement of the spine and sacroiliac joint; extra-articular manifesta-
tions of spondylitis include iritis, anterior uveitis, aortic insufficiency, and conduc-
tion defects; erosive condylar changes; TMJ ankylosis may occur
Yes Yes Usually sudden onset; often monarticular; commonly involves great toe, ankle, and
wrist; joint swollen, red, and tender; increased serum uric acid; late radiographic
changes
Yes Yes Generally unilateral; TMJ may be only joint involved; joint frequently swollen; pres-
ence of intra-articular calcification; may be a history of trauma
Yes Yes History of trauma; radiograph normal except for possible widening of joint space;
local tenderness; usually unilateral
Yes Yes Unilateral joint tenderness; often crepitus; TMJ may be only joint involved; radio-
graph may be normal or show condylar flattening, lipping, spurring, or erosion
No Yes Usually unilateral, but can be bilateral; may be history of trauma; young patient
may show retarded mandibular growth; radiographs show loss of normal joint
architecture
Yes Yes Pain exacerbated by function; clicking on opening or opening limited to <25 mm
with no click; positive magnetic resonance imaging findings; may be history of
trauma; usually unilateral

be seen on plain radiographs, sagittal and coronal computed
tomography (CT) scans are the preferred modality for imag-
ing the bony structures.
Diagnosis
The diagnosis of degenerative arthritis is based on the
patient’s history and clinical and radiographic findings.
There is often a history of trauma or parafunctional oral
habits. The involvement is generally unilateral, and there
are no significant changes in any of the other joints. The
pain tends to be well localized, and the TMJ is often tender
to palpation.
Treatment
The treatment of degenerative arthritis of the TMJ is usually
medical, just as in other joints in the body. It involves the
use of nonsteroidal anti-inflammatory drugs, application of
heat, eating a soft diet, limitation of jaw function, and use
of a bite appliance to control parafunction if the patient has
a chronic habit of clenching or grinding the teeth. Physical
therapy with thermal agents, ultrasound, and iontophoresis
also can be beneficial, and isotonic and isometric exercises
are used to improve joint stability after the acute symptoms
have subsided. The use of intra-articular steroid injections is
controversial, and they should be used only in patients with
acute symptoms that do not respond to other forms of medical
management. Because of the potential damaging effects of
long-acting steroids,4 they should be limited to no more than
three or four single injections given at 3-month intervals.
Intra-articular injection of high-molecular-weight sodium
hyaluronate given twice, 2 weeks apart, has been shown to
have essentially the same therapeutic effect as a steroid injec-
tion, without the potential adverse side effects.5
When the acute symptoms have been controlled, therapy
is directed toward control of the factors possibly contributing
to the degenerative process. Unfavorable loading of the joint
 668 LASKIN  |  Temporomandibular Joint Pain
is eliminated by replacement of missing teeth to establish a
good, functional occlusion; by correction of any severe den-
tal malrelationships through orthodontics or orthognathic
surgery; and by continued use of a bite appliance at night to
control any teeth-clenching or teeth-grinding habits.6
In patients in whom medical management for 3 to
6 months fails to relieve the symptoms, surgical manage-
ment may be indicated. Surgery involves removal of only
the minimal amount of bone necessary to produce a smooth
articular surface. The unnecessary removal of the entire
cortical plate, as occurs with the so-called condylar shave
procedure or high condylotomy, can lead to a continuation
of the resorptive process in some instances and should be
avoided if possible.
Rheumatoid Arthritis
Of patients with rheumatoid arthritis, 50% have involve-
ment of the TMJ. Although the TMJ may be affected early in
the course of the disease, other joints in the body usually are
involved first. Children and adults are affected, with a female-
to-male ratio of 3:1. In children, destruction of the mandibu-
lar condyle by the disease process results in growth retardation
and facial deformity characterized by a severely retruded chin.
Fibrous or bony ankylosis is a possible sequela at all ages.
Clinical Findings
Patients with rheumatoid arthritis of the TMJ have bilat-
eral pain, tenderness, swelling in the preauricular region,
and limitation of mandibular movement. These symptoms
are characterized by periods of exacerbation and remission.
Joint stiffness and pain are usually worse in the morning
and decrease during the day. The limitation in mandibu-
lar movement worsens as the disease progresses; the patient
also may develop an anterior open bite.
Imaging Findings
Although there may not be any radiographic changes in the
early stages of the disease, about 50% to 80% of patients
show bilateral evidence of demineralization, condylar flat-
tening, and bone erosion as the disease progresses, so the
articular surface appears irregular and ragged. Erosion of the
glenoid fossa also is seen sometimes. As a result of destruc-
tion of the intra-articular disk, there also is narrowing of the
joint space. With continued destruction of the condyle, the
loss of ramus height can lead to contact of only the posterior
teeth and an anterior open bite.
Diagnosis
Rheumatoid arthritis is diagnosed on the basis of the clinical
and radiographic findings and confirmatory laboratory tests.
The distinguishing features for rheumatoid arthritis and
degenerative arthritis of the TMJ are shown in Table 45-3.
Treatment
The treatment of rheumatoid arthritis of the TMJ is similar
to that for other joints.7 Anti-inflammatory drugs are used
during the acute phases, and mild jaw exercises are used to
prevent excessive loss of motion when the acute symptoms
subside. In severe cases, disease-modifying drugs, such as
methotrexate, etanercept, infliximab, adalimumab, abata-
cept, and rituximab, also are used. Surgery may be necessary
in patients with an anterior open bite after the disease goes
into remission or in paients in whom ankylosis develops.
Spondyloarthropathies
In addition to the adult and juvenile forms of rheumatoid
arthritis, psoriatic arthritis and ankylosing spondylitis also
can involve the TMJ.8-10
Psoriatic Arthritis
Psoriatic arthritis occurs in a small percentage of patients who
have long-standing cutaneous psoriasis. It can have a sudden
onset, can be episodic in nature, and may show spontane-
ous remission.9 Often only one TMJ is involved. Symptoms
include TMJ pain and tenderness, restricted jaw movement,
and crepitation, mimicking the symptoms of rheumatoid
arthritis.9 The radiographic changes are nonspecific and can-
not be distinguished easily from other types of arthritis, par-
ticularly rheumatoid arthritis and ankylosing spondylitis.10
They usually involve erosive changes in the condyle and
glenoid fossa associated with extreme narrowing of the joint
space.11 In severe cases, ankylosis may develop.12
The diagnosis usually is based on the triad of psoriasis,
radiographic evidence of erosive arthritis, and a negative
serologic test for rheumatoid factor. Even in the presence
of a rash, however, the diagnosis cannot be absolutely con-
firmed. The differential diagnosis always should include
rheumatoid arthritis, Reiter’s syndrome, ankylosing spondy-
litis, and gout.
The treatment of psoriatic arthritis of the TMJ is similar
to that of rheumatoid arthritis, involving the use of disease-
modifying drugs.13 Surgery is necessary if ankylosis occurs.
Ankylosing Spondylitis
About one third of patients with ankylosing spondylitis
develop TMJ involvement several years after the onset of
the disease. Pain and limitation of jaw movement are the
most common symptoms, and ankylosis can develop in
­adv­anced cases.8,14 On radiographic examination, about
30% of patients show erosive changes in the condyle and
fossa and narrowing of the joint space.15 In long-standing
cases, there is sometimes a more florid osteophytic response
during quiescent periods. The severity of the changes seems
to be related to the severity of the disease.
The treatment of ankylosing spondylitis of the TMJ is
generally medical and is part of the total management of
the patient. Physical therapy is used to improve jaw mobil-
ity, and bite appliances are used, when indicated, to reduce
parafunctional stress on the joint. If ankylosis develops,
­surgery is the treatment of choice.
Traumatic Arthritis
Acute trauma to the mandible that does not result in a
fracture still can produce injury to the TMJ. When this
occurs in a child, it is essential to warn the parents about
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
the possibility of future retardation of mandibular growth
and associated facial deformity resulting from damage
to the articular cartilage, which is an important growth
site.16
Traumatic arthritis is characterized by TMJ pain and
tenderness and limitation of jaw movement. The resultant
inflammation and occasional hemarthrosis also can result in
loss of tooth contact on the affected side. Frequently, there
are bruises or lacerations at the site of the initial injury. No
radiographic changes may be seen, or there may be wid-
ening of the joint space owing to intra-articular edema or
hemorrhage. In some instances, radiographs may show an
intracapsular fracture that was not recognized on clinical
examination.
The treatment of traumatic arthritis consists of the use of
nonsteroidal anti-inflammatory drugs, application of heat, a
soft diet, and initial restriction of jaw movement. When the
acute symptoms subside, range-of-motion exercises should
be used to avoid fibrous ankylosis.
Infectious Arthritis
Infectious arthritis rarely involves the TMJ. Although it can
affect the joint as part of such systemic diseases as gonorrhea,
syphilis, tuberculosis, and Lyme disease,17,18 the most com-
mon way is by direct extension of an adjacent infection of
dental, parotid gland, or otic origin. 19 Occasionally, it also
may occur from the localization of blood-borne organisms in
the joint after a traumatic injury or by direct involvement
through a penetrating wound.20
Clinical Findings
Infectious arthritis generally results in unilateral pain, ten-
derness, swelling, and redness in the region of the TMJ.
Chills, fever, sweating, and systemic findings characteristic
of the specific type of infection also are present. There is
often an inability to occlude the teeth because of the swell-
ing within the joint. In pyogenic forms of infectious arthri-
tis, fluctuation may be present in the joint region. Patients
with Lyme disease show characteristic skin lesions.18
Imaging Findings
The radiographic findings are usually normal in the early
stages of the disease because of the lack of bony involve-
ment, but the intra-articular accumulation of pus or inflam-
matory exudate may cause separation of the articulating
surfaces, which can be detected on magnetic resonance
imaging (MRI). Later, depending on the severity and chro-
nicity of the infection, varying degrees of bony destruction,
ranging from damage to the articular surface of the mandib-
ular condyle to extensive osteomyelitis, may be seen. In the
late stages, fibrous or bony ankylosis may occur. In children,
infectious arthritis can affect the growth of the condyle and
result in facial asymmetry.
Treatment
The treatment of infectious arthritis includes the use of
appropriate antibiotics, proper hydration, control of pain,
and limitation of jaw movement. Suppurative infections
669
may require aspiration, incision and drainage, or seques-
trectomy. When there has been extensive bone loss, recon-
structive procedures may be necessary. In children in whom
mandibular growth has been affected, a costochondral graft
can be used to correct the facial asymmetry and re-establish
growth of the mandible.
Metabolic Arthritis
Metabolic arthritis, which can accompany gout or pseudo­
gout (calcium pyrophosphate dehydrate arthropathy), is
rare in the TMJ.21
Gout
Gouty arthritis of the TMJ occurs most frequently in men
older than 40 years and usually is preceded by involvement
of one or more joints of the feet or hands. The attack usu-
ally occurs suddenly, and the joint becomes swollen, pain-
ful, red, and tender. Recovery may occur in a few days, and
remission can last for months to years.
When the attacks are infrequent, there may not be any
radiographic changes for a long time. Because there have
been so few cases reported, the precise radiographic changes
that occur have not been well documented. Calcified areas
in the disk, destruction of the hard tissues of the joint, con-
dylar exostoses and spurring, and the presence of tophi have
been described.21
The initial approach to treatment of gout involving the
TMJ is medical. If the symptoms are not controlled, how-
ever, surgical débridement of the joint and arthroplasty may
be indicated.
Pseudogout
Calcium pyrophosphate dehydrate arthropathy (pseudo­
gout) in the TMJ clinically mimics gout, and the mandib-
ular condyle may show degenerative and erosive changes
radiographically. In the primary form, which usually is
seen in older patients, there is intra-articular calcification
(chondrocalcinosis), and diffuse calcification occurs in the
intra-articular disk.21-25 Similar changes are seen in the sec-
ondary form, but it occurs in younger patients and is fre-
quently preceded by a history of trauma. Just as in gout of
the TMJ, the initial treatment of pseudogout is medical,
and surgery is reserved for patients in whom such treatment
is ineffective.
INTERNAL DERANGEMENTS
Internal derangements are a common cause of pain in the
TMJ. They represent a disturbance in the normal anatomic
relationship between the intra-articular disk and the con-
dyle, resulting in an interference with the smooth move-
ment of the joint.
Clinical Findings
There are three stages of internal derangement: a pain-
less incoordination phase, in which there is a momentary
catching sensation during mouth opening; anterior disk dis-
placement with reduction into the normal position during
 670 LASKIN  |  Temporomandibular Joint Pain
mouth opening, which is characterized by a clicking or
popping sound (Fig. 45-1); and anterior disk displacement
without reduction on attempted mouth opening, which is
characterized by a restriction of jaw movement, or locking
(Fig. 45-2). The joint pain in patients with anterior disk
displacement, with or without reduction, is caused by con-
dylar compression of the highly innervated retrodiskal tis-
sue that occupies the glenoid fossa as the intra-articular disk
assumes a more forward position, and by the accompanying
inflammation.
ETIOLOGY
The three main causes of internal derangement of the intra-
articular disk are trauma, abnormal functional loading of the
joint, and degenerative joint disease.26 It also has been sug-
gested that spasm in the lateral pterygoid muscle, a portion
of which attaches to the anterior aspect of the disk, can lead
to a disk derangement, but the evidence for this theory is cir-
cumstantial. Although some clinicians believe that occlusal
factors also play a role in causing internal derangements, no
conclusive studies have shown such a relationship.
Acute macrotrauma is probably the most common cause
of internal derangement. Among the incidents that have
been implicated are a blow to the jaw, endotracheal intuba-
tion, cervical traction, and iatrogenic stretching of the joint
during dental or oral surgical procedures. Although whip-
lash injuries frequently have been implicated in the etiology
of internal derangement, a study of 155 patients with this
type of injury showed that only one developed clicking in
the TMJ immediately after the automobile accident.27 At
1 month of follow-up, two additional patients of the 129
A structures. In patients in whom the condition causing the
Closing click
F A
B
Opening click
E
C
D
Figure 45-1  Anterior displacement of the intra-articular disk with re-
duction on opening of the mouth. A clicking or popping sound occurs
as the disk returns to its normal position in relation to the condyle. Dur-
ing closure, the disk again becomes anteriorly displaced, sometimes ac-
companied by a second sound (reciprocal click). (Modified from McCarty
W: Diagnosis and treatment of internal derangements of the articular disc
and mandibular condyle. In Solberg WK, Clark GT [eds]: Temporomandibular
Joint Problems: Biologic Diagnosis and Treatment. Chicago, Quintessence,
1980, p 155.)
contacted experienced clicking, but at 1 year, no additional
patients of the 104 contacted had developed clicking.
Although internal derangements of the TMJ can be caused
by a whiplash injury, the incidence seems to be low.
Whether a patient merely develops alterations in the
articular surface leading to a catching or binding sensation,
anterior disk displacement with reduction on mouth opening
(clicking or popping), or anterior disk displacement without
reduction during mouth opening (locking) after trauma to
the TMJ depends on the severity of the injury. Although the
associated traumatic arthritis causes pain during function in
each of these instances, the pain is more severe in the last
two conditions because of compression of the retrodiskal tis-
sue, which is now located in the articular zone.
The functional overloading of the TMJ associated with
the habit of chronic teeth clenching is another frequent
cause of internal derangements. Although the TMJ is con-
structed for eccentric movements, it is not constructed for
the constant isometric loading and unloading that occurs
during this activity. Such parafunction affects the lubrica-
tion of the joint, introducing friction between the disk and
the condyle that leads to degenerative changes in the artic-
ular surfaces and results in gradual anterior displacement of
the disk.26,28
Degenerative joint disease may precede the development
of an internal derangement, or it may occur after the devel-
opment of an internal derangement. In the first instance,
the changes in the character of the articulating surfaces
result in an inability of the parts to glide smoothly over each
other, gradually leading to a forward displacement of the
disk, which normally rotates posteriorly during mouth open-
ing. In the second instance, the displaced disk results in an
altered relationship between the articulating components of
the joint, which leads to the degenerative changes in these
E
B
D
C
Figure 45-2  Anterior displacement of the intra-articular disk without
reduction on attempted mouth opening. The displaced disk acts as a bar-
rier and prevents full translation of the condyle. (Modified from McCarty
W: Diagnosis and treatment of internal derangements of the articular disc
and mandibular condyle. In Solberg WK, Clark GT [eds]: Temporomandibular
Joint Problems: Biologic Diagnosis and Treatment. Chicago, Quintessence,
1980, p 151.)
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN 671

Management of internal with a history of clicking treated by such conservative non­

derangements of the TMJ surgical modalities, which are not directed specifically to
the problems of joint noise or disk displacement, showed
Clicking Locking that the condition worsened in only 1%, indicating that it is
permissible to observe individuals with painless clicking as
long as they remain otherwise asymptomatic.30
Arthrocentesis or In patients with pain and clicking in the TMJ that is
Analgesics arthroscopic lysis
Soft diet unresponsive to nonsurgical management, the disk should
and lavage
Limited jaw function be repositioned arthroscopically or by open surgery (dis-
Bite appliance patients
Bite appliance
with parafuction
koplasty). Patients with parafunctional habits should con-
tinue the use of a bite appliance when sleeping. In patients
with locking (anterior disk displacement without reduc-
Pain Pain Pain Pain tion), whether painful or not, treatment is urgent because
continues relieved continues relieved if the condition is left untreated for a long time, the subse-
Diskoplasty Continue bite Diskoplasty or Continue bite quent management can be complicated by further degenera-
appliance in diskectomy appliance tive changes in the disk and condyle that make disk salvage
patients with Periodic (diskoplasty) impossible. The initial treatment involves
parafunction follow-up joint lavage and lysis of adhesions either arthroscopically
Periodic or by arthrocentesis. The latter involves establishing inlet
follow-up
and outlet portals in the upper joint space with hypodermic
Figure 45-3  Management of internal derangements of the temporo- needles, irrigation with lactated Ringer’s solution to remove
mandibular joint. Patients with painful clicking or locking are treated inflammatory tissue breakdown products and cytokines,
medically initially, whereas patients with locking require surgical inter- and lysis of adhesions by hydraulic distention and manual
vention. manipulation of the joint (Fig. 45-4).31
The results of arthrocentesis parallel the results achieved
with arthroscopic lysis and lavage, and the procedure is less
degenerative joint disease is still active, whether primarily invasive. Although neither of these procedures restores the
or secondarily, it—and the disk derangement—must be disk to its normal position, they do restore disk and joint
treated to resolve the problem completely.

Imaging Findings
Depending on the cause of the internal derangement and its
duration, the radiographs may or may not show any evidence
of degenerative joint disease. Magnetic resonance imaging
shows anterior disk displacement in the closed mouth posi-
tion, however, and a return to a normal disk relationship dur-
ing mouth opening in patients with clicking and popping;
in patients with locking, the disk remains in the anterior
position on attempted mouth opening, and there is limited
movement of the condyle. There also is a small group of
patients with locking who show the intra-articular disk in
normal position when the teeth are in occlusion, rather
than anteriorly displaced, and there is no change in disk A
position when the patient attempts to open the mouth.29 In
such cases, there is adhesion of the disk to the articular emi-
nence preventing translation of the condyle. These patients
differ from patients with anteriorly displaced, nonreducing
disks in that they do not have a history of TMJ clicking
preceding the sudden onset of locking.

Treatment
The initial treatment of patients with painful clicking
or popping in the TMJ consists of a nonsteroidal anti-
inflammatory drug; a soft, nonchewy diet; and the use of
a bite-opening appliance to reduce compression of the ret-
rodiskal tissue (Fig. 45-3). A muscle relaxant drug can be
added to the regimen if the patient has associated myofas- B
cial pain. When the pain has stopped, no further treatment Figure 45-4  Temporomandibular joint arthrocentesis. A, Hypodermic
is necessary, although the joint noise still may be present. needles inserted into the upper joint space to allow lavage of the joint. 
A long-term follow-up study (1 to 15 years) of 190 patients B, Joint being irrigated with lactated Ringer’s solution.
 672 LASKIN  |  Temporomandibular Joint Pain
mobility, and reduce pain and improve function in most
patients.32,33 In these patients, the retrodiskal tissue within
the joint undergoes fibrosis and acts as a pseudodisk. It is
important that patients who have teeth-grinding or teeth-
clenching habits are prescribed a bite appliance postopera-
tively to wear while sleeping.
In patients who do not respond favorably to arthroscopy
or arthrocentesis, repositioning of the displaced disk by an
open operation should be done. If the disk is extremely
deformed and cannot be repositioned, or if there is a large,
nonreparable perforation in the disk or tear in the retrodis-
kal tissue, the disk should be removed. Although autogenous
auricular cartilage or dermal grafts, or temporalis muscle
flaps, have been used as a disk replacement, the results have
been unpredictable.31 More recent long-term studies have
shown that most patients can tolerate a diskless joint.34
Currently, there are no acceptable alloplastic substitutes for
the disk.
NEOPLASMS
Although primary neoplasms involving the TMJ are uncom-
mon, they still need to be considered in the differential
diagnosis of painful conditions affecting this region.35,36
Chondroma, osteochondroma, and osteoma are the most
frequently encountered benign tumors, but isolated cases of
fibro-osteoma, myxoma, fibrous dysplasia, giant cell repara-
tive granuloma, aneurysmal bone cyst, synovioma, synovial
chondromatosis, chondroblastoma, osteoblastoma, glomus
tumor, and synovial hemangioma have been reported.
­Mali­gnant tumors of the TMJ are even rarer, with infrequent
reports of fibrosarcoma, chondrosarcoma, synovial fibrosar-
coma, osteosarcoma, malignant fibrous histiocytoma, malig-
nant schwannoma, leiomyosarcoma, and multiple myeloma.
The TMJ also can be invaded by neoplasms from the cheek,
the parotid gland, the external auditory canal, and the adja-
cent ramus of the mandible. Metastasis to the condyle from
distant neoplasms in the breast, lung, prostate, colon, and
thyroid gland also has been described.
Tumors of the TMJ can cause pain, limitation of jaw
movement, deviation of the mandible to the affected side
on attempted mouth opening, and difficulty in occluding
the teeth. Depending on the nature of the condition, the
radiographs may show bony deformation, apposition, or
resorption. A biopsy is necessary to establish the definitive
diagnosis.
MYOFASCIAL PAIN AND DYSFUNCTION
MPD, or masticatory myalgia, is a psychophysiologic disease
that primarily involves the muscles of mastication and not
the TMJ. Women are affected more frequently than men; the
ratio in various reports ranges from 3:1 to 5:1. Although the
condition can occur in children, the greatest incidence
seems to be in adults 20 to 40 years old. MPD frequently is
confused with painful conditions affecting the TMJ, such
as degenerative arthritis or internal derangements, because
patients with primary MPD can develop these diseases sec-
ondarily, and patients with primary joint disease can develop
secondary MPD. Better understanding of the causes and
pathogenesis of this condition now makes its diagnosis eas-
ier, however, and its treatment more effective.37,38
Etiology
Stress seems to be an important factor in the development
of MPD.39 It is hypothesized that centrally induced increases
in muscle activity, frequently combined with the presence of
parafunctional habits such as clenching or grinding of the
teeth, result in the associated muscle fatigue, pain, and dys-
function.40 Similar symptoms also occasionally can result,
however, from muscle overextension, muscle overcontrac-
tion, or trauma (Fig. 45-5).
Clinical Findings
Pain of unilateral origin is the most common symptom of
MPD. In contrast to the pain associated with joint disease,
which is well localized, the pain of muscle origin is more dif-
fuse. The patient generally is unable to identify accurately
the specific site involved, which can serve as an important
diagnostic criterion in distinguishing between muscle and
joint disorders.
Depending on the muscle involved, the pain associated
with MPD may be described by the patient in various ways.
The masseter is the most frequent muscle involved, and the
patient usually refers to the pain as a jaw ache. The temporalis
is the next most commonly involved muscle, and it produces
pain on the side of the head and is interpreted by the patient
as a headache. Involvement of the lateral pterygoid muscle
produces earache or a deep pain behind the eye, whereas
medial pterygoid involvement causes discomfort on swal-
lowing and the feeling of a painful, swollen gland beneath
the angle of the mandible. Medial pterygoid involvement
also can cause stuffiness or a full feeling in the ear.
The pain associated with MPD is usually constant, but it
is often more severe on arising in the morning or may worsen
gradually as the day progresses. Pain generally is exacerbated
by jaw function, especially during such activities as eating
and excessive talking. Myofascial pain tends to be regional
rather than local, and patients with a long-standing problem
may complain that the pain in the facial region has spread
to the cervical area and later to the shoulders and back.
Muscular
Stress “Dental irritation”
hyperactivity
Muscular Muscular Muscular
overextension fatigue overcontraction
Myofascial pain-dysfunction
Occlusal Internal
Contracture Degenerative
disharmony derangement arthritis
Altered chewing pattern
Figure 45-5  Causes of myofascial pain and dysfunction. Although the
diagram shows three pathways, the one involving psychological stress is
most common. The mechanism by which stress leads to myofascial pain
and dysfunction is termed the psychophysiologic theory. (Modified from
Laskin DM: Etiology of the pain-dysfunction syndrome. J Am Dent Assoc
79:147-153, 1969. Copyright © 1969 American Dental Association. Reprinted
by permission of ADA Publishing Co., Inc.)
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
Tenderness in the muscles of mastication is another com-
mon finding, and the presence of tenderness can be used to
confirm the source of the pain in muscles that are acces-
sible to palpation (masseter, temporalis, and medial ptery-
goid). Although muscle tenderness usually is not reported
by the patient, this symptom can be elicited easily by the
examiner. The most frequent sites of tenderness are near
the angle of the mandible, in the belly and posterosupe-
rior aspect of the masseter, in the anterior temporal region,
and over the temporal crest on the anterior aspect of the
coronoid process. The location of some of the tender areas
suggests that the tendons also may be a source of the pain
and tenderness.
Limitation of mandibular movement is the third cardinal
symptom of MPD. It manifests as an inability to open the
mouth as wide as usual and as a deviation of the mandible to
the affected side when mouth opening is attempted. Lateral
excursion to the unaffected side also is reduced. The limita-
tion of mandibular movement usually is correlated with the
amount of pain present.
A clicking or popping sound in the TMJ is another find-
ing in some patients with MPD. This is not a cardinal sign,
however, because it occurs only in patients with a chronic
teeth-clenching habit, which gradually produces frictional
changes in the joint and subsequent disk displacement.26
The presence of joint sounds alone is insufficient to make
a diagnosis of MPD. The joint sounds must be accompa-
nied by myofascial pain and tenderness in the masticatory
muscles that began before the onset of the joint noise. Such
patients must be distinguished from patients with a primary
internal derangement, in whom muscle splinting produces
myofascial pain and tenderness after the onset of the joint
noise. The history and the difference in physical findings are
helpful in making this distinction.
In addition to having the three cardinal symptoms of
pain, muscle tenderness, and limitation of mouth opening,
patients with MPD usually have no clinical or radiographic
evidence of pathologic changes in the TMJ. These negative
characteristics are important in establishing the diagnosis
because they confirm that the primary site of the problem is
not the articular structures.
Diagnosis
Because the cardinal signs and symptoms of MPD are similar
to those produced by such organic problems involving the
TMJ as degenerative joint disease and internal disk derange-
ment and by a variety of nonarticular conditions (see Tables
45-1 and 45-2), the diagnosis of this condition can be diffi-
cult, requiring a careful history and a thorough clinical eval-
uation. Periapical radiographs of the teeth and screening
radiographs (transcranial, transpharyngeal, or panoramic)
of the TMJs can be helpful in eliminating dental problems
or gross joint disease. If the screening views of the TMJs
show some abnormality, CT scans are usually advisable for
confirmation. MRI also can be useful in determining the
position of the disk when an internal derangement of the
TMJ is being considered. Depending on the suspected con-
dition, other radiographic views of the head and neck and
scintigraphy may be needed to establish a final diagnosis.
Certain laboratory tests may be helpful in some instances.
These include a complete blood cell count if an infection is
673
Table 45-4  Distinguishing Features of Myofascial  
Pain and Fibromyalgia
Myofascial Pain Fibromyalgia
Age distribution 20-40 years 20-50 years
Gender distribution Mainly women Mainly women
Distribution of pain Localized; usually Generalized; bilater-
unilateral ally symmetric
Tender points Few Multiple
Trigger points Uncommon Common
Fatigue Localized muscle Generalized fatigue
fatigue
Sleep disturbance Common Common
suspected; serum calcium, phosphorus, and alkaline phos-
phatase measurements for possible bone disease; serum uric
acid determination for gout; serum creatinine and creatinine
kinase levels as indicators of muscle disease; and erythrocyte
sedimentation rate, rheumatoid factor, latex fixation, and
antinuclear antibody tests for suspected rheumatoid arthri-
tis. Electromyography can be used to evaluate muscle func-
tion. Psychological evaluation and psychometric testing are
good research tools, but have little diagnostic value other
than for determining the presence of any associated abnor-
mal behavioral characteristics.
A condition that sometimes is confused with myofascial
pain is fibromyalgia, particularly when MPD involves sev-
eral regions in addition to the face. Although a small subset
of patients with MPD eventually may develop fibromyal-
gia, they are probably distinct conditions.41 Table 45-4 lists
the distinguishing characteristics of myofascial pain versus
fibromyalgia.
Treatment
The treatment of MPD is divided into four phases.42 When
a definitive diagnosis is made, phase I therapy should be
started (Fig. 45-6). Phase I therapy initially involves provid-
ing the patient with some understanding of the problem.
Because patients often have difficulty accepting a psycho-
physiologic explanation for their condition, the discussion
should deal with the issue of muscle fatigue as the cause of
the pain and dysfunction, delaying consideration of the role
of stress and psychological factors until the symptoms have
improved, and the patient’s confidence has been gained.
Relating the symptoms to the specific masticatory muscles
from which they arise helps the patient understand the rea-
son for the type and location of the pain—headache from
the temporalis muscle, jaw ache from the masseter muscle,
discomfort on swallowing and stuffiness in the ear from the
medial pterygoid muscle, and earache and pain behind the
eye from the lateral pterygoid muscle.
In addition to the initial explanation, the patient should
be counseled regarding home therapy; this includes rec-
ommendations about avoidance of clenching and grind-
ing of the teeth, eating a soft diet, use of moist heat and
massage on the masticatory muscles, and limitation of jaw
movement. A nonsteroidal anti-inflammatory drug should
be prescribed for the pain. In patients who have problems
sleeping, a small dose of amitriptyline at bedtime is helpful
in improving sleep and reducing parafunction.
 674 LASKIN  |  Temporomandibular Joint Pain

About 50% of these patients experience resolution of

their symptoms within 2 to 4 weeks with phase I therapy.
For patients whose symptoms persist, phase II therapy is ini-
tiated. Home therapy and medications are continued, and a
bite appliance is made for the patient. Although numerous
types have been used, the Hawley-type maxillary appliance
is probably most effective because it prevents contact of the
posterior teeth and prevents most forms of parafunctional
activity (Fig. 45-7).43 The appliance generally is worn at

Management of Myofascial Pain and Dysfunction

Phase I Therapy (2-4 weeks)

Initial explanation of the problem

Home therapy
Figure 45-7  Hawley-type maxillary bite appliance. Only the anterior
Medication for pain and sleep teeth contact the appliance, and there is space between the posterior
Symptoms teeth (arrow).
Symptoms persist eliminated

Phase II Therapy
night, but it can be worn for 5 to 6 hours during the day,
(2-4 weeks) if necessary. The appliance should not be worn continu-
Phase out therapy
ously, however, because the posterior teeth may supraerupt
Reevaluate diagnosis Symptoms Final explanation in some patients.
eliminated of problem
Check for compliance With phase II therapy, another 20% to 25% of patients
Instructions for become symptom-free in 2 to 4 weeks. When the patient
Continue home therapy
and medications
self-management becomes symptom-free, the medications are stopped first,
Follow-up and wearing the bite appliance is discontinued next. If the
Prescribe a bite appointments patient has a return of symptoms, and the appliance is worn
appliance
only at night, its use can be continued indefinitely.
Patients who do not respond to the use of a bite appli-
Symptoms persist ance are entered into phase III treatment for 4 to 6 weeks.
In this phase, either physical therapy (heat, massage, ultra-
Symptoms
sound, electrogalvanic stimulation) or relaxation therapy
eliminated
Phase III Therapy (4-6 weeks) (electromyographic biofeedback, conditioned relaxation) is
Continue home therapy
added to the regimen. There is no evidence to show that
and medications one form of treatment is better than the other, and either
can be used first. If one is unsuccessful, the other can be
Reevaluate the bite appliance
tried. Phase III therapy usually helps another 10% to 15%
Initiate physical therapy of the patients.
or relaxation therapy If all of these approaches fail, and there is no question
about the correctness of the diagnosis, psychological coun-
Symptoms persist seling is recommended. This counseling involves helping
patients identify possible stresses in their lives and learning
to cope with such situations. If the diagnosis is in doubt, the
Phase IV Therapy patient should be referred first for appropriate dental and
neurologic consultation and re-evaluation. Another alter-
Consultation Pain center
native is to refer patients with recalcitrant MPD to a TMJ
center or pain clinic because such patients generally require
a multidisciplinary approach for successful treatment.
Psychological
counseling
SUMMARY
Figure 45-6  Management of myofascial pain and dysfunction. The
treatments are divided into four phases. If the symptoms are eliminated The successful management of patients with temporo-
in any of the first three phases, the ongoing therapy is gradually phased mandibular disorders depends on establishing an accurate
out, and the patient is instructed in continued self-management of the
condition. (Modified from Laskin DM, Block S: Diagnosis and treatment of
diagnosis and using proper therapy based on an under-
myofascial pain dysfunction [MPD] syndrome. J Prosthet Dent 56:75-84, standing of the etiology of the condition being treated.
1986.) Of particular importance is separating patients with MPD,
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
who constitute the major group encountered and who are
not surgical candidates, from patients with TMJ disease,
who frequently require surgical treatment. Even in the lat-
ter group, many commonly encountered conditions, such
as arthritis and internal disk derangements, often respond
to nonsurgical therapy, and this type of treatment should
be given a fair trial before more aggressive management is
considered.
REFERENCES
  1. Merjersjo C: Therapeutic and prognostic considerations in TMJ osteo-
arthrosis: A literature review and a long-term study in 11 subjects.
J Craniomandib Pract 5:70, 1987.
  2. Madsen B: Normal variations in anatomy, condylar movements and
arthrosis frequency of the TMJs. Acta Radiol 4:273, 1966.
  3. Milam SB, Zardeneta G, Schmitz JP: Oxidative stress and degen-
erative temporomandibular joint disease. J Oral Maxillofac Surg
56:214, 1998.
  4. Haddad IK: Temporomandibular joint osteoarthritis: Histopathologi-
cal study of the effects of intra-articular injection of triamcinolone
acetonide. Saudi Med J 21:675, 2000.
  5. Bjornland T, Gjaerum AA, Moystad A: Osteoarthritis of the tem-
poromandibular joint: An evaluation of the effects and complica-
tions of corticosteroid injection compared with injection with
sodium hyaluronate. J Oral Rehabil 34:583, 2007.
  6. Abubaker AO, Laskin DM: Nonsurgical management of arthritis of
the temporomandibular joint. Oral Maxillofac Surg Clin N Am 7:1,
1995.
  7. Zide MF, Carlton D, Kent JH: Rheumatoid arthritis and related
arthropathies: Systemic findings, medical therapy, and peripheral joint
surgery. Oral Surg Oral Med Oral Pathol 61:119, 1986.
  8. Davidson C, Wojtulewsky JA, Bacon PA, et al: Temporomandibular
joint disease in ankylosing spondylitis. Ann Rheum Dis 34:87, 1975.
  9. Wilson A, Braunwald E, Issilbacker KJ: Psoriatic arthropathy of the
temporomandibular joint. Oral Surg Oral Med Oral Pathol 70:555,
1990.
10. Kononen M: Radiographic changes in the condyle of the temporo-
mandibular joint in psoriatic arthritis. Acta Radiol 28:185, 1987.
11. Koorbusch GF, Zeitler DL, Fotos PG, et al: Psoriatic arthritis of the
temporomandibular joint with ankylosis. Oral Surg Oral Med Oral
Pathol 71:267, 1991.
12. Miles DA, Kaugers GA: Psoriatic involvement of the temporoman-
dibular joint: Literature review and report of two cases. Oral Surg Oral
Med Oral Pathol 71:770, 1991.
13. Wenneberg B: Inflammatory involvement of the temporomandibular
joint: Diagnostic and therapeutic aspects and a study of individuals
with ankylosing spondylitis. Swed Dent J Suppl 20:1, 1983.
14. Salvarini C, Cantini F, Olivieri I: Disease-modifying antirheumatic
drug therapy for psoriatic arthritis. Clin Exp Rheumatol 20(Suppl
28):S71, 2002.
15. Wenneberg B, Hollender L, Kopp S: Radiographic changes in the
temporomandibular joint in ankylosing spondylitis. Dentomaxillofac
Radiol 12:25, 1983.
16. Harris S, Rood JP, Testa HJ: Post-traumatic changes of the temporo-
mandibular joint by bone scintigraphy. Int J Oral Maxillofac Surg
17:173, 1988.
17. Hanson TL: Pathological aspects of arthritides and derangements. In
Sarnat BG, Laskin DM (eds): The Temporomandibular Joint: A Bio-
logical Basis for Clinical Practice, 4th ed, Philadelphia, WB Saunders,
1992, pp 165-182.
18. Lesnicar DG, Zerdoner D: Temporomandibular joint involvement
caused by Borrelia burgdorferi. J Craniomaxillofac Surg 35:397, 2007.
20. Leighly SM, Spach DH, Myall RW, et al: Septic arthritis of the tem-
poromandibular joint: Review of the literature and report of two cases
in children. Int J Oral Maxillofac Surg 22:292, 1993.
21. Gross BD, Williams RB, DiCosimo CT, et al: Gout and pseudogout
of the temporomandibular joint. Oral Surg Oral Med Oral Pathol
63:551, 1987.
22. Nakagawa Y, Ishibashi K, Kobayoshi K, et al: Calcium phosphate
deposition disease in the temporomandibular joint: Report of two
cases. J Oral Maxillofac Surg 57:1357, 1999.
675
23. Chuong R, Piper MA: Bilateral pseudogout of the temporomandibular
joint: Report of a case and review of the literature. J Oral Maxillofac
Surg 53:691, 1995.
24. Aoyama S, Kino K, Amagosa T, et al: Differential diagnosis of calcium
pyrophosphate dihydrate deposition of the temporomandibular joint.
Br J Oral Maxillofac Surg 38:550, 2000.
25. Ascani G, Pieramici MD, Fiosa A, et al: Pseudogout of the temporo-
mandibular joint: A case report. J Oral Maxillofac Surg 66:386, 2008.
26. Laskin DM: Etiology and pathogenesis of internal derangements
of the temporomandibular joint. Oral Maxillofac Surg Clin N Am
6:217, 1994.
27. Heise AP, Laskin DM, Gervin AS: Incidence of temporomandibu-
lar joint symptoms following whiplash injury. J Oral Maxillofac Surg
50:825, 1992.
28. Nitzan DW: The process of lubrication impairment and its involve-
ment in temporomandibular disk displacement: A theoretical con-
cept. J Oral Maxillofac Surg 59:36, 2001.
29. Nitzan DW, Samson B, Better H: Long-term outcome of arthrocente-
sis for sudden-onset persistent, severe closed lock of the temporoman-
dibular joint. J Oral Maxillofac Surg 55:151, 1997.
30. Greene CS, Laskin DM: Long-term status of TMJ clicking in patients
with myofascial pain and dysfunction. J Am Dent Assoc 117:461,
1988.
31. Laskin DM: Surgical management of internal derangements. In Laskin
DM, Greene CS, Hylander WL (eds): Temporomandibular Disor-
ders: An Evidence-Based Approach to Diagnosis and Treatment.
Chicago, Quintessence, 2006, pp 469-481.
32. Dimitroulis G: A review of 55 cases of chronic closed lock treated with tem-
poromandibular joint arthroscopy. J Oral Maxillofac Surg 60:519, 2002.
33. Carvajal W, Laskin DM: Long-term evaluation of arthrocentesis for
treatment of internal derangement of the temporomandibular joint.
J Oral Maxillofac Surg 58:852, 2000.
34. Eriksson L, Westesson PL: Discectomy as an effective treatment for
painful temporomandibular joint internal derangement: A 5 year
clinical and radiographic follow-up. J Oral Maxillofac Surg 59:750,
2001.
35. Stern D: Benign and malignant tumors. In Laskin DM, Greene CS,
Hylander WL (eds): Temporomandibular Disorders: An Evidence-
Based Approach to Diagnosis and Treatment. Chicago, Quintessence,
2006, pp 319-333.
36. Clayman L: Surgical management of benign and malignant neo-
plasms. In Laskin DM, Greene CS, Hylander WL (eds): Temporo-
mandibular Disorders: An Evidence-Based Approach to Diagnosis
and Treatment. Chicago, Quintessence, 2006, pp 509-532.
37. Laskin DM: Diagnosis and etiology of myofascial pain and dysfunc-
tion. Oral Maxillofac Surg Clin N Am 7:73, 1995.
38. Clark GT: Treatment of myogenous pain and dysfunction. In Laskin
DM, Greene CS, Hylander WL (eds): Temporomandibular Disorders:
An Evidence-Based Approach to Diagnosis and Treatment. Chicago,
Quintessence, 2006, pp 483-500.
39. Dworkin SF: Psychological and psychosocial assessment. In Laskin
DM, Greene CS, Hylander WL (eds): Temporomandibular Disor-
ders: An Evidence-Based Approach to Diagnosis and Treatment.
Chicago, Quintessence, 2006, pp 203-217.
40. Laskin DM: Etiology of the pain-dysfunction syndrome. J Am Dent
Assoc 59:147, 1969.
41. Cimino R, Michelotti A, Stradi R, et al: Comparison of the clinical
and psychologic features of fibromyalgia and masticatory myofascial
pain. J Orofac Pain 12:35, 1998.
42. Laskin DM, Block S: Diagnosis and treatment of myofascial pain-
dysfunction (MPD) syndrome. J Prosthet Dent 56:75, 1986.
43. Clark GT, Minakuchi H: Oral appliances. In Laskin DM, Greene CS,
Hylander WL (eds): Temporomandibular Disorders: An Evidence-
Based Approach to Diagnosis and Treatment. Chicago, Quintes-
sence, 2006, pp 377-390.
Internet Links
1. www.nicdr.nih.gov. General information, clinical trials, and sponsored
research in TMJ and related areas
2. www.aaoms.org. General information about TMJ surgery
3. www.tmj.org. Advocate group that provides general information for
patients
 46
Key Points
The symptoms of uveitis vary widely based on the location of
the inflammation within the eye and the suddenness of onset.
Ankylosing spondylitis is the systemic disease most often
associated with uveitis in North America and Europe.
During a lifetime, about 40% of patients with ankylosing
spondylitis develop acute anterior uveitis.
The uveitis associated with HLA-B27 tends to be unilateral,
recurrent, and sudden in onset. Recurrences sometimes affect
the opposite eye.
Sarcoidosis frequently manifests as a uveitis.
Most patients with retinal vasculitis do not have a systemic
vasculitis.
Many patients with scleritis have a systemic disease, such as
rheumatoid arthritis.
Antineutrophilic cytoplasmic antibody testing helps to
­identify a subset of patients with severe scleritis.
Wegener’s granulomatosis is the rheumatic disease that most
frequently involves the orbit.
Anterior ischemic optic neuropathy is the most common
ocular manifestation of temporal arteritis.
Most patients with visual loss secondary to optic nerve
­ischemia do not have arteritis.
Virtually all of the systemic inflammatory diseases that
require rheumatologic care tend to affect the eye or its sur-
rounding structures. Table 46-1 presents the prototypic ocu-
lar manifestations of rheumatoid arthritis, systemic lupus
erythematosus, Sjögren’s syndrome, spondyloarthropathies,
vasculitides including Wegener’s granulomatosis and tem-
poral arteritis, scleroderma, Behçet’s syndrome, relapsing
polychondritis, and dermatomyositis. Each of these diseases
is addressed elsewhere in this text; this chapter focuses on
specific ocular structures—the uvea, cornea, orbit, and optic
nerve—and illustrates how inflammation of each might
relate to an autoimmune or inflammatory process.
OCULAR ANATOMY AND PHYSIOLOGY
A diagram of the eye is shown in Figure 46-1. The eye is a
tiny, but elegantly complex structure. The anterior segment
of the eye includes the cornea, which is avascular and trans-
parent when healthy. The lens also is an avascular structure.
The anterior chamber is filled with aqueous humor, which
has homology to cerebrospinal fluid. When the blood-
­aqueous barrier is intact, the aqueous humor contains no
  Supplemental images available on the Expert Consult Premium Edition website.
The Eye and Rheumatic
Diseases
James T. Rosenbaum
leukocytes and very little protein. The blood-aqueous bar-
rier, which resembles the blood-synovial barrier, is disrupted
in anterior uveitis. In this case, a routine, noninvasive bio-
microscopic or slit lamp examination would reveal leuko-
cytes and increased protein in the anterior chamber. An
ophthalmologist has the opportunity to observe two univer-
sal hallmarks of inflammation noninvasively.
The term uvea derives from the Latin word for “grape.”
The anterior uvea includes the iris and the ciliary body. The
aqueous humor is synthesized by the ciliary body. The pos-
terior portion of the uvea is the choroid, which is a highly
vascular tissue just posterior to the retina. Any portion of
the uveal tract could become inflamed; adjacent tissue also
is frequently inflamed. Anatomic subsets of uveitis include
anterior uveitis, which includes iritis or iridocyclitis (ciliary
body inflammation); intermediate uveitis, in which leuko-
cytes are present within the vitreous humor; and posterior
uveitis, in which the choroid and retina are inflamed. A
panuveitis means that all portions of the uveal tract are
inflamed. An attempt has been made to standardize the
nomenclature used to describe uveitis by the Standardiza-
tion of Uveitis Nomenclature working group,1 although
ambiguities still persist because not all ophthalmologists fol-
low these definitions as yet.
Signs and symptoms of uveitis depend on the portion of
the uveal tract that is affected. An anterior uveitis, espe-
cially if it begins suddenly, is associated with redness, pain,
and photophobia. Visual loss varies and is often due to
macular edema if present (Figs. 46-2 and 46-3). An inter-
mediate uveitis usually causes floaters owing to the leuko-
cytes that enter the visual axis, although most floaters are
due to aging or other changes within the vitreous humor.
A posterior uveitis by itself does not usually produce pain or
­redness. Visual loss depends on the location and extent of
the inflammatory process.
The outer tunic of the eye is known as the sclera. At
the front of the eye, the sclera meets the cornea at a tissue
known as the limbus. The most interior layer of the eye is
an extension of the brain that responds to visual signals,
the retina. The eye shares some common features with the
joint, including the presence of hyaluronic acid primarily
in the vitreous humor and the presence of type II collagen,
although ocular inflammation is not a reported accompani-
ment of collagen-induced arthritis.
OCULAR IMMUNE RESPONSE
The eye generally is regarded as an immune privileged site.2
From a teleologic perspective, many scientists believe that
the eye has evolved mechanisms to avoid becoming inflamed
because of the consequences this has for visual acuity.
677
 678 Rosenbaum  |  The Eye and Rheumatic Diseases

Table 46-1  Most Characteristic Ocular Findings  

of Selected Rheumatic Diseases
Disease
Rheumatoid arthritis
Systemic lupus   Sicca
erythematosus
Sjögren’s syndrome
Spondyloarthritis
Wegener’s granulomatosis
Temporal arteritis
Scleroderma
Behçet’s disease
Relapsing polychondritis
Dermatomyositis
Most Characteristic Eye Findings
Sicca
Scleritis
Cotton-wool spots
Sicca
Acute anterior uveitis
Scleritis
Orbital inflammation
Anterior ischemic optic neuropathy
Sicca
Uveitis, retinal arteritis
Scleritis, episcleritis, uveitis
Heliotrope eyelids
Figure 46-2  Fluorescein angiogram. The normal macula is avascular
and does not stain with the fluorescein dye. This patient has macular
edema indicated by the donut-shaped pattern of dye in the center of the
Retina
photo. The optic nerve is at the 3-o’clock position in the photo. Macular
edema can complicate uveitis, even an anterior uveitis.
Macula

Cornea

Lens Optic nerve

Figure 46-1  Diagram of the eye.
­ imilar to the brain, the internal portion of the eye has no
S
lymphatics, although the conjunctiva on the ocular surface
has lymphatic drainage. Portions of the eye are avascular—
the cornea and lens. The aqueous humor contains several
factors that are known to be immunosuppressive, including
transforming growth factor-β and α-melanocyte-stimulat-
ing hormone. Several tissues within the eye express ligands
that promote apoptosis, including TRAIL and Fas ligand.
If a soluble antigen is injected into the anterior chamber, a
cellular immune response is suppressed. This phenomenon
is known as ACAID, anterior chamber–associated immune
deviation. These factors are important to consider in the
effort to understand why the eye is sometimes targeted as
part of an immune or inflammatory disease.
UVEITIS
Rheumatologists may be consulted to identify a systemic
disease in a patient with uveitis, and a rheumatologist often
is asked to assist in the management of immunosuppression
in selected patients with uveitis. In some referral practices
for patients with uveitis, 40% of patients might have an
associated systemic illness. Table 46-2 lists the differential
diagnoses of uveitis. The immunologic diseases most likely
to be associated with uveitis are listed in Table 46-3.
Figure 46-3  Ocular coherence tomography produces precise imaging
of retinal structure. The ovoid black hole in the center of the image is due
to macular edema, a major cause of visual loss in patients with uveitis.
Table 46-2  Differential Diagnosis of Uveitis
Infections—toxoplasmosis, syphilis, herpes simplex, herpes zoster,
and cytomegalovirus
Systemic, immune-mediated diseases
Masquerade syndromes such as lymphoma
Syndromes confined to the eye, such as pars planitis, birdshot  
chorioretinopathy, and serpiginous choroiditis
The most common systemic illness associated with uveitis
in most North American practices is ankylosing spondylitis.
From an epidemiologic perspective, anterior uveitis is more
common than posterior or intermediate uveitis.3 About 50%
of individuals who develop an anterior uveitis are HLA-
B27+.4 The uveitis associated with HLA-B27 is almost
always unilateral, is recurrent, is of relatively short dura-
tion (<3 months per attack), resolves completely between
attacks, and is associated with reduced intraocular pressure
(in contrast to herpes simplex, which can cause a recur-
rent anterior uveitis associated with increased ­ intraocular
pressure).5 Hypopyon or pus in the anterior chamber is
sometimes present in patients with HLA-B27–associated
uveitis (Fig. 46-4). Recurrent episodes can affect the contra-
lateral eye, but simultaneous bilateral involvement is rare.
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
Table 46-3  Immune-Mediated Diseases Most Often  
Associated with Uveitis
Ankylosing spondylitis
Behçet’s disease
Drug/hypersensitivity reactions
Familial granulomatosus synovitis
Inflammatory bowel disease
Interstitial nephritis
Juvenile idiopathic arthritis
Multiple sclerosis
Neonatal onset multisystem inflammatory disease
Psoriatic arthritis
Reactive arthritis
Sarcoidosis
Sweet’s syndrome
Systemic lupus erythematosus
Vasculitis, especially Cogan’s syndrome and Kawasaki disease
Vogt-Koyanagi-Harada syndrome
Figure 46-4  The creamy material at the bottom of the pupil is an
a­ ccumulation of leukocytes or hypopyon.
Many studies have tried to address the question of how fre-
quently a patient with HLA-B27–associated anterior uve-
itis has an associated spondyloarthropathy. A wide range of
answers have been suggested, and the percentage depends on
the definition of spondyloarthropathy, but one reasonable
estimate is that 80% of HLA-B27+ patients with acute ante-
rior, unilateral uveitis have associated spondyloarthropathy.5
The uveitis associated with reactive arthritis is indistin-
guishable from the uveitis associated with ankylosing spondy-
litis. In either of these entities, about 40% of patients develop
acute anterior uveitis during a lifetime. Although conjunctivi-
tis is part of the classic triad of reactive arthritis (in association
with arthritis and nongonococcal urethritis), conjunctivitis is
uncommon. A genome-wide screen for susceptibility genes
for acute anterior uveitis identified loci that also predispose to
ankylosing spondylitis and loci that seem to be unassociated
with susceptibility to ankylosing spondylitis.6
Approximately 5% of patients with inflammatory bowel
disease and 7% of patients with psoriatic arthritis also
679
Figure 46-5  Keratic precipitates. The white dots result from concre-
tions of cells depositing against the corneal endothelium. These keratic
precipitates are large and usually described as granulomatous even
though a granuloma is not present histologically.
develop uveitis. Although some of these patients have dis-
ease that is unilateral, anterior, and recurrent, many have
disease that is bilateral, chronic in duration, and posterior to
the lens.7,8 About half of all patients with Crohn’s disease or
psoriatic arthritis and uveitis are HLA-B27+.
Sarcoidosis is the second most common systemic dis-
ease associated with uveitis, at least in North America, and
in some geographic areas it might be more common than
spondyloarthritis. Sarcoidosis is promiscuous within the eye,
meaning that it can affect a wide range of structures, includ-
ing the orbit, lacrimal gland, anterior uvea, vitreous humor,
choroid, retina, or optic nerve. The ocular inflammation
with sarcoidosis frequently is termed granulomatous because
large collections of cells deposit on the back of the cornea
(Fig. 46-5). A retinal vasculitis is frequently a prominent fea-
ture of sarcoidosis, even though systemic vasculitis is not a
typical systemic feature of the disease; this results partly from
the manner in which vasculitis is diagnosed in the retina.
Histologic evidence of vessel wall destruction is rarely
obtained because of the morbidity of a retinal biopsy. Instead,
a retinal vasculitis is diagnosed on the basis of perivascular
sheathing along a vessel as seen on funduscopic examina-
tion (Fig. 46-6), intraretinal hemorrhages that must be
secondary to vascular injury, and fluorescein angiography
indicating increased vascular permeability.9 The term reti-
nal vasculitis is misleading to most rheumatologists because
the classic systemic vasculitides, such as polyarteritis nodosa
and Wegener’s granulomatosis, rarely are associated with
retinal vasculitis.
Sarcoidosis frequently manifests initially as an ocular
problem.10 An ocular symptom is the initial manifestation
almost as frequently as a lung symptom. Sarcoidosis fre-
quently involves the conjunctiva, which is an accessible tis-
sue for biopsy confirmation of the diagnosis. In most series of
patients with uveitis, about 30% of the patients have uveitis
that defies placement within a diagnostic category.11 Many
of these patients may have sarcoidosis that is difficult to find
outside the eye. The sensitivity and specificity of studies
such as a serum angiotensin-converting enzyme level or gal-
lium scan for sarcoid that is primarily ocular are unknown.
The author routinely obtains a chest computed tomography
 680 Rosenbaum  |  The Eye and Rheumatic Diseases
Figure 46-6  Retinal vasculitis. Arrows indicate areas of vascular sheathing
or occlusion.
(CT) scan to look for symmetric hilar adenopathy in any
patient who has a uveitis of unknown etiology.12
Juvenile idiopathic arthritis comprises several ­ different
diseases. Patients with juvenile ankylosing spondylitis
resemble their adult counterparts in that they can develop
a sudden-onset, unilateral anterior uveitis. The subset of
juvenile idiopathic arthritis that is most classically associ-
ated with uveitis tends to be girls with the onset of arthritis
between 2 and 8 years old.13 The joint disease is pauciarticu-
lar, and most patients are antinuclear antibody positive. The
uveitis tends to have an insidious onset such that pain and
redness are almost always absent. The joint disease can be
minimal as well, so that some patients are not diagnosed
until a visual screening examination is performed when
starting school. The eye disease is usually bilateral and very
persistent, although remissions are now well described. Band
keratopathy, which is the deposition of calcium superficially
in the cornea, is a well-known and frequent complication
of this form of uveitis (Fig. 46-7). Patients also may develop
glaucoma and posterior synechiae, a term that describes
adhesions of the iris to the lens.
Other forms of uveitis associated with joint disease
include Behçet’s syndrome, relapsing polychondritis, and
vasculitis such as Cogan’s syndrome and Kawasaki disease.
In Behçet’s syndrome, uveitis is often the symptom that
“drives” the therapy; that is, it is often the manifestation
that most requires systemic immunotherapy.14 Eye inflam-
mation is usually bilateral and recurrent. In contrast to the
recurrences typical of ankylosing spondylitis, recurrences of
uveitis with Behçet’s syndrome usually do not have com-
plete resolution between attacks. A hallmark of Behçet’s
syndrome–associated uveitis is a retinal vasculitis. Retinal
arteries are especially prone to be affected. The visual prog-
nosis with Behçet’s syndrome can be grim, and blindness is a
frequent concomitant of untreated ocular disease.
Relapsing polychondritis can have an impact on almost
any portion of the eye, including the episclera, sclera, and
uveal tract.15 Ocular inflammation is common.
Cogan’s syndrome is classically defined as sensorineural
hearing loss with corneal disease, especially an interstitial
keratitis. This definition is usually broadened to include
any ocular inflammatory process, such as uveitis or scleritis.
Although uveitis can occur with polyarteritis or Wegener’s
Figure 46-7  Band keratopathy is illustrated by the calcific patches
stretching across the cornea.
granulomatosis, scleral disease is more typical. In contrast,
anterior uveitis in association with conjunctivitis is present
in most patients with Kawasaki’s disease.
Uveitis and arthritis occasionally can result from an infec-
tion such as Whipple’s disease or Lyme disease. Uveal involve-
ment with Lyme disease is described, but extremely rare.
Some autoinflammatory diseases are associated with
uveitis. Autoinflammatory diseases are characterized by
widespread inflammation in the absence of detectable auto-
antibodies. Many autoinflammatory syndromes respond
dramatically to inhibition of interleukin-1. Blau syndrome,
which also is known as familial granulomatous synovitis,
results from a single base change in the nucleotide bind-
ing domain of the CARD15 gene, which is also known as
NOD2.16 Polymorphisms elsewhere in this same gene pre-
dispose to Crohn’s disease. Blau syndrome is characterized
by the childhood onset of uveitis, arthritis, and dermatitis.
Inflammation in additional organ systems also has been
described. The disease is autosomal dominant. The histo-
pathology of affected skin or joint can show noncaseating
granuloma as in sarcoidosis. Lung involvement has not
been described in Blau syndrome, however. Many patients
thought to have so-called early-onset sarcoid have now
been shown by gene sequencing to have new mutations in
the NOD2 gene.17
Neonatal-onset multisystem inflammatory disease
(NOMID), which also is known as chronic infantile neu-
rologic cutaneous articular syndrome, is an autosomal domi-
nant autoinflammatory syndrome. Ocular involvement in
NOMID is more variable than in Blau syndrome. Charac-
teristic findings include papilledema and uveitis.18
The therapy of uveitis depends on multiple factors, such
as severity, location within the eye, patient preference, and
the specific diagnosis (e.g., Behçet’s syndrome might be espe-
cially responsive to either infliximab19 or interferon alfa20).
For noninfectious causes of uveitis that involve the anterior
portion of the eye, treatment usually begins with topical
corticosteroids and often dilating drops to prevent posterior
synechiae and to relieve spasm of the ciliary muscle. Peri-
ocular corticosteroid injections, usually with triamcinolone,
are given for inflammation posterior to the lens that is not
responding to topical medication. Local corticosteroids can
increase intraocular pressure, induce cataracts, interfere
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
Figure 46-8  Scleral nodule. Active scleritis is present superior to the
limbus. In this patient, the scleritis has taken on a nodular configuration.
Figure 46-9  Scleromalacia has resulted in a bluish ulceration in the
sclera.
with the response to infection, and delay wound healing. A
long-lasting corticosteroid implant containing fluocinolone
has been approved by the Food and Drug Administration.21
All patients who elect this type of therapy develop a cata-
ract if the lens has not already been surgically removed, and
most patients develop glaucoma, many requiring surgery to
control the intraocular pressure.
Systemic immunosuppressive therapy generally is reserved
for patients with active, noninfectious causes of inflam-
mation. For systemic immunosuppression to be indicated,
usually the inflammation is bilateral and severe enough to
interfere with activities of daily living. A variety of immu-
nomodulatory medications have been tried to treat intra-
ocular inflammation, including azathioprine, chlorambucil,
cyclophosphamide, cyclosporine, daclizumab, infliximab,
methotrexate, mycophenolate mofetil, and tacrolimus. The
optimal choice depends on many factors, not the least of
which is the empiric result of any therapeutic approach.
Maintaining a treatment for uveitis usually requires some
efficacy in association with good tolerability. In this regard,
at least one study found methotrexate to be superior to other
antimetabolites.22 Because of the range of diseases being
treated and the range of clinical response, there is room for
681
Figure 46-10  Corneal melt. The white light of the slit lamp beam nar-
rows over the peripheral cornea where the tissue is thin.
all of these options in the therapeutic armamentarium of a
uveitis clinic.
SCLERITIS AND CORNEAL MELT
Scleritis often is divided into five categories: diffuse anterior,
nodular, necrotizing, scleromalacia perforans, and posterior
(Fig. 46-8). The first three categories each result in a red,
painful eye. Pain is more variable in scleromalacia perforans,
in which a nodule pathologically similar to a rheumatoid
nodule forms in the sclera (Fig. 46-9). Pain also varies with
posterior scleritis, and because the sclera extends back to
the optic nerve, posterior scleritis can occur in a localized
fashion such that the eye is not red. Because of the risk of
perforation, the sclera is not normally biopsied, but biopsy
studies have shown that scleritis is often a granulomatous
inflammation of scleral tissue.23
Patients with scleritis can develop complications within
the eye, including uveitis, glaucoma, optic nerve edema, and
retinal or choroidal distortion. A corneal melt or peripheral
thinning of the cornea sometimes develops in individuals
with severe scleritis and represents a potentially blinding
complication of the disease (Fig. 46-10).
About 50% of patients with scleritis have an associated
systemic illness.24 The most common such illness is rheuma-
toid arthritis. Generally, this is long-standing, seropositive
rheumatoid arthritis. These patients may have associated
nodules, vasculitis, or pleuropericarditis. They have a short-
ened life expectancy compared with other patients with
rheumatoid arthritis.25 It is unusual for scleritis to be an ini-
tial manifestation of rheumatoid arthritis.
Wegener’s granulomatosis is probably the second most
common disease associated with scleritis. In contrast to
rheumatoid arthritis, scleritis can be the initial manifes-
tation of Wegener’s granulomatosis, and our routine is to
obtain antineutrophilic cytoplasmic antibody serology on
any patient who presents with scleritis without an obvious
systemic disease association. Other systemic associations
with scleritis include inflammatory bowel disease, relapsing
polychondritis, other vasculitides including temporal arte-
ritis, and ankylosing spondylitis. Infections are a rare but
possible cause of scleritis. Tophaceous gout also has been
reported as a rare cause of scleritis.
 682 Rosenbaum  |  The Eye and Rheumatic Diseases
Scleritis tends to be a painful and persistent disease that
often lasts for years. In contrast, episcleritis involves more
superficial tissue and is usually transient. Episcleritis may be
a feature of rheumatoid arthritis, although many patients
with episcleritis may not have any associated systemic illness.
Complications within the eye, such as glaucoma or uveitis,
are absent. Mild discomfort, rather than frank pain, is the
usual presenting symptom. In contrast to scleritis, patients
with episcleritis have vessels that constrict completely after
2.5% phenylephrine (Neo-Synephrine) is placed on the sur-
face of the eye.
Some patients with scleritis, especially patients who do
not have an associated systemic illness, are treated ade-
quately by an oral nonsteroidal anti-inflammatory drug.
Some experts treat scleritis with locally injected corticoste-
roids, but this should be avoided if the sclera is thin (necro-
tizing disease). In addition, corticosteroid has the theoretical
risk of promoting thinning. The usual option for patients
who do not respond to nonsteroidal anti-inflammatory
drugs is oral prednisone. Some patients can be maintained
on low doses of prednisone, but many require the addition
of an antimetabolite as a steroid-sparing drug. A substan-
tial subset of patients with scleritis, especially patients who
are positive for antineutrophilic cytoplasmic antibody, are
best managed with an alkylating agent, such as cyclophos-
phamide. Because of the risk of this therapeutic approach,
this drug is often given until the disease is in remission for
several months, and then the therapy is switched to an anti-
metabolite to maintain the disease-free state.
ORBITAL DISEASE
Graves’ disease is the most common orbital inflammatory
disease and generally results in an orbital myositis that can
be identified on imaging such as CT, ultrasound, or mag-
netic resonance imaging (MRI). From a rheumatologic
perspective, Wegener’s granulomatosis is the disease that
most commonly affects the orbit. The inflammation can be
extremely painful and may result in blindness. Inflamma-
tion is sometimes more recalcitrant to therapy than other
aspects of Wegener’s granulomatosis. A small series sug-
gested that rituximab may be efficacious in patients with
Wegener’s granulomatosis, including patients with orbital
involvement.26
Orbital pseudotumor, or nonspecific orbital inflammatory
disease, is a diagnosis of exclusion that is made on the basis
of objective orbital swelling as documented by imaging and
a biopsy showing an inflammatory process that cannot be
ascribed to another process such as Graves’ disease. A biopsy
specimen of the orbit is not always obtained, but it can be
useful in ruling out lymphoma or a metastatic malignancy
as the cause of the proptosis. Methotrexate is a therapeutic
option to treat nonspecific orbital inflammation.27 Other
systemic diseases that affect the orbit commonly include
sarcoidosis.
OPTIC NEURITIS
Optic nerve disease can result from many insults, includ-
ing toxins (some of which are medications), vascular insuf-
ficiency as occurs from atherosclerotic disease or giant cell
arteritis, and immunologic attack. The immune-mediated
disease that most commonly affects the optic nerve is
­multiple sclerosis. This demyelinating condition generally
starts suddenly in one eye with pain, an afferent pupillary
defect, a loss in color vision, and visual field loss typical
of optic nerve disease. Initially, the optic nerve may show
papilledema, or it may appear normal if the inflammation is
retrobulbar. Over several weeks, the affected nerve usually
becomes pale.
Demyelinating disease affecting the optic nerve generally
is not treated by a rheumatologist, but a rare patient with
optic nerve disease has inflammation that might require
long-term immunosuppression. These patients carry a diag-
nosis labeled variously as autoimmune optic neuropathy or
sometimes steroid-sensitive optic neuropathy. This diagno-
sis is clinically distinct from optic neuritis associated with
multiple sclerosis in that MRI of the head should not indi-
cate a demyelinating process, the disease is often bilateral,
the kinetics of the inflammation are different from multiple
sclerosis, and the disease usually responds to oral cortico-
steroids. In most centers, a neuro-ophthalmologist would
be involved in establishing this diagnosis. Systemic lupus
erythematosus and sarcoidosis may affect the optic nerve in
this way, but many patients with this diagnosis do not have
an associated systemic illness. Either alkylating agent ther-
apy or an antimetabolite can be beneficial for many patients
with this entity.28
Sudden blindness is arguably the most feared conse-
quence of temporal arteritis. This disease is characterized by
granulomatous inflammation of multiple vessels above the
waist. These frequently include the temporal artery and the
posterior ciliary arteries. Inflammation in these latter vessels
leads to anterior ischemic optic neuropathy (AION), which
is ischemia of the optic nerve that manifests as sudden visual
loss (Fig. 46-11). Temporal arteritis also can affect the cen-
tral retinal artery, which may result in blindness. For this
condition, the funduscopic appearance of the eye shows
markedly reduced arteriolar flow and a cherry-red spot in
the macula. Temporal arteritis can cause diplopia by affect-
ing circulation to extraocular muscles.
The visual loss associated with temporal arteritis is fre-
quently labeled arteritic AION to distinguish it from the
more common nonarteritic AION that is usually ­attributable
Figure 46-11  Anterior ischemic optic neuropathy resulting from 
giant cell arteritis. The optic nerve is swollen, and there are surrounding 
hemorrhages.
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
to small vessel atherosclerosis. Patients with arteritic AION
are typically older than 50 years and have an erythrocyte
sedimentation rate greater than 50 mm/hr. Many patients
with arteritic AION have associated symptoms of poly-
myalgia rheumatica, jaw claudication, scalp tenderness, or
temporal artery tenderness. The biopsy specimen of the
temporal artery shows vasculitis in about 80% of patients
with temporal arteritis if an adequate length of vessel is
sampled. Twenty percent of patients with temporal arteritis
might have a biopsy specimen of the artery that is negative
because it either has spared this vessel or has affected it in
a sufficiently patchy distribution such that the biopsy speci-
men did not reveal the pathology. Patients with nonarteritic
AION tend to have small optic nerve cups.
SUMMARY
In some ways, from a rheumatologist’s perspective, the eye is
a microcosm of the body. Its complex structures frequently
reflect inflammation elsewhere in the body. The treatment
of many forms of ocular inflammation requires collaboration
between a rheumatologist and an ophthalmologist.
REFERENCES
  1. Jabs DA, Nussenblatt RB, Rosenbaum JT: Standardization of uve-
itis nomenclature for reporting clinical data. Results of the First
International Workshop. Am J Ophthalmol 140:509-516, 2005.
  2. Niederkorn JY: See no evil, hear no evil, do no evil: the lessons of
immune privilege. Nat Immunol 7:353-359, 2006.
  3. Gritz DC, Wong IG: Incidence and prevalence of uveitis in North-
ern California; the Northern California Epidemiology of Uveitis
Study. Ophthalmology 111:491-500, 2004.
  4. Brewerton DA, Caffrey M, Nicholls A, et al: Acute anterior uveitis
and HL-A 27. Lancet 2:994-996, 1973.
  5. Rosenbaum JT: Characterization of uveitis associated with spondy-
loarthritis. J Rheumatol 16:792-796, 1989.
  6. Martin TM, Zhang G, Luo J, et al: A locus on chromosome 9p pre-
disposes to a specific disease manifestation, acute anterior uveitis, in
ankylosing spondylitis, a genetically complex, multisystem, inflamma-
tory disease. Arthritis Rheum 52:269-274, 2005.
  7. Paiva ES, Macaluso DC, Edwards A, et al: Characterisation of uve-
itis in patients with psoriatic arthritis. Ann Rheum Dis 59:67-70,
2000.
  8. Lyons JL, Rosenbaum JT: Uveitis associated with inflammatory
bowel disease compared with uveitis associated with spondyloar-
thropathy. Arch Ophthalmol 115:61-64, 1997.
683
  9. Rosenbaum JT, Robertson JE, Watzke RC: Retinal vasculitis: A
primer. West J Med 154:182-185, 1991.
10. Obenauf CD, Shaw HE, Sydnor CF, et al: Sarcoidosis and its ophthal-
mic manifestations. Am J Ophthalmol 86:648-655, 1978.
11. Rosenbaum JT: Uveitis: An internist’s view. Arch Intern Med
149:1173-1176, 1989.
12. Kaiser PK, Lowder CY, Sullivan P, et al: Chest computerized
tomography in the evaluation of uveitis in elderly women. Am J
Ophthalmol 133:499-505, 2002.
13. Petty RE, Smith JR, Rosenbaum JT: Arthritis and uveitis in chil-
dren: A pediatric rheumatology perspective. Am J Ophthalmol 135:
879-884, 2003.
14. Yazici H, Pazarli H, Barnes CG, et al: A controlled trial of azathio-
prine in Behçet’s syndrome. N Engl J Med 322:281-285, 1990.
15. Isaak BL, Liesegang TJ, Michet CJJ: Ocular and systemic findings in
relapsing polychondritis. Ophthalmology 93:681-689, 1986.
16. Miceli-Richard C, Lesage S, Rybojad M, et al: CARD15 mutations
in Blau syndrome. Nat Genet 29:19-20, 2001.
17. Rose CD, Wouters CH, Meiorin S, et al: Pediatric granulomatous arthri-
tis: An international registry. Arthritis Rheum 54:3337-3344, 2006.
18. Dollfus H, Hafner R, Hofmann HM, et al: Chronic infantile neurological
cutaneous and articular/neonatal onset multisystem inflammatory dis-
ease syndrome: Ocular manifestations in a recently recognized chronic
inflammatory disease of childhood. Arch Ophthalmol 8:386-392, 2000.
19. Sfikakis PP, Theodossiadis PG, Katsiari CG, et al: Effect of inflix­
imab on sight-threatening panuveitis in Behçet’s disease. Lancet
358:295-296, 2001.
20. Kotter I, Zierhut M, Eckstein AK, et al: Human recombinant inter-
feron alfa-2a for the treatment of Behçet’s disease with sight threaten-
ing posterior or panuveitis. Br J Ophthalmol 87:423-431, 2003.
21. Lim LL, Smith JR, Rosenbaum JT: Retisert (Bausch & Lomb/Control
Delivery Systems). Curr Opin Investig Drugs 6:1159-1167, 2005.
22. Baker KB, Spurrier NJ, Watkins AS, et al: Retention time for corti-
costeroid-sparing systemic immunosuppressive agents in patients with
inflammatory eye disease. Br J Ophthalmol 90:1481-1485, 2006.
23. Riono WP, Hidayat AA, Rao NA: Scleritis: A clinicopathologic study
of 55 cases. Ophthalmology 106:1328-1333, 1999.
24. Akpek EK, Thorne JE, Qazi FA, et al: Evaluation of patients with
scleritis for systemic disease. Ophthalmology 111:501-506, 2004.
25. Foster CS, Forstot SL, Wilson LA: Mortality rate in rheumatoid
arthritis patients developing necrotizing scleritis or peripheral ulcer-
ative keratitis. Ophthalmology 91:1253-1263, 1984.
26. Keogh KA, Wylam ME, Stone JM, et al: Induction of remission by
B lymphocyte depletion in eleven patients with refractory antineu-
trophil cytoplasmic antibody-associated vasculitis. Arthritis Rheum
52:262-268, 2005.
27. Smith JR, Rosenbaum JT: A role for methotrexate in the management
of non-infectious orbital inflammatory disease. Br J Ophthalmol 85:
1220-1224, 2001.
28. Myers TD, Smith JR, Wertheim MS, et al: Use of corticosteroid
sparing systemic immunosuppression for treatment of corticosteroid
dependent optic neuritis not associated with demyelinating disease.
Br J Ophthalmol 88:673-680, 2004.
 47
KEY POINTS
Many systemic rheumatologic conditions present with skin
findings.
The differential diagnosis for skin findings may require a skin
biopsy for clinical-pathologic correlation.
Systemic steroids should be avoided in psoriasis, because
­flaring may occur with tapering of systemic glucocorticoids.
New biologic therapies have substantially improved the care
of patients with severe and resistant psoriasis.
The neutrophilic dermatoses are a group of inflammatory
diseases, including pyoderma gangrenosum and Sweet’s
­syndrome, that can be associated with autoimmune diseases.
Patients with lupus erythematosus can have a wide variety of
lupus-specific and -nonspecific skin lesions.
Lupus-nonspecific skin lesions are more frequently seen in
patients with systemic lupus erythematosus.
Patients with dermatomyositis frequently have pathogno-
monic skin lesions that may be seen in the absence of muscle
disease.
Antimalarials may be of benefit in patients with morphea.
THE DIAGNOSIS OF SKIN LESIONS
ASSOCIATED WITH RHEUMATIC DISEASES
The skin is a highly visible organ that is frequently affected
in rheumatic diseases, and the presence of skin lesions may
be helpful diagnostically. However, certain caveats must be
considered before proceeding to a more specific discussion of
these diseases. First, a major pitfall for the nondermatologist
is incomplete knowledge of the entities in the differential
diagnosis. For example, malar erythema occurs frequently in
patients with systemic lupus erythematosus (SLE), but the
differential diagnosis for malar erythema is rather extensive
and includes both conditions that are much more prevalent
than lupus (e.g., rosacea) and those that are far less preva-
lent (e.g., Rothmund-Thomson syndrome). Patients fre-
quently have more than one skin condition, which often
makes the diagnosis more challenging.
Another caveat has to do with skin biopsy. Being able
to determine when a skin biopsy is likely to be diagnosti-
cally useful often requires specialized knowledge, as does
the interpretation of pathology reports. With inflamma-
tory skin conditions, the microscopic findings are often
less diagnostically specific than is the clinical examination.
Unfortunately, it is common for a pathology report to list
a diagnosis without indicating how definitive the findings
were. For example, a skin biopsy report may list psoriasis
as the final diagnosis, but, depending on the specific case,
  Supplemental images available on the Expert Consult Premium Edition website.
The Skin and Rheumatic
Diseases
LELA A. LEE  •  VICTORIA P. WERTH
other unstated possibilities may include nummular dermati-
tis, atopic dermatitis, seborrheic dermatitis, lichen simplex
chronicus, dermatophytosis, and drug eruption. Thus, a
working knowledge of both dermatopathology and derma-
tology, and the ability to place the histologic findings in the
context of the clinical presentation, may be necessary to
arrive at the correct diagnosis.
These considerations notwithstanding, it is clearly useful
for the physician caring for patients with rheumatic diseases
to be well versed in their cutaneous manifestations. In this
chapter, we provide an overview of these manifestations,
as well as a perspective on their diagnosis and differential
diagnosis. Therapy is discussed briefly when the treatment
may be specifically directed toward the skin lesions. Etiology
and pathogenesis of these diseases are covered elsewhere in
this text.
PSORIASIS
Psoriasis is one of the most common inflammatory skin dis-
eases, affecting about 2% of the general population. There is
a wide range of severity, from a few relatively asymptomatic
plaques to extensive, disabling disease. The onset may occur
ar any time during life. Once the disease is present, there
may be exacerbations and remissions, but it does not tend
to resolve permanently. In general, onset in childhood por-
tends more severe disease.
The characteristic skin lesions of psoriasis are sharply
demarcated plaques with underlying erythema and silvery
scales, although there may be a paucity of scales if the lesions
have been partially treated or if they occur in intertriginous
areas. When the scale is removed, pinpoint bleeding may
be observed (Auspitz sign). Lesions may occur in areas of
trauma (Koebner’s phenomenon), such as in surgical scars.
In some cases, lesions contain small pustules.
General phenotypes of psoriasis are chronic plaque, gut-
tate, localized pustular, generalized pustular, and erythro-
derma.1 Chronic plaque and guttate psoriasis are the most
common; generalized pustular psoriasis and erythroderma
are typically the most disabling and even life threatening.
Chronic plaque psoriasis lesions are often relatively large
in diameter and occur preferentially on the elbows, knees,
scalp, genitalia, lower back, and gluteal cleft, although
they may occur at many other locations. It is quite com-
mon for only one area of skin, such as the scalp, to be
affected. Guttate lesions are relatively small in diameter
and usually quite numerous, distributed preferentially on
the trunk and proximal extremities (Fig. 47-1). Guttate
psoriasis occurs relatively commonly in children and young
adults, often manifesting a few weeks after a ­streptococcal
infection.
685
 686 LEE  |  The Skin and Rheumatic Diseases
Figure 47-1  Guttate psoriasis resembles “drops” of discrete scaly
­ apules with erythema, often on the trunk. (Courtesy of Dr. Nicole Rogers,
p
­Tulane University Department of Dermatology, New Orleans.)
Nail changes are common, occurring in about half of
patients, and are often mistaken for fungal infection. Charac­
teristic changes include pitting, onycholysis (“oil spots”),
dystrophy of nails, and loss of the nail plate. These changes
are not specific for psoriasis. Notably, pitting may occur as a
result of trauma, and the finding of a few pits in the nails may
not be helpful diagnostically. Nail changes are more frequent
in patients with arthritis of the distal interphalangeal joints.2
Arthritis occurs more often in patients with severe cuta-
neous disease, but cutaneous disease need not be present at
all. Remissions and exacerbations of arthritis do not correlate
well with remissions and exacerbations of skin disease. The
presence of psoriatic skin lesions may be helpful in support-
ing a diagnosis of psoriatic arthritis, although many patients
with psoriasis have joint disease unrelated to psoriasis.
The diagnosis of psoriatic skin disease is usually made on
clinical grounds alone, based in large part on the morphol-
ogy and distribution of lesions. The differential diagnosis
may be extensive and includes in selected cases nummular
eczema, seborrheic dermatitis, candidiasis (in intertriginous
areas), pityriasis rubra pilaris, Bowen’s disease or Paget’s dis-
ease (for isolated plaques), drug eruption, pityriasis rosea,
pityriasis lichenoides, dermatophytosis, lichen planus, sec-
ondary syphilis, parapsoriasis, cutaneous lupus (especially
subacute cutaneous lupus), and dermatomyositis. In cases in
which the diagnosis is not clear-cut, biopsy may be helpful.
The histologic findings may range from virtually diagnostic
for psoriasis to merely consistent with but not diagnostic.
Histologically, psoriasis generally cannot be distinguished
from Reiter’s syndrome.
Common topical therapies include corticosteroids, tar,
anthralin, calcipotriene, and tazarotene.3 Phototherapy
using sunlight, broad-band ultraviolet B (UVB), narrow-
band UVB, or psoralen plus ultraviolet A (PUVA) is still
a mainstay for many patients. Common systemic therapies
include methotrexate, acitretin, cyclosporine, and the rela-
tively new biologic agents. Although topical corticosteroids
are an acceptable treatment for many patients, systemic
corticosteroids are avoided for the treatment of cutaneous
­disease because of the severe flaring of psoriasis that can
occur following their withdrawal.
REITER’S SYNDROME
The diagnosis of Reiter’s syndrome may be rather straight-
forward in a young male who develops urethritis, conjunc-
tivitis, and arthritis following an episode of nongonococcal
urethritis. However, in many cases, the clinical features are
not fully expressed, and cutaneous lesions may be helpful in
establishing the diagnosis.4
Circinate balanitis is the most common of the characteris-
tic mucocutaneous lesions. Small erythematous papules and
pustules coalesce to form serpiginous erosive or crusted plaques
on the glans penis. In uncircumcised men, the appearance is
often that of erosion rather than crust, because the moisture
and trauma minimize the formation of crust. In circumcised
men, crusting may be more obvious than erosion.
The palms and, particularly, the soles may develop lesions
that are initially similar to the small erythematous papules
and pustules of the genital region. With time, these lesions,
termed keratoderma blennorrhagica, tend to become markedly
hyperkeratotic (Fig. 47-2). They may coalesce into large
plaques or generalized hyperkeratosis involving the entire
plantar surface, or they may remain discrete, erythematous,
hyperkeratotic papules a few millimeters in diameter.
Erythematous, scaly plaques indistinguishable from pso-
riasis may appear elsewhere on the skin, including the scalp,
elbows, and knees. When lesions occur around the nails, it
is common for there to be hyperkeratosis underneath the
nails. Pitting is not typical of Reiter’s syndrome, but thick-
ening, ridging, or shedding of the nail plate may occur.
Erosions of the oral mucosa are relatively common on the
tongue, buccal mucosa, and palate.
The cutaneous lesions are usually diagnosed on a clini-
cal basis. Skin biopsy may be helpful in excluding many
entities in the differential diagnosis but generally cannot
exclude psoriasis—the major condition in the differential
­diagnosis. One somewhat distinguishing feature is that the
older lesions of keratoderma blennorrhagica may have a
considerably thickened stratum corneum, corresponding to
the markedly hyperkeratotic papules seen grossly.
Figure 47-2  Reiter’s syndrome with keratoderma blennorrhagica of
the feet.
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
For the genital lesions, conditions to consider in the
differential diagnosis may include candidiasis, psoriasis,
dermatitis, Bowen’s disease, Paget’s disease, squamous cell
carcinoma, Zoon’s balanitis, erosive lichen planus, lichen
sclerosus (balanitis xerotica obliterans), aphthosis, fixed drug
eruption, and certain infectious diseases. The differential
diagnosis for lesions on the soles and palms may include pso-
riasis, hereditary or acquired hyperkeratosis of the palms and
soles, pustular eruption of the palms and soles, pompholyx,
scabies, and dermatophytosis. The differential diagnosis for
oral lesions may include geographic tongue, lichen planus,
candidiasis, aphthae, and autoimmune bullous ­diseases.
The approach to the treatment of skin lesions is simi-
lar to that for psoriasis, particularly in cases in which the
lesions are persistent. Choice of topical therapies may be
somewhat limited owing to the sites involved. It is difficult
to deliver medication topically to the oral mucosa, and the
genital area may develop irritant reactions to certain topi-
cal medications. Often, topical corticosteroids are preferred
for both these areas because of the low potential for irrita-
tion and the availability of topical preparations specifically
designed for these sites. In the genital area, suprainfection
with Candida may occur, and concurrent therapy with a top-
ical or systemic anticandidal medication may be needed.
RHEUMATOID ARTHRITIS
The major skin manifestations associated with rheumatoid
arthritis (RA) can generally be divided into granulomatous
lesions, exemplified by the rheumatoid nodule, and neutro-
philic lesions, exemplified by vasculitis and pyoderma gan-
grenosum.
Rheumatoid nodules are the most common cutaneous
manifestations of RA.5 They occur more often in seroposi-
tive patients and correlate somewhat with higher rheuma-
toid factor titers, more severe arthritis, and increased risk
for vasculitis. Nodules are usually relatively deep, firm, and
painless, and they tend to develop over areas of pressure and
trauma, such as the extensor forearms, fingers, olecranon
processes, ischial tuberosities, sacrum, knees, heels, and pos-
terior scalp (Fig. 47-3). In patients who wear glasses, nod-
ules may develop under the bridge or nosepiece. In most
cases, rheumatoid nodules are in the subcutaneous tissue
or deep dermis, but they occasionally occur more deeply or
more superficially.
Clinically, depending on the presentation, numerous
entities may be considered in the differential diagnosis,
including infections, inflammatory disorders, and benign
tumors. If necessary, biopsy of a nodule may be quite helpful
in establishing the diagnosis. Rheumatoid nodules exhibit
a distinctive histologic finding called necrobiosis, a fibrinoid
degeneration of the connective tissue, surrounded by pali-
saded histiocytes. Necrobiosis is also a characteristic feature
of granuloma annulare and necrobiosis lipoidica diabetico-
rum. Although necrobiosis lipoidica diabeticorum is easily
distinguished from rheumatoid nodule clinically, the subcu-
taneous variant of granuloma annulare may be difficult to
distinguish both clinically and histologically.
The term rheumatoid nodulosis has been used to describe
an entity characterized by subcutaneous rheumatoid nod-
ules, cystic bone lesions, rheumatoid factor positivity,
and arthralgias in patients with little or no evidence of
687
Figure 47-3  Rheumatoid nodule over the extensor tendon of the distal
interphalangeal joint.
systemic manifestations of RA or erosive joint disease.6
Older males are preferentially affected.
The development of new nodules in RA patients under-
going treatment with methotrexate has been noted by sev-
eral observers and termed accelerated rheumatoid nodulosis.7
The nodules occur preferentially on the hands. There are
also case reports of the phenomenon in RA patients treated
with etanercept.
The other major type of cutaneous lesion associated
with RA is neutrophil predominant. Rheumatoid vasculi-
tis occurs more frequently in patients who are seropositive
and have rheumatoid nodules, and it often occurs relatively
late in the course of the disease.8 Vessels of any size may be
affected. In the skin, vasculitis may appear as purpuric pap-
ules and macules, nodules, ulcerations, or infarcts. Bywaters’
lesions are periungual or digital pulp purpuric papules rep-
resenting a small vessel vasculitis; they are not necessarily
associated with vasculitic lesions elsewhere.
The differential diagnosis of purpuric or petechial lesions
may include stasis dermatitis, Schamberg’s purpura, plate-
let dysfunction, petechial drug eruptions, viral exanthems,
emboli, thromboses, and sludging. Of these, Schamberg’s
purpura, a relatively common condition unassociated with
systemic disease, is probably most frequently confused with
small vessel vasculitis. Skin biopsy may be quite helpful in
establishing the diagnosis of vasculitis, particularly if an
early lesion is sampled, although rheumatoid vasculitis can-
not be distinguished histologically from many other causes
of small vessel vasculitis. Immunofluorescent examination of
an early lesion may be helpful in ruling out immunoglobulin
(Ig) A–predominant vasculitis. The differential diagnosis of
ulcers and infarcts is extensive. Biopsy is often unrewarding,
because nonspecific changes present in established lesions
may make interpretation difficult. On occasion, however,
biopsy of ulcers or infarcts may result in a definitive diagno-
sis of vasculitis.
The neutrophilic dermatoses are a group of diseases that
are inflammatory rather than infectious in origin, typified
by pyoderma gangrenosum and Sweet’s syndrome. These
conditions have been associated with a variety of extracu-
taneous diseases, including RA. The classic pyoderma gan-
grenosum lesion is a very rapidly appearing large, destructive
ulcer in which the border is undermined. The classic lesion
 688 LEE  |  The Skin and Rheumatic Diseases
of Sweet’s syndrome is an erythematous, edematous plaque
with a surface that is often described as mammillated, pseu-
dovesicular, or microvesicular. Intermediate clinical appear-
ances between these two entities have been described. For
pyoderma gangrenosum, the differential diagnosis usually
includes conditions causing leg ulcers, and the diagnosis
is mainly clinical; biopsy serves primarily to exclude some
of the other entities under consideration. For Sweet’s syn-
drome, the differential diagnosis may include infections,
halogenoderma, and other neutrophilic dermatoses; biopsy
often provides helpful supporting evidence. The mainstay
of therapy for acute lesions of both conditions is systemic
corticosteroids. For more persistent lesions, a variety of
options may be considered; cyclosporine and infliximab are
two of the more common. Colchicine or potassium iodide
may be useful therapies for Sweet’s syndrome, particularly
in patients with infections or contraindications to cortico-
steroids.
The term rheumatoid neutrophilic dermatitis has been used
to describe chronic, erythematous, urticaria-like plaques
that occur primarily on the distal arms.9 Clinically and his-
tologically, rheumatoid neutrophilic dermatitis is quite simi-
lar to Sweet’s syndrome and may be a variant of it.
Palisaded neutrophilic and granulomatous dermatitis of
connective tissue disease is an unusual condition or set of
conditions for which consistent terminology is still evolv-
ing. As the name implies, the major bases for diagnosis of
this entity are the histologic appearance and the occur-
rence in a patient with connective tissue disease, often
RA.10 The clinical appearance ranges from erythematous
or flesh-­colored papules that appear primarily on the fingers
and elbows to erythematous or flesh-colored linear cords
on the trunk. Some authors classify the latter as interstitial
granulomatous dermatitis with cutaneous cords or intersti-
tial granulomatous dermatitis with arthritis. Treatment can
be challenging. Palisaded neutruophilic and granulomatous
dermatitis may respond to dapsone or sulfapyridine. Inter-
stitial granulomatous dermatits with arthritis can be treated
with antimalarials or immunosuppressives; however, this is
both a newly described and a relatively infrequent condi-
tion, and all evidence is based on case reports and small case
series. Patients can progress to a severe deforming arthritis.
In some cases, granuloma annulare and rheumatoid nodule
may be in the differential diagnosis.
JUVENILE RHEUMATOID ARTHRITIS
AND STILL’S DISEASE
The majority of patients with classic Still’s disease mani-
fest an exanthematous eruption coincident with daily fever
spikes.11 The lesions are evanescent, usually nonpruritic,
erythematous macules occurring over the trunk, extremities,
and face. The differential diagnosis includes viral exanthem,
drug eruption, familial periodic fever syndromes, and rheu-
matic fever. It is not unusual for exanthems of any type to
be more prominent during fevers, but viral exanthems and
drug eruptions would not be expected to clear completely
between fever spikes. However, it should be noted that the
eruption of erythema infectiosum (fifth disease) due to par-
vovirus B19 may resolve completely but reappear when the
skin temperature rises, such as during warm baths or exer-
cise. Skin biopsy is unlikely to be helpful in ­establishing the
diagnosis of Still’s disease or in excluding other entities in
the differential diagnosis. Adult-onset Still’s disease is also
typified by an evanescent, erythematous, sometimes salmon-
colored eruption over the trunk and extremities, associated
with high fever.
Subcutaneous nodules may develop in both juvenile-
onset and adult-onset Still’s disease. The lesions tend to
occur at the same sites as the rheumatoid nodules in RA,
but histologically, they appear similar to the nodules of
rheumatic fever.
LUPUS ERYTHEMATOSUS
The skin becomes involved during the course of the disease
in the majority of patients with lupus erythematosus (LE),
and skin lesions may be important in establishing the diag-
nosis. Some skin lesions are highly likely to be associated
with systemic (i.e., extracutaneous) disease, whereas others
may or may not be associated with extracutaneous disease.
The phenomenon of lupus skin lesions in the absence of
systemic disease was previously termed discoid lupus by some;
however, discoid lupus is a term used by dermatologists to
denote a specific type of skin lesion, regardless of the pres-
ence or absence of systemic disease. We use the latter mean-
ing in this chapter.
LUPUS-SPECIFIC SKIN LESIONS
Gilliam classified cutaneous lesions as being specific or non-
specific for lupus, with discoid lupus lesions being an exam-
ple of the former, and palpable purpura being an example of
the latter.12 Although this division is quite useful, a lupus-
specific lesion sometimes occurs in a patient whose primary
autoimmune disease is something other than LE. For exam-
ple, subacute cutaneous lupus lesions may occur in patients
whose primary condition is Sjögren’s syndrome, and discoid
lesions may be seen in a variety of conditions, including
mixed connective tissue disease. Many of the lupus-specific
skin lesions can occur in patients who have no evidence of
extracutaneous disease.
The characteristic morphologies of the various lupus-
specific skin lesions are in large part a function of the depth
and intensity of the inflammatory infiltrate, presence or
absence of epidermal basal cell damage, involvement of hair
follicles, abundance of dermal mucin, and tendency to scar.
In practice, these features may overlap, and a patient may
have more than one type of lesion, making classification dif-
ficult. Because therapy for most of the lupus-specific lesions
is similar, it is not always important to distinguish among
the various types of lesions. However, it can be useful to
identify conditions that are more likely to scar, and thus
warrant more aggressive therapy, and to identify conditions
that are highly likely or highly unlikely to be associated with
systemic disease.
Acute cutaneous lupus erythematosus (ACLE) lesions
are typified by malar erythema, the classic butterfly rash
(Fig. 47-4). The inflammation tends to be superficial, with
little propensity to scar. Precipitation or exacerbation of
lesions by sun exposure is common, and lesions tend to be
distributed on the sun-exposed face, neck, extensor arms,
and dorsal hands, where the skin over the knuckles is rela-
tively spared. Often the lesions are transient, but they may
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN 689

Figure 47-5  Subacute cutaneous lupus erythematosus—annular-

Figure 47-4  Acute malar rash in a butterfly distribution in systemic
polycyclic type.
l­upus erythematosus.

be ­ persistent. When the face is severely affected, facial multiforme. Skin biopsy for routine histology is often helpful
edema may be prominent. Oral lesions are often present in establishing the diagnosis. The characteristic finding of
concurrently. Acute eruptions with considerable focal basal skin biopsy for immunofluorescence is a particulate deposi-
cell damage can result in erythematous papules with dusky tion of IgG in the epidermis of both lesions and uninvolved
centers that clinically mimic erythema multiforme. The skin (Fig. 47-6).15 This pattern can be reproduced in animal
major importance of recognizing ACLE is its strong asso- models by infusing anti-Ro; thus, immunofluorescence pro-
ciation with systemic disease. The differential diagnosis of vides information that duplicates serologic testing for anti-
malar rash may include several conditions. In some cases, Ro.16 The particulate epidermal pattern seen in normal skin
the facial rash of ACLE may be difficult to distinguish from does not carry the same implication for an increased risk
rosacea. Seborrheic dermatitis, atopic dermatitis, and pho- of SLE as does the finding of granular deposits of IgG at
tosensitive eruptions such as polymorphous light eruption the dermal-epidermal junction (the nonlesional lupus band
and drug-induced photosensitivity should also be consid- test). It should be noted that many immunofluorescence
ered. Dermatomyositis may cause a photosensitive facial laboratories do not routinely report epidermal findings.
erythema with edema very similar to ACLE, although the Discoid lupus erythematosus (DLE) lesions are the most
erythema tends to be more violaceous. Persistent lesions common of the persistent lupus-specific skin lesions. Active
on the neck and arms may be indistinguishable from sub- lesions are erythematous papules and plaques that feel indu-
acute cutaneous lupus erythematosus (SCLE). Discoid lupus rated to palpation because of the substantial numbers of
lesions occasionally appear in a butterfly distribution, and inflammatory cells infiltrating the dermis. Involvement of
they can result in disfiguring scarring. Skin biopsy is usually hair follicles may be grossly evident as follicular plugs and
not performed on malar erythema because of its transient scarring alopecia. Dyspigmentation is common, often with
character, the scar resulting from biopsy, and the availabil- hypopigmentation or even depigmentation in the center
ity of other means of establishing the diagnosis of SLE. If a and hyperpigmentation at the periphery (Fig. 47-7). Vis-
biopsy is done, it should be noted that dermatomyositis and ible scale is common; it is occasionally pronounced in a
SCLE cannot be distinguished from ACLE by histology, and clinical variant called hypertrophic DLE. In established
skin biopsy findings are sometimes nonspecific.
SCLE is a photosensitive eruption usually associated
with anti-Ro/SS-A autoantibodies.13 Lesions are of two
main types: annular erythematous plaques and scaly ery-
thematous psoriatic plaques. They are distributed over the
sun-exposed skin of the arms, upper trunk, neck, and sides
of the face (Fig. 47-5). Inexplicably, the midfacial area is
usually uninvolved. Fair-skinned individuals are preferen-
tially affected. Lesions may resolve with hypopigmenta-
tion or even depigmentation, but they rarely scar. Several
drugs, particularly hydrochlorothiazide, have been reported
to induce SCLE.14 The risk for developing systemic disease
is not fully known, but perhaps 15% of patients with SCLE
have or will develop significant systemic disease—often
SLE, Sjögren’s syndrome, or an overlap. Depending on the
morphology of the lesions and the clinical presentation,
the differential diagnosis may include psoriasis, tinea, poly-
morphous light eruption, reactive erythema, and erythema Figure 47-6  Direct immunofluorescence (lupus band test).
 690 LEE  |  The Skin and Rheumatic Diseases
Figure 47-7  Discoid lupus erythematosus of the scalp, with scarring
alopecia and central hypopigmentation. (Courtesy of Dr. Nicole Rogers,
Tulane University School of Medicine, New Orleans.)
lesions, ­ scarring may be disfiguring. Lesions tend to occur
on the scalp, ears, and face, but they may be widespread and
occasionally involve mucosal surfaces. It is unusual to have
lesions below the neck in the absence of lesions above the
neck.17 Sun exposure may exacerbate DLE in some cases, but
the presence of lesions in sun-protected areas of the scalp
and ears and the frequent absence of a history of photosensi-
tivity indicate that sun exposure is probably not a trigger in
every instance. There are case reports of squamous cell car-
cinoma developing in established DLE lesions. In a patient
who presents with DLE lesions, the risk for developing SLE
is probably about 5% to 10%, although mild systemic symp-
toms such as arthralgias are relatively common. The differ-
ential diagnosis of DLE lesions often consists of conditions
exhibiting intense lymphocytic or granulomatous infiltrates,
such as sarcoid, Jessner’s lymphocytic infiltrate, granuloma
faciale, polymorphous light eruption, lymphocytoma cutis,
and lymphoma cutis. In the scalp, lichen planopilaris and
other scarring alopecias may be considered. Skin biopsy for
routine histology often establishes the diagnosis definitively.
In more difficult cases, biopsy for immunofluorescence may
provide additional supporting diagnostic information.
Lesions are expected to have granular deposits of immuno-
globulins at the dermal-epidermal junction. Unless there
is concomitant systemic disease, normal skin would not be
expected to have immunoglobulin deposits.
Tumid lupus erythematosus (TLE) skin lesions are simi-
lar to DLE lesions, in that they are erythematous, indurated
papules and plaques with a substantial lymphocytic infiltrate.
Unlike DLE, however, the lesions do not exhibit epidermal
abnormalities, follicular involvement, or scarring. Consider-
able mucin is present in the dermis, giving the lesions a some-
what boggy look and feel. In some reports, lesions are most
common on the face and may be reproduced by phototesting.18
The risk for SLE appears to be very low, and immunoglobulin
deposits are not generally present in skin biopsies. Jessner’s
lymphocytic infiltrate and other lymphocytic and granuloma-
tous infiltrative conditions (noted earlier) are included in the
differential diagnosis. Skin biopsy for routine histology is valu-
able in establishing the diagnosis, with the exception of reli-
ably distinguishing TLE from Jessner’s ­lymphocytic ­infiltrate.
Some believe that Jessner’s lymphocytic infiltrate and TLE
are the same entity, and it might reasonably be argued that
TLE should not be classified as a form of chronic cutaneous
LE. However, the presence of TLE lesions in some patients
with lupus is evidence to the contrary.
Lupus erythematosus panniculitis (LEP) lesions have
inflammation in the subcutaneous tissue, resulting in deep,
indurated plaques that become disfiguring, depressed areas
(Fig. 47-8). Usual sites of involvement are the face, upper
trunk, breasts, upper arms, buttocks, and thighs. The risk for
SLE is not known precisely, but it is clear that some patients
with LEP have or will develop SLE. The differential diagno-
sis is that of the panniculitides, but the distribution exhib-
ited in LEP is unusual for most other conditions that cause
panniculitis. The combination of clinical presentation and
skin biopsy for histology usually establishes the diagnosis.
Some unusual variants of cutaneous lupus are chilblain
lupus (red or dusky plaques on colder areas of skin such as
the fingers, toes, nose, elbows, knees, and lower legs), cuta-
neous lupus–lichen planus overlap, and a bullous eruption
due to autoantibodies to type VII collagen or other base-
ment membrane zone proteins. Not all bullae related to
lupus are due to autoantibodies to basement membrane pro-
teins, however. It is not unusual to develop bullae simply
from intensive destruction of the basal cell layer in ACLE,
SCLE, or, rarely, DLE.
Treatment of the lupus-specific lesions is similar for most
of the subtypes, with some exceptions and modifications.
Sun protection is critical for lesions that are initiated or
exacerbated by sun exposure. Many or most patients under-
estimate the amount of sunscreen that should be applied,
the potential damage of seemingly minimal exposure dur-
ing the course of day-to-day activities, and the value of pro-
tective clothing. Topical therapy is often used to avoid the
side effects of systemic medications or to provide adjunctive
therapy, although topical agents are unlikely to be benefi-
cial if the disease process is deep, as in panniculitis. Topical
or intralesional corticosteroids are the most commonly used
local therapy, but there are some reports that topical cal-
cineurin inhibitors (e.g., tacrolimus) and topical ­ retinoids
are beneficial.19,20 The first-line systemic medication for
Figure 47-8  Lupus profundus (panniculitis) with extensive atrophy.
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
c­ utaneous lupus is antimalarial therapy. Several reports
indicate that smoking tobacco decreases the likelihood of
a response to antimalarials.21 For antimalarial-resistant skin
disease, a wide variety of medications have been used, but
there is no clear second choice when antimalarials do not
work. Although dapsone is arguably not helpful in most
types of cutaneous lupus, it may be helpful in neutrophil-
predominant bullous eruptions.22 Measures to keep the skin
warm may be useful for chilblain lupus.
NONSPECIFIC CUTANEOUS LESIONS
A wide variety of lupus-nonspecific skin lesions have been
reported. Many of these, such as vasculitic lesions, are cuta-
neous clues to the possibility of extracutaneous disease.
Noteworthy in this regard is livedo reticularis. The netlike
erythema of livedo reticularis is a vascular phenomenon
caused by reduced oxygenation at the periphery of the area
supplied by a particular vessel. This might simply be due to
vasoconstriction, such as occurs in a cold environment, in
which case it is a benign finding. If livedo is more prominent
than usual, not corrected by warming, and persistent, it can
be an indication of decreased flow due to pathology such
as vasculitis, atherosclerotic disease, or sludging. In lupus,
livedo reticularis may be a sign of the presence of antiphos-
pholipid antibodies.23
Other nonspecific skin lesions include Raynaud’s phe-
nomenon, palmar erythema, periungual telangiectasia,
alopecia, erythromelalgia, papulonodular mucinosis, and
anetoderma. Sclerodactyly, calcinosis, and rheumatoid nod-
ules have been reported but may be more likely in overlap
syndromes than in SLE.
NEONATAL LUPUS SYNDROME
Neonatal lupus erythematosus (NLE) is associated with
maternal IgG autoantibodies to Ro/SS-A and La/SS-B.24
Affected children may have cutaneous lesions, cardiac dis-
ease (complete heart block, cardiomyopathy, or both), hep-
atobiliary disease, or hematologic cytopenias. Most children
have only one or two features of the disease. Similar to the
anti-Ro/SS-A–associated SCLE of adults, the skin lesions
are often photosensitive, have relatively superficial inflam-
matory infiltrates, and do not tend to scar. The lesions usu-
ally appear at a few weeks of age but have been noted at
birth in several cases. The natural history of the skin disease
is that the lesions last for weeks or months and resolve spon-
taneously, usually leaving no residuum. In a few cases, per-
sistent telangiectasias have been noted. Individual lesions
appear as erythematous annular papules or plaques. Lesions
are usually more numerous and more intensely inflamed
on the face and scalp but may also occur on the trunk and
extremities. Confluent periorbital erythema, giving the
appearance of an erythematous mask, is common and diag-
nostically helpful. Even though the skin disease resolves and
most children without extracutaneous involvement remain
otherwise healthy, there is a possibility that children who
have had NLE are at increased risk for the development of
autoimmune disease later in childhood.25
Differential diagnosis of the skin lesions may include
reactive erythema, drug eruption, erythema multiforme,
and urticaria. Annular NLE lesions usually have little or
691
no scale, unlike the annular lesions of tinea. In areas where
there is intense destruction of the basal cell layer, lesions
may be crusted and look similar to bullous impetigo. Treat-
ment of skin lesions consists largely of sun protection and
mild topical steroids.
The pathogenesis of lupus is covered elsewhere, but it is
noteworthy that SCLE-like, anti-Ro/SS-A–associated skin
lesions may occur in neonates; however, other lupus-specific
skin lesions do not appear to be maternally transmissible.
SJÖGREN’S SYNDROME
The most common mucocutaneous findings of Sjögren’s syn-
drome are related to glandular dysfunction. Lacrimal gland
dysfunction causes dryness and irritation of the eyes and can
lead to keratitis and corneal ulceration. Salivary gland dys-
function causes dry mouth and may result in angular chei-
litis and numerous dental caries. Vaginal xerosis may cause
burning and dyspareunia. The skin may be dry, cracked, and
pruritic. Mildly dry mucous membranes and even severely
dry skin may be present in a substantial percentage of nor-
mal individuals who live in dry climates, so the findings
should be interpreted in the context of the setting. In Japa-
nese patients with Sjögren’s syndrome, an annular erythema
has been described that is somewhat reminiscent of annular
SCLE or the annular lesions of neonatal lupus, although
they are more indurated.26
Vasculitis is a relatively common finding. In one series
of 558 patients with primary Sjögren’s syndrome, 52 had
vasculitis, typically involving small vessels. In most cases,
lesions were purpuric, but in some, urticarial vasculitis was
the clinical presentation. Patients with cutaneous evidence
of vasculitis generally had more severe systemic disease.27
DERMATOMYOSITIS
The current American College of Rheumatology criteria
for dermatomyositis (DM) do not recognize the existence
of amyopathic dermatomyositis (ADM). This has led to a
problem in diagnosing patients with predominantly skin
involvement. ADM, or dermatomyositis sine myositis, refers
to classic cutaneous manifestations of DM without evidence
of inflammatory myopathy. ADM has also been defined as
the presence of biopsy-proven cutaneous findings of classic
DM for 6 months or longer without any clinical evidence
of proximal muscle weakness, serum muscle enzyme abnor-
malities, or abnormal muscle testing. This latter definition
excludes any patient treated with systemic immunosuppres-
sive therapy for 2 or more consecutive months during the
first 6 months of cutaneous manifestations of DM, because
such therapy could suppress clinically significant myositis,
or any patient using drugs that are associated with DM-like
skin changes (e.g., hydroxyurea).28
The most common cutaneous manifestations of active
DM include Gottron’s papules (Fig. 47-9) and Gottron’s
sign, which are pathognomonic of DM. Other characteristic
findings of DM include heliotrope rash of the periorbital and
upper eye area (Fig. 47-10), V- or shawl-shaped macular ery-
thema over the chest and back, cuticular overgrowth, and
periungual telangiectasias. Patients with active disease can
have widespread erythema over the trunk and ­extremities,
with accentuation on the extensor arms and legs and the
 692 LEE  |  The Skin and Rheumatic Diseases
lateral thighs. Erythema and scale of the scalp can result in
extensive alopecia. Hyperkeratosis of the palmar and lateral
surfaces of the fingers, called mechanic’s hands, can be asso-
ciated with anti-Jo-1 autoantibodies and interstitial lung
disease. Rarely, patients can have a panniculitis. Vasculopa-
thy, with livedo reticularis and ulceration, can occur. Itch-
ing can result in excoriations and lichenification. Damage
from lesions includes postinflammatory hyperpigmentation,
poikiloderma, calcinosis, lipoatrophy, and depressed scars.29
Poikiloderma is a descriptive term for a pattern of finely mot-
tled white areas and brown pigmentation, telangiectasia,
and atrophy.
Skin biopsy from a patient with cutaneous DM is iden-
tical to that seen with cutaneous lupus erythematosus. A
diagnosis of DM is made by clinical-pathologic correlation
and need not include muscle disease. In the absence of
clinical muscle findings, the workup for DM should include
muscle enzyme testing, pulmonary function testing, chest
radiograph, electrocardiogram, and evaluation for underly-
ing malignancy. There has been an increased association
between DM, including ADM, and underlying malignancy.
The most frequent malignancies are lung, ovarian, pancre-
atic, stomach, colorectal, and non-Hodgkin’s lymphoma.30
An increased risk of malignancy is present for at least 5 years
after the diagnosis of DM, so patients should receive routine
cancer screening during that time.31 Patients with DM fre-
quently experience a delay in diagnosis, and the presence of
photosensitivity, malar rash, oral ulcers, and a positive anti-
nuclear antibody test means that they are frequently misdi-
agnosed as having SLE, having met the current criteria for
that disease.
In patients with both skin and muscle disease, the mus-
cle disease frequently resolves with aggressive treatment
with glucocorticoids, with or without immunosuppressives.
Patients are sometimes treated with intravenous immuno-
globulin (IVIG), cyclosporine, or tacrolimus. Patients with
AMD or residual skin disease after treatment often benefit
from hydroxychloroquine. Patients who do not improve on
a single antimalarial can benefit from the addition of quina-
crine or a switch from hydroxychloroquine to chloroquine.32
Immunosuppressives such as methotrexate, azathioprine,
or mycophenolate mofetil can be of additional benefit in
patients with resistant skin disease. IVIG can be of benefit
for the skin disease of DM, and studies related to the role of
biologics are ongoing.33
Figure 47-9  Gottron’s papules over the interphalangeal joints in
dermatomyositis.
Figure 47-10  Heliotrope eruption of dermatomyositis, with character-
istic edema.
SCLERODERMA AND OTHER SCLEROSING
CONDITIONS
MORPHEA
Scleroderma can occur as localized or systemic disease. The
localized form of the disease occurs as localized or gener-
alized morphea, linear scleroderma, or facial hemiatrophy,
otherwise known as Parry-Romberg syndrome. Linear sclero-
derma can occur over the forehead in a variant called en
coup de sabre (Fig. 47-11). Morphea is seen more commonly
in adults, with an increased incidence with advancing age;
linear scleroderma occurs more frequently in children and
adolescents.34 Although remissions are reported to occur in
3 to 5 years, ongoing clinical activity or reactivation is not
unusual.
Localized scleroderma patients typically lack sclerodac-
tyly, Raynauds’ phenomenon, or internal organ involvement.
The level of involvement in localized scleroderma can be in
the dermis (morphea), fat (subcutaneous morphea), fat and
fascia (morphea profundus), and fascia (eosinophilic fasci-
itis). Morphea typically has round or oval, irregular plaques
that are initially dull red or violaceous, smooth, and indu-
rated. They frequently progress to chalky white, atrophic
lesions, although some patients have residual hyperpigmen-
tation overlying the lesions. Morphea can have different
presentations. When it overlaps with lichen sclerosus et
atrophicus, flat-topped papules coalesce to form a white
plaque, sometimes combined with a deeper morphea lesion.
Some lesions are small and oval, known as guttate morphea.
There are many mimickers of morphea, including radiation-
induced morphea, injection-induced morphea-like lesions,
morphea-like Lyme disease (seen in Europe), eosinophilia-
myalgia syndrome, toxic oil syndrome, and, more recently,
nephrogenic fibrosing dermopathy.
Linear scleroderma is frequently located on the lower
limbs, upper limbs, frontal head area, and anterior trunk.
It is frequently unilateral and can result in joint deformity,
joint contractures, and limb atrophy. Some cases are asso-
ciated with seizures or other focal neurologic symptoms.
Parry-Romberg syndrome can occur in the first or second
decade of life and leads to unilateral facial atrophy in 95%
of cases. Half of patients start as en coup de sabre and progress
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
Figure 47-11  Linear scleroderma of the forehead. (Courtesy of
Dr. Victoria Werth, University of Pennsylvania Department of Dermatology
and Philadelphia VAMC, Philadelphia.)
to soft tissue involvement in the upper face. Patients can
have seizures, headaches, visual changes, and atrophy of the
salivary glands and hemiatrophy of the tongue on the same
side as the facial atrophy. Any reparative surgical treatment
should be timed to occur no sooner than 1 year after cessa-
tion of the ongoing atrophic process.
Treatment of localized scleroderma includes protection
from trauma and cold, antimalarials (hydroxychloroquine
[Plaquenil] alone, hydroxychloroquine and quinacrine
together, or chloroquine with or without quinacrine), low-
dose prednisone for eosinophilic fasciitis, topical calcipot-
riene, and methotrexate.35 Phototherapy with narrow-band
UVB, UVA1, and PUVA and topical photodynamic therapy
have also been used.36,37
SYSTEMIC SCLERODERMA
Patients with early limited cutaneous scleroderma have
­Raynaud’s phenomenon for many years, minimal consti-
tutional symptoms, puffy fingers, limited skin thickening,
and anticentromere antibody. Patients with early diffuse
cutaneous scleroderma frequently have delayed Raynaud’s
phenomenon, acute onset, many constitutional symptoms,
arthralgias, tendon friction rubs, swollen puffy hands, and
early diffuse skin thickening; they may have anti-Scl-70
antibody as well as anti-RNA polymerase III. The major
and minor criteria for diffuse cutaneous scleroderma include
many skin findings. Major criteria include proximal sclero-
derma. Minor criteria include sclerodactyly (Fig. 47-12),
digital pitting and scars of the fingertips, loss of substance of
the finger pad, and ­bibasilar pulmonary fibrosis.38 Ischemia
and skin changes in systemic scleroderma can result in skin
ulcers. Therapies used in the cutaneous treatment of systemic
scleroderma include D-penicillamine, methotrexate, cyclo-
phosphamide, photopheresis, and bone marrow transplan-
tation.39-41 Treatment of Raynaud’s phenomenon is covered
693
elsewhere. There are reports that bosentan, an endothelin
receptor antagonist, and phosphodiesterase inhibitors such
as sildenafil have been helpful in the treatment of skin
ulcers.42,43 Patients who have pruritus may benefit from sys-
temic ­antihistamines.
EOSINOPHILIC FASCIITIS
Eosinophilic fasciitis (EF) involves inflammation of the fas-
cia overlying muscle and results in swelling of the extremi-
ties, followed by fibrosis and contractures. The digits are
typically spared. There is often a rapid onset of disease activ-
ity, particularly following physical exertion. A contaminant
of l-tryptophan was associated with an EF-like disease in
the early 1990s.44 Approximately 30% of EF patients have
morphea concurrently. Occasionally, localized scleroderma
overlaps with other autoimmune diseases, such as SLE.
Diagnosis is based on a deep, usually excisional biopsy of
the skin that includes fascia. There are inflammatory cells
among collagen bundles, thickening of collagen, sclerosis of
the dermis and fat or fascia, and absent sweat glands and
hair. Antinuclear antibodies are positive 46% of the time in
localized scleroderma; this correlates with disease severity.45
A peripheral eosinophilia and hypergammaglobulinemia
can be seen.
POEMS SYNDROME
POEMS syndrome includes polyneuropathy, organomeg-
aly, endocrinopathy, monoclonal gammopathy, and skin
changes. Polyneuropathy and a monoclonal plasma prolifer-
ative disorder must be present, along with one other minor
criterion, including sclerotic bone lesions, Castleman’s dis-
ease, organomegaly, edema, endocrinopathy, papilledema,
or skin changes. Other associated findings may include asci-
tes, pleural effusions, thrombocytosis, fingernail clubbing,
and white nails. The sensorimotor polyneuropathy has both
demyelinating and axonal features and is slowly progressive
and debilitating in most cases. Organomegaly may consist of
hepatosplenomegaly or lymph node enlargement. Endocri-
nopathies include diabetes, impotence, gynecomastia, and
hypothyroidism. The monoclonal (M) protein abnormality
consists of IgA or IgG heavy chains with λ light chains. The
Figure 47-12  Sclerodactyly with flexion contractures.
 694 LEE  |  The Skin and Rheumatic Diseases
skin changes may consist of hyperpigmentation, hypertri-
chosis, hyperhidrosis, skin thickening, telangiectasia, and
glomeruloid hemangiomas.46 Treatment with corticoste-
roids, low-dose alkylators, high-dose melphalan, and autol-
ogous peripheral blood stem cell transplantation has been
reported to be successful.47,48
SCLEROMYXEDEMA
Scleromyxedema is a rare disorder frequently characterized
by a dysproteinemia and widespread skin changes. The para-
protein has been shown to stimulate fibroblast production
of mucin, suggesting a causal role and a rationale for lower-
ing it. Patients have waxy papules on the face, neck, upper
trunk, forearms, hands, and thighs that become confluent
and occur in association with underlying sclerosis, which
can lead to joint contractures, sclerodactyly, and carpal
tunnel syndrome. Common extracutaneous manifestations
include upper gastrointestinal dysmotility, muscle weak-
ness, joint contractures, and neurologic symptoms such as
seizures, encephalopathy, coma, and obstructive or restric-
tive pulmonary disease. Eighty percent of patients have a
monoclonal gammopathy, most frequently IgG-λ, but occa-
sionally IgG-κ.
Skin biopsy shows mucin deposition and a prolifera-
tion of fibroblasts in the upper dermis. Treatment has
included prednisone, IVIG, PUVA, systemic retinoids,
thalidomide, interferon-α, plasmapheresis, photophere-
sis, low-dose melphalan, and high-dose dexametha-
sone.49,50 Chemotherapeutic agents such as melphalan,
cyclosporine, cladribine, cyclophosphamide, methotrex-
ate, and chlorambucil have been used. Successful remis-
sion after autologous stem cell transplantation has been
reported.51
NEPHROGENIC FIBROSING DERMOPATHY
Nephrogenic fibrosing dermopathy (NFD) is a relatively
new illness that occurs in patients with renal disease; most
patients have undergone dialysis for renal failure.52 NFD
presents as either a morphea-like disease or a more dif-
fuse acral sclerosois. Morphea-like presentations include
ill-defined indurated plaques, with islands of sparing and
finger-like projections, that involve the lower more than
the upper extremities. More diffuse confluent acral sclero-
sis, sometimes with trunk involvement, can occur. There
are often yellow plaques on the conjunctiva. Patients may
experience pain, severe itching, joint contractures, and
fibrosis and calcification of the skin, subcutaneous tissue,
fascia, muscle, myocardium, lung, renal tubule, and testis.
Patients typically do not have Raynaud’s phenomenon.
Skin biopsy is identical to that seen with scleromyx-
edema, with stellate fibroblasts, glycosaminoglycans, and
thickening of collagen.53 Transforming growth factor-β is
increased in lesional skin.54 There is no proven effective
therapy, and prognosis depends on the extent and rapidity
of skin involvement and the severity of systemic disease.54
Treatments with potential benefit include plasmapheresis,
IVIG, immunosuppressives, glucocorticoids, interferon-
α, thalidomide, PUVA, UVA1, and photopheresis.55 The
critical issue is to determine the inciting agent in this
disease.
PRIMARY NECROTIZING VASCULITIS
INVOLVING THE SKIN
In cutaneous diseases, vasculitis is a term typically reserved
for lesions characterized by damage to vessel walls and a
neutrophilic or granulomatous infiltrate. Classification,
pathogenesis, diagnostic evaluation, and therapy are cov-
ered elsewhere. Here, the focus is on the skin findings and
differential diagnosis. The primary determinant of the
appearance is the size of the vessel affected.
LEUKOCYTOCLASTIC SMALL VESSEL VASCULITIS
AND ITS VARIANTS
Leukocytoclastic small vessel vasculitis is a rather common
condition with characteristic histologic findings of fibrinoid
necrosis of vessel walls, a neutrophil-predominant infiltrate,
and leukocytoclasis (i.e., fragmented nuclei resulting from
the degeneration of neutrophils).56 In the skin, the ves-
sels involved are in the dermis, and damage to these ves-
sels results in lesions of a characteristic size. The lesions are
both erythematous and purpuric and are distinctly palpable
if there are sufficient numbers of neutrophils in the lesion.
The usual diameter of the purpuric papules is about 0.3 to
0.6 cm, although smaller and larger lesions may be observed
(Fig. 47-13). Discrete lesions have a round shape. The cen-
ter may look dusky, pustular, or ulcerated, or it may appear as
a hemorrhagic vesicle. Larger ulcerations may occur when
lesions coalesce. Particularly in larger lesions, the devital-
ized tissue may be a focus for secondary bacterial infection.
Lesions tend to occur in dependent areas. Thus, for ambula-
tory patients, lesions are most numerous on the lower legs.
Koebnerization—the appearance of lesions along lines of
trauma, such as scratches—is sometimes observed.
As mentioned under the discussion of rheumatoid
­vasculitis, the differential diagnosis of purpuric or pete-
chial lesions may include stasis dermatitis, Schamberg’s
purpura, platelet dysfunction, petechial drug eruptions,
viral exanthems, emboli, thromboses, and sludging. Skin
biopsy is often helpful in establishing the diagnosis of small
vessel vasculitis, especially if an early lesion is sampled.
Figure 47-13  Leukocytoclastic vasculitis demonstrating nonblanch­
ing, purpuric macules.
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
Immunofluorescence of an early lesion may establish
whether the vasculitis is IgA predominant.
Small vessel vasculitis unassociated with connective tis-
sue disease and not IgA predominant is sometimes called
hypersensitivity vasculitis. It is often apparently confined to
the skin and therefore has a good prognosis. In some cases,
infections or drugs may be implicated, but often, the ini-
tiating event is unknown. Therapy is directed first toward
treating the underlying cause, if one is found. If there is
no significant extracutaneous disease, treatment is often
symptomatic. Leg elevation, compression stockings, and
reduction of activity may be helpful. Nonsteroidal anti-
inflammatory agents or antihistamines are sometimes used.
Systemic corticosteroids are not routinely indicated for skin-
limited disease. For patients with persistent disease confined
to the skin, colchicine and dapsone have each been used
with some success.57
A common variant of small vessel vasculitis is Henoch-
Schönlein (HS) purpura. HS purpura occurs commonly in
children and is often associated with extracutaneous find-
ings of gastrointestinal or renal involvement. The typi-
cal lesion of IgA-predominant small vessel vasculitis is an
erythematous or urticarial macule or papule that evolves
rapidly into palpable purpura. It has been noted that IgA-
predominant vasculitis in particular may display superficial
plaques of palpable purpura or a retiform configuration of
lesions.58 The most reliable means of establishing that the
vasculitis is IgA predominant is biopsy for immunofluores-
cence. IgA-predominant vasculitis is not unusual in adults.
Compared with the presentation in children, there is a lower
association with a preceding upper respiratory infection and
a higher association with medication use.
Mixed cryoglobulinemia can present as a small vessel vas-
culitis. Additional skin findings include livedo reticularis,
urticarial papules, cold urticaria, Raynaud’s phenomenon,
acrocyanosis, leg ulcers, and digital ulceration or gangrene.
Hepatitis C is a frequent association. Patients with type I
monoclonal cryoglobulinemia may have purpura due to
cryogelling rather than a true vasculitis.
A distinct subset of patients with small vessel vasculitis
has lesions that are primarily urticarial rather than purpuric.
The main clinical entity in the differential diagnosis is urti-
caria. Individual lesions of urticaria tend to be short-lived,
usually lasting less than 24 hours, whereas individual lesions
of urticarial vasculitis tend to last for several days. Addi-
tional skin findings may include angioedema, livedo reticu-
laris, nodules, and bullae. In some lesions, foci of purpura
may be observed. Urticarial vasculitis has been classified
into two groups: normocomplementemic and hypocomple-
mentemic. Extracutaneous disease is more likely to occur in
the hypocomplementemic group, and some of these patients
have underlying SLE. It has been proposed that there is a
distinct subset of the hypocomplementemic group char-
acterized by IgG antibodies to C1q, angioedema, ocular
inflammation, arthritis, obstructive pulmonary disease, and
renal disease. The pulmonary disease tends to be severe and
life threatening. This entity has been termed hypocomple-
mentemic ­urticarial vasculitis syndrome.59,60
Erythema elevatum diutinum is an unusual form of small
vessel vasculitis that is characterized by erythematous or vio-
laceous papules, plaques, and nodules over the dorsal hands,
ears, knees, heels, and buttocks. In the clinical differential
695
diagnosis are Sweet’s syndrome, multicentric reticulohis-
tiocytosis, sarcoidosis, and lymphoma, among others. With
time, fibrosis often occurs. Established lesions may be disfig-
uring and may have an appearance somewhat reminiscent of
keloids. Although significant extracutaneous involvement
is not expected, and many patients are otherwise well, ery-
thema elevatum diutinum has been reported in association
with various autoimmune, infectious, and hematologic con-
ditions, including streptococcal infection, paraproteinemia,
inflammatory bowel disease, RA, SLE, and HIV. For active
skin lesions, dapsone is the usual treatment.61 Intralesional
corticosteroids are sometimes used for fibrotic lesions.
Acute hemorrhagic edema of childhood is an uncommon
but generally benign and self-limited form of vasculitis usu-
ally occurring in children younger than 2 years, commonly
preceded by an upper respiratory infection or medication.62
The clinical appearance may be dramatic, with large purpu-
ric plaques on the face, ears, and extremities. Based on the
appearance of the skin lesions, meningococcemia is some-
times suspected, but a child with acute hemorrhagic edema
appears relatively healthy. Generally, there is no extracuta-
neous involvement. Treatment is symptomatic.
GRANULOMATOUS VASCULITIDES
Skin lesions are occasionally the presenting feature of
Wegener’s granulomatosis.63 The most common type of
lesion is palpable purpura, with or without necrosis. Many
other types of lesions have been noted, including papules,
ecchymoses, hemorrhagic bullae, necrotic papules, subcuta-
neous nodules, and ulcers. Ulcerative lesions may be similar
in appearance to pyoderma gangrenosum, though they lack
an undermined border. Oral ulcers are relatively common
but nonspecific in appearance. A more specific oral finding
is hypertrophic gingival inflammation with petechiae.
Skin biopsy is sometimes helpful diagnostically, but
unfortunately, biopsies are often nonspecific or show leuko-
cytoclastic vasculitis. True granulomatous vasculitis is not
often observed in skin specimens. Extravascular granulo-
matous inflammation is sometimes noted and may be more
likely in nonpurpuric papules or nodules than in palpable
purpura.
Differential diagnosis of the skin lesions includes other
small vessel vasculitides, particularly when cutaneous
lesions consist of palpable purpura and the biopsy finding is
leukocytoclastic vasculitis. If granulomatous inflammation
is observed, the differential diagnosis may include Churg-
Strauss syndrome, polyarteritis nodosa, microscopic poly-
angiitis, RA, SLE, infection, lymphoproliferative disorders,
chronic active hepatitis, erythema nodosum, granuloma
annulare, and inflammatory bowel disease.
Patients with Churg-Strauss syndrome characteristically
present with respiratory symptoms, but skin lesions are com-
mon during the vasculitic phase of the disease.64 Hemor-
rhagic lesions ranging from petechiae to palpable purpura to
ecchymoses, cutaneous nodules with or without ulceration,
subcutaneous nodules, and nonspecific erythematous erup-
tions are most common. As with Wegener’s granulomatosis,
hemorrhagic lesions tend to show small vessel vasculitis on
biopsy, and nodules are more likely to demonstrate granulo-
mas. The clinical and histologic differential diagnosis often
includes polyarteritis nodosa, Wegener’s granulomatosis,
 696 LEE  |  The Skin and Rheumatic Diseases
and microscopic polyangiitis. As alluded to earlier, the his-
tologic finding of Churg-Strauss granuloma is not specific
and may be seen in several entities. Large numbers of eosin-
ophils in the biopsy may be diagnostically helpful but not
definitive. Hypereosinophilic syndrome may share clinical
and laboratory features with Churg-Strauss syndrome, but
vasculitis is not characteristic.
POLYARTERITIS NODOSA AND RELATED
CONDITIONS
Historically, classic polyarteritis nodosa (PAN) and micro-
scopic polyangiitis were classified together as PAN, but
they appear to be distinct conditions distinguishable in part
by the size of the vessels affected. Classic PAN involves
medium-sized vessels, whereas microscopic polyangiitis
involves primarily vessels ranging in size from capillaries to
arterioles.
Cutaneous findings of classic PAN represent damage
downstream of the affected vessel and consist of ulceration,
digital gangrene, ecchymoses from vessel rupture, livedo
reticularis, and subcutaneous nodules that may follow the
course of arteries. Because the affected vessel is proximal to
the skin, skin biopsy is likely to show nonspecific findings.
The skin findings of microscopic polyangiitis reflect the
smaller size of the vessels affected. Petechiae, palpable pur-
pura, purpuric plaques, erythematous nodules, and ulcer-
ations may be observed. There have also been some reports
of livedo reticularis. The cutaneous pathology is that of a
leukocytoclastic vasculitis, but in contrast to many of the
small vessel vasculitides previously discussed, small arteri-
oles may be involved. The differential diagnosis is mainly
other small vessel vasculitides. The size of affected vessels,
antineutrophil cytoplasmic autoantibody (ANCA) positiv-
ity, paucity or absence of antibody deposits in vessels, and
spectrum of extracutaneous involvement aid in distinguish-
ing microscopic polyangiitis from other vasculitides.
There is a variant of PAN termed benign cutaneous PAN
or, perhaps more accurately, primarily cutaneous PAN. In this
condition, arterioles in the subcutaneous fat and lower der-
mis are affected, and the presentation is often that of tender
subcutaneous nodules and livedo reticularis. Associations
with Crohn’s disease, hepatitis B, and hepatitis C have been
reported. Clinically, panniculitides such as erythema nodo-
sum and erythema induratum may be considered, although
livedo reticularis is not expected. Biopsy for diagnosis should
include subcutaneous fat. Even after biopsy, microscopic
polyangiitis may be difficult to exclude. Although relatively
little information is available concerning ANCA in cutane-
ous PAN, a negative ANCA is more consistent with cuta-
neous PAN than with microscopic polyangiitis. Although
the outcome is benign, the course is often chronic and
relapsing. Therapy is typically conservative and may consist
of intralesional corticosteroids, nonsteroidal anti-inflamma-
tory drugs, low-dose methotrexate, dapsone, or, occasion-
ally, systemic corticosteroids.
Kawasaki’s disease is considered a PAN variant due to the
involvement of coronary arteries. Skin findings ­ constitute
several of the criteria for diagnosis. None of the findings
is specific for Kawasaki’s disease, but the constellation of
findings establishes the diagnosis. Kawasaki’s disease usu-
ally affects young children but has been reported in adults.
Criteria for diagnosis are otherwise unexplained fever
for at least 5 days and four of the following five ­ findings:
(1) bilateral nonexudative conjunctivitis; (2) injected
pharynx, strawberry tongue, or injected or fissured lips; (3)
erythema of palms or soles, hand and foot edema, and, in
the convalescent phase, desquamation; (4) erythematous,
polymorphous, generalized skin eruption; and (5) cervical
lymphadenopathy. In addition, erythema of the perineal
region is quite common, and transverse lines across the fin-
gernail beds have been noted in a few cases.
LARGE VESSEL VASCULITIS
Skin findings of temporal arteritis (giant cell arteritis) con-
sist mainly of palpable temporal arteries, skin tenderness in
the area, and scalp nodules or ulcerations. Skin lesions in
patients with Takayasu’s arteritis may include Raynaud’s
phenomenon, livedo reticularis, ulcerated nodules, subcu-
taneous nodules, and pyoderma gangrenosum–like ulcers.
Skin biopsy is generally not performed in these conditions.
INFECTIONS
There are many infectious diseases that present with both
skin and rheumatologic findings.65 This section highlights
a few examples.
LYME BORRELIOSIS
Borrelia burgdorferi, the causative agent of Lyme disease in
North America, is associated with erythema migrans (EM).
In Europe, the related genospecies Borrelia afzelii is associ-
ated with both EM and acrodermatitis chronica atrophicans
(ACA), and several European studies have found compel-
ling evidence of B. afzelii infection in patients with mor-
phea. There is no similar association between Borrelia and
morphea in the United States.66 Hematogenous dissemina-
tion from the initial skin site is believed to cause secondary
skin lesions and extracutaneous manifestations, and only
certain subtypes of B. burgdorferi are associated with dis-
semination.
EM is the first manifestation of Lyme disease in 60%
to 80% of people and occurs at the site of the tick bite.67
At the time of the skin lesion, which occurs within a few
days to a month after the bite, the spirochetes enter the cir-
culation and disseminate. The skin findings may be asso-
ciated with fever, chills, fatigue, headache, neck stiffness,
myalgias, arthralgias, conjunctivitis, erythematous throat,
and regional or generalized lymphadenopathy. The lesions
of EM begin as red macules that become papular and then
expand into an erythematous, annular plaque (Fig. 47-14).
There are two forms of EM. In one, there is an expanding
red plaque with varying intensities of redness within the
plaque. In the second, there is a target-like appearance, with
a central red plaque surrounded by normal-appearing skin,
which in turn is surrounded by another band of erythema.
The lesions can enlarge very rapidly, and multiple lesions
due to hematogenous spread are seen in 17% of cases. As
the lesion enlarges, the central erythema may fade. The
central portion of the lesion may be edematous, vesicular,
urticarial, or crusted. Triangular and elongated oval lesions
have been described, but circular lesions are most frequent.
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
Figure 47-14  Lyme disease with characteristic erythematous, annular
plaques. (Courtesy of Dr. Joshua Levin, University of Pennsylvania Depart-
ment of Dermatology, Philadelphia.)
The most common locations are the inguinal region, axil-
lae, abdomen, and behind the knees. EM lesions are usually
­asymptomatic but can be pruritic or painful. Untreated EM
lesions resolve in a median of 28 days, with a range from
1 day to 14 months. Resolution occurs within a few days
after treatment with antibiotics such as doxycycline or peni­
cillin.
ACA is associated with late Lyme disease. It occurs mainly
in women between 40 and 70 years old. The lesions begin on
an extremity, usually the lower leg or foot, as a bluish red
edematous plaque. Fibrous bands may develop, especially in
the ulnar and tibial regions, and fibrous nodules may form
near joints. Regional lymphadenopathy is often present.
Over many years, the skin becomes atrophic.68 B. burgdorferi
has been isolated from the skin of patients with ACA.69
PARVOVIRUS
Presentation of parvovirus B19 includes an erythematous
“slapped cheeks” appearance; a lacy, reticulated proximal
extremity rash; a febrile petechial eruption; and papular-
purpuric gloves and socks syndrome. The infection is self-
­limited and generally resolves spontaneously within 1 to
2 weeks. Laboratory findings may include mild or severe
leukopenia, transient neutropenia or relative neutrophilia,
eosinophilia, and mild thrombocytopenia. Adults often
contract the virus from infected children and commonly
present with systemic disease, including arthropathy and a
flulike illness.70
ATYPICAL INFECTIONS: MycoBACTERIUM
MARINUM
There are many types of mycobacterial, atypical mycobac-
terial, and deep fungal infections that can affect the skin
and joints. Mycobacterium marinum is one example; it can
be acquired through exposure to fresh water, salt water, fish
tanks, swimming pools, fish or other aquatic organisms,
timber cuts, or splinters. The incubation period is usually
about 3 weeks, although much longer periods are possible.
The disease often occurs after inoculation into abrasions or
697
after penetrating injuries to the fingers and hands. This is an
indolent disease consisting of nodules or ulcerated plaques,
occasionally with extension to deep tissue. Common areas
of involvement are the fingers, dorsum of the hands, and
knees. The lesions can be localized or sporotrichoid (25%),
with dissemination 2% of the time.
PANNICULITIS
Panniculitis refers to a group of diseases that manifest as
inflammation or alterations in the subcutaneous fat. The
complexity of causes for even one form of panniculitis, such
as erythema nodosum; the relative rarity of most forms of
panniculitis; and the number of different panniculitides has
slowed the acquisition of scientific knowledge. The causes
of many panniculitides are still poorly understood.
Panniculitis may be primary, without an identifiable
cause, or secondary. Common secondary causes of pannicu-
litis include infection, trauma, pancreatic disease, immu-
nodeficiency states, malignancies, and connective tissue
disease. Erythema nodosum is the most common form of
panniculitis, and although there is a long list of diseases
and medications that have been associated with it, there is
often no identifiable underlying condition. Underlying con-
ditions that have been associated with erythema nodosum
include inflammatory bowel disease, sarcoidosis, malignan-
cies such as leukemia and lymphoma, infections (bacterial,
Yersinia, rickettsial, chlamydial, spirochetal, and protozoal
disease), pregnancy, drugs (sulfonamides, contraceptives),
and autoimmune diseases such as Behçet’s disease, Sjögren’s
syndrome, Reiter’s syndrome, and SLE.71
As the understanding of lobular panniculitis has
expanded, cases that were once grouped under the catch-
all of Weber-Christian disease are now recognized to be
clearly definable and separate entities such as lupus pan-
niculitis, cytophagic histiocytic panniculitis, α1-antitrypsin
deficiency, factitial panniculitis, traumatic panniculitis, and
calciphylaxis.72-74
Infections are recognized as a trigger of panniculitis, as
exemplified by erythema nodosum due to streptococcal
infection, hepatitis B or C associated with PAN, infec-
tious panniculitides in immunocompromised hosts, and,
most recently, some cases of erythema induratum or nodu-
lar ­vasculitis associated with Mycobacterium tuberculosis. In
addition, atypical infections can cause lesions that look like
panniculitis.
An understanding of the heterogeneity of lymphomas
that involve the fat is still evolving, but advances are being
made in differentiating various histologic and clinical out-
comes.75 For example, some patients thought to have lupus
panniculitis based on cytopenia and laboratory tests were
found to actually have subcutaneous lymphoma, after a
careful review of their pathology.
Patients with panniculitis frequently have erythematous
tender nodules, and the clinical presentation is seldom spe-
cific enough to allow a determination of the exact subtype of
panniculitis without a biopsy. Patients with panniculitis can
have associated symptoms such as low-grade fever, fatigue,
arthralgia, and myalgias.
An adequate skin biopsy, often involving an elliptical
excision, is essential to properly diagnose the various enti-
ties that fall under the category of panniculitis (Table 47-1).
 698 LEE  |  The Skin and Rheumatic Diseases
Table 47-1  Classification of Panniculitis
I.  Without prominent vasculitis
  A.  Septal inflammation
    1.  Lymphocytic and mixed: erythema nodosum and variants
    2. Granulomatous: palisaded granulomatous diseases, sarcoid-
osis, subcutaneous infection (tuberculosis, syphilis)
    3. Sclerotic: scleroderma, eosinophilic fasciitis, lipodermato-
sclerosis, toxins
  B.  Lobular inflammation
    1. Neutrophilic: infection, ruptured folliculitis and cysts, pan-
creatic fat necrosis
    2. Lymphocytic: lupus panniculitis, poststeroid panniculitis,
lymphoma, leukemia
    3. Macrophagic: histiocytic cytophagic panniculitis
    4. Granulomatous: erythema induratum, nodular vasculitis, pal-
isaded granulomatous diseases, sarcoidosis, Crohn’s disease
    5. Mixed inflammation with many foam cells: α1-antitrypsin de-
ficiency, Weber-Christian disease, traumatic fat necrosis
    6. Eosinophilic: eosinophilic panniculitis, arthropod bites,  steroid-sparing agents. There have been reports of response
parasites
    7.  Enzymatic fat necrosis: pancreatic enzyme panniculitis
    8. Crystal deposits: sclerema neonatorum, subcutaneous fat
necrosis of the newborn, gout, oxalosis
    9.  Embryonic fat pattern: lipoatrophy, lipodystrophy
II.  With prominent vasculitis (septal or lobular)
  A. Neutrophilic: leukocytoclastic vasculitis, subcutaneous poly-
arteritis nodosa, thrombophlebitis, erythema nodosum lepro-
sum (ENL)
  B. Lymphocytic: nodular vasculitis, perniosis, angiocentric lym-
phomas
  C. Granulomatous: nodular vasculitis, erythema induratum, ENL,
Wegener’s granulomatosis, Churg-Strauss allergic granuloma-
tosis
III. Mixed patterns
Panniculitis is typically divided into four main subgroups:
septal, lobular, mixed, and panniculitis with vasculitis.
Determining the exact nature of the cellular infiltrate also
contributes to arriving at a proper diagnosis. There is no
question that these categorizations help narrow the differ-
ential diagnosis in any given case, but there may be overlap-
ping features or reaction patterns that do not allow a specific
diagnosis. Clinical-pathologic correlation is important, as
emphasized by a published review that includes an expanded
and useful classification of panniculitis.76
There are anecdotal reports of the efficacy of combina-
tion antimalarials such as hydroxychloroquine and quina-
crine in treating subcutaneous sarcoid, but no studies exist,
so definitive recommendations cannot be made. Reports
about the use of mycophenolate mofetil and thalidomide to
treat inflammatory causes of panniculitis, such as nodular
panniculitis and erythema nodosum, have already been pub-
lished, indicating that newer drugs will likely evolve for the
treatment of these conditions.77,78 The efficacy of these and
more established drugs, such as nonsteroidal anti-inflamma-
tory agents, antimalarials, and methotrexate, needs to be
studied, and it is hoped that outcomes will be more system-
atically evaluated.
RELAPSING POLYCHONDRITIS
The diagnosis of relapsing polychondritis is based on the
typical clinical manifestations, with auricular findings seen
in 90% of patients. Nasal and respiratory tract ­ chondritis
can occur, along with nonerosive inflammatory arthritis,
cardiac valvular insufficiency, vasculitis, and eye and audio-
vestibular involvement. The estimated prevalence of 3.5
per million makes controlled trials nearly impossible. The
cause is unknown, but the pathogenesis appears to be medi-
ated by an immune reaction to type II collagen. Clinical
skin manifestations include inflammation of the ear, with
sparing of the earlobe. Diagnosis includes the presence of
a positive serum antibody test to type II collagen and a
wedge biopsy that shows cartilage necrosis and perichondral
inflammation with lymphocytes and histiocytes. Involve-
ment of other cartilage areas, including the upper airway,
should be assessed. Glucocorticoids are the therapeutic
choice for reducing the inflammatory process in patients
with relapsing polychondritis. For patients with sustained
disease, many immunosuppressive drugs have been used as
to tumor necrosis factor-α (TNF-α) inhibitors in patients
­otherwise refractory to therapy.79
INFILTRATIVE DISEASES
AMYLOID
Type AL amyloidosis is rare, with an incidence of less than
1 per 100,000. Skin lesions may occur in up to 40% of these
patients. Skin lesions can be an early sign of the disease and
include purpura, petechiae, and ecchymoses due to infiltra-
tion of blood vessels by amyloid. Other skin findings include
alopecia, plaques, and nodules, often found on flexor sur-
faces, the face, or the buccal mucosa. Bullae and nail dys-
trophy are occasionally seen. Diagnosis is confirmed by
biopsy of lesional or nonlesional skin, along with urine and
serum immunoelectrophoresis to confirm the presence of a
circulating monoclonal protein. Skin biopsy shows Congo
red–­positive, homogeneous, hyaline, fibrillary deposits.
Treatment includes autologous stem cell transplantation,
with approximately 50% of patients achieving prolonged
remission with such therapy. Other effective therapies
include the combination of melphalan with high-dose
dexamethasone or the use of thalidomide.80 The prognosis
depends on the stage at the time of diagnosis, emphasizing
the importance of recognizing the disease.
SARCOIDOSIS
Cutaneous involvement occurs in 20% to 25% of cases
of sarcoidosis and is most likely to be seen early in the
disease. Cutaneous lesions can be classified as nonspe-
cific, typically erythema nodosum, and specific or granu-
lomatous. Erythema nodosum occurs frequently as part
of Löfgren’s syndrome, with bilateral hilar lymphade-
nopathy and acute iridocyclitis. This variant has a good
­prognosis and resolves in 80% of patients within 2 years.
The skin lesions of sarcoidosis generally have no prognos-
tic significance or ­ correlation with disease activity. Skin
involvement has no effect on the course of the disease,
and the number of skin lesions does not correlate with
systemic disease. Skin plaques tend to be more persistent
and commonly associated with chronic forms of the dis-
ease. Lupus pernio (Fig. 47-15), with violaceous plaques
on the nose, ears, cheeks, lips, and fingers, is often seen
in long-standing sarcoidosis and is associated with upper
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
Figure 47-15  Sarcoidosis with “apple-jelly” plaques on the face and
lupus pernio, or nasal rim lesions.
airway involvement and pulmonary fibrosis.81 Other forms
of cutaneous sarcoidosis include papules, follicular papules,
subcutaneous nodules, ulcerative lesions, alopecia, and
icthyosis. Cutaneous sarcoid can arise in scars. Because of
the many different presentations, diagnosis can be chal-
lenging, and a skin biopsy is necessary to confirm the clin-
ical suspicion. Mimickers of papules include xanthelasma,
rosacea, trichoepithelioma, syphilis, lupus erythemato-
sus, and granuloma annulare. Plaques can resemble lupus
vulgaris, necrobiosis lipoidica, morphea, leprosy, leish-
maniasis, or lupus erythematosus. Nodules can resemble
lymphoma or other types of panniculitis. Treatment of
cutaneous sarcoidosis depends on the degree of systemic
involvement. Clearly, patients who need prednisone for
systemic disease often experience improvement of their
cutaneous sarcoid. Patients with isolated skin disease or
systemic disease not requiring aggressive therapy can ben-
efit from topical or intralesional corticosteroids, topical
tacrolimus, hydroxychloroquine, combination antimalari-
als with hydroxychloroquine and quinacrine, or chloro-
quine. If antimalarials are not adequate, methotrexate or
oral retinoids can be used. There have been case reports
and small case series of successful therapy with thalido-
mide and TNF-α inhibitors, as well as laser remodeling of
lupus pernio.82,83 There is some concern that ­interferon-α
therapy may induce sarcoidosis.
MISCELLANEOUS SKIN DISEASES
AND ARTHRITIS
BEHÇET’S DISEASE
The criteria for Behçet’s disease include recurrent oral and
genital ulcers, eye lesions (uveitis or retinal vasculitis),
characteristic skin lesions, and a positive pathergy test.84
The pathergy test involves using a sterile needle to prick the
forearm. The results are positive when the puncture causes
an aseptic erythematous nodule or pustule that is more than
699
2 mm in diameter at 24 to 48 hours. A diagnosis is made
if patients have recurrent oral ulcerations plus at least two
of the other findings without other clinical explanations.
Skin lesions include erythema nodosum, pseudofolliculitis,
and papulopustular lesions or acneiform nodules in postado-
lescents. Oral ulcers are painful and occur on the gingiva,
tongue, and buccal and labial mucosa. Genital ulcers, usu-
ally larger and deeper than oral ulcers, are typically on the
scrotum and penis in men and the vulva in women. Venous
involvement, including superficial thrombophlebitis and
deep venous thrombosis, can occur. On skin biopsy, small
vessel vasculitis is common. Ulcer treatment includes topi-
cal corticosteroids, colchicine, and thalidomide. Systemic
corticosteroids are prescribed for unresponsive erythema
nodosum.
FAMILIAL MEDITERRANEAN FEVER
Familial Mediterranean fever is an autosomal recessive
disease that tends to affect certain ethnic groups, includ-
ing Sephardic Jews, Arabs, Armenians, and Turks. There is
a mutation on the short arm of chromosome 16, and the
mutant protein pyrin likely plays an inhibitory role in the
control of inflammation.85,86 The disease is characterized by
recurrent, self-limited attacks of peritonitis, pleuritis, and
synovitis. Erysipelas-like erythema is the pathognomonic
skin manifestation. This is characterized by tender, erythem-
atous, well-demarcated plaques, usually located on the lower
legs.87 They may be triggered by physical effort and subside
spontaneously within 48 to 72 hours of bed rest. Fever and
leukocytosis may accompany this condition. Other associ-
ated skin findings include HS purpura; nonspecific purpura;
erythema of the face, trunk, or palm; angioneurotic edema;
Raynaud’s phenomenon; pyoderma; and subcutaneous nod-
ules. Secondary generalized amyloidosis may lead to chronic
renal failure and death if not recognized. Skin biopsy shows
edema of the superficial dermis and sparse perivascular
infiltrate composed of a few lymphocytes, neutrophils, and
nuclear dust, without vasculitis. Direct immunofluorescence
shows deposits of C3 in the wall of small superficial vessels.
Early treatment with colchicine, which prevents or dimin-
ishes the frequency and severity of inflammatory episodes,
can be beneficial.
MULTICENTRIC RETICULOHISTIOCYTOSIS
Multicentric reticulohistiocytosis is a rare condition of
unknown cause that most frequently occurs in Cauca-
sian women in the fifth and sixth decades of life. There
is destructive symmetric arthritis, with arthritis mutilans
developing in about 45% of cases, associated with cutane-
ous papulonodular lesions. Skin findings include cutaneous
red to brown papules or nodules, typically on the face, on
the dorsum of the fingers, and over the proximal and distal
­interphalangeal joints, but they can occur in a more gen-
eralized distribution. A rarer presentation includes photo-
distributed erythema, often with targeting over joints, that
masquerades as DM.88 Diagnosis is made by skin biopsy,
which shows infiltration of histiocytes and multinucleated
giant cells. These changes can be seen in a variety of ­tissues,
including the heart, lungs, skeletal muscle, and gastroin-
testinal tract. The differential diagnosis of the skin disease
 700 LEE  |  The Skin and Rheumatic Diseases
includes other infiltrative processes such as sarcoidosis and
even leprosy. Treatment recommendations, based mostly on
small case reports, include glucocorticoids and methotrex-
ate, with the use of cyclophosphamide and finally chloram-
bucil if the condition is unresponsive.89 Cyclosporine, TNF
inhibitors, and bisphosphonates have also been used with
reported benefit.90 In about one third of patients, multicen-
tirc reticulohistiocytosis may precede or follow an underly-
ing malignancy. Reported associated malignancies include
cancer of the breast, cervix, colon, stomach, lung, larynx,
and ovary; lymphoma; leukemia; sarcoma; melanoma;
mesothelioma; and metastatic cancer of unknown primary
source.
CHRONIC INFANTILE NEUROLOGIC CUTANEOUS
AND ARTICULAR SYNDROME
Mutations in cryopyrin (CIAS1, NALP3, PYPAF1), associ-
ated with increased proinflammatory cytokines, especially
interleukin-1β, have been found in about 50% of patients
with chronic infantile neurologic cutaneous and articular
(CINCA) syndrome.91,92 CINCA syndrome is characterized
by a neonatal urticarial eruption, fever, and arthritis, and
leukocytosis. Other findings include chronic meningitis,
papilledema, hearing loss, and growth retardation. Recent
isolated reports suggest that TNF-α inhibitors, anakinra,
and thalidomide may be beneficial in these patients.
Acknowledgments
The authors want to acknowledge Dr. Nicole Rogers for her assistance with
the figures.
REFERENCES
  1. Biondi Oriente C, Scarpa R, Pucino A, et al: Psoriasis and psoriatic
arthritis: Dermatological and rheumatological co-operative clinical
report. Acta Derm Venereol Suppl 146:69-71, 1989.
  2. Jones SM, Armas JB, Cohen MG, et al: Psoriatic arthritis: Outcome
of disease subsets and relationship of joint disease to nail and skin dis-
ease. Br J Rheumatol 33:834-839, 1994.
  3. Lebwohl M: Psoriasis. Lancet 361:1197-1204, 2003.
  4. Gladman DD: Clinical aspects of the spondyloarthropathies. Am J
Med Sci 316:234-238, 1998.
  5. Sayah A, English JC: Rheumatoid arthritis: A review of the cutane-
ous manifestations. J Am Acad Dermatol 53:191-209, 2005.
  6. Ginsberg MH, Genant HK, Yu TF, et al: Rheumatoid nodulosis: An
unusual variant of rheumatoid disease. Arthritis Rheum 18:49-58,
1975.
  7. Kremer JM, Lee JK: The safety and efficacy of the use of methotrex-
ate in long-term therapy for rheumatoid arthritis. Arthritis Rheum
29:822-831, 1986.
  8. Vollertsen RS, Conn DL, Ballard DJ, et al: Rheumatoid vasculitis: Sur-
vival and associated risk factors. Medicine 65:365-375, 1986.
  9. Brown TS, Fearneyhough PK, Burruss JB, et al: Rheumatoid neutro-
philic dermatitis in a woman with seronegative rheumatoid arthritis.
J Am Acad Dermatol 45:596-600, 2001.
10. Sangueza OP, Caudell MD, Mengesha YM, et al: Palisaded neutro-
philic granulomatous dermatitis in rheumatoid arthritis. J Am Acad
Dermatol 47:251-257, 2002.
11. Schneider R, Passo MH: Juvenile rheumatoid arthritis. Rheum Dis
Clin North Am 28:503-530, 2002.
12. Gilliam JN, Sontheimer RD: Distinctive cutaneous subsets in the
spectrum of lupus erythematosus. J Am Acad Dermatol 4:471-475,
1982.
13. Sontheimer RD, Maddison PJ, Reichlin M, et al: Serologic and HLA
associations in subacute cutaneous lupus erythematosus, a clinical sub-
set of lupus erythematosus. Ann Intern Med 97:664-671, 1982.
14. Reed BR, Huff JC, Jones SK, et al: Subacute cutaneous lupus erythe-
matosus associated with hydrochlorothiazide therapy. Ann Intern Med
103:49-51, 1985.
15. David-Bajar KM, Bennion SD, DeSpain JD, et al: Clinical, histologic,
and immunofluorescent distinctions between subacute cutaneous
lupus erythematosus and discoid lupus erythematosus. J Invest Derma-
tol 99:251-257, 1992.
16. Lee LA, Gaither KK, Coulter SN, et al: Pattern of cutaneous immu-
noglobulin G deposition in subacute cutaneous lupus erythematosus
is reproduced by infusing purified anti-Ro (SSA) autoantibodies into
human skin-grafted mice. J Clin Invest 83:1556-1562, 1989.
17. Prystowsky SD, Herndon JH Jr, Gilliam JN: Chronic cutaneous lupus
erythematosus (DLE)—a clinical and laboratory investigation of 80
patients. Medicine 55:183-191, 1976.
18. Kuhn A, Richter-Hintz D, Oslislo C, et al: Lupus erythematosus tumi-
dus—a neglected subset of cutaneous lupus erythematosus: Report of
40 cases. Arch Dermatol 136:1033-1041, 2000.
19. Graf J, Webb A, Davis J: The use of topical tacrolimus (FK506/Pro-
topic) in cutaneous manifestations of autoimmune diseases. J Clin
Rheum 9:310-315, 2003.
20. Edwards KR, Burke WA: Treatment of localized discoid lupus ery-
thematosus with tazarotene. J Am Acad Dermatol 41:1049-1050,
1999.
21. Rahman P, Gladman DD, Urowitz MB: Smoking interferes with
efficacy of antimalarial therapy in cutaneous lupus. J Rheumatol
25:1716-1719, 1998.
22. Hall RP, Lawley TJ, Smith HR, et al: Bullous eruption of systemic
lupus erythematosus: Dramatic response to dapsone therapy. Ann
Intern Med 97:165-170, 1982.
23. Frances C, Piette JC: The mystery of Sneddon syndrome: Relationship
with antiphospholipid syndrome and systemic lupus erythematosus.
J Autoimmun 15:139-143, 2000.
24. Lee LA: Transient autoimmunity related to maternal autoantibod-
ies: Neonatal lupus. Autoimmun Rev 4:207-213, 2005.
25. Martin V, Lee LA, Askanase AD, et al: Long-term followup of chil-
dren with neonatal lupus and their unaffected siblings. Arthritis
Rheum 46:2377-2383, 2002.
26. Nishikawa T, Provost TT: Differences in clinical, serologic, and
immunogenetic features of white versus Oriental anti-SS-A/Ro-posi-
tive patients. J Am Acad Dermatol 25:563-564, 1991.
27. Ramos-Casals M, Anaya JM, Garcia-Carrasco M, et al: Cutaneous
vasculitis in primary Sjogren syndrome: Classification and clinical sig-
nificance of 52 patients. Medicine 83:96-106, 2004.
28. Gerami P, Schope JM, et al: A systematic review of adult-onset clini-
cally amyopathic dermatomyositis (dermatomyositis sine myositis):
A missing link within the spectrum of the idiopathic inflammatory
myopathies. J Am Acad Dermatol 54:597-613, 2006.
29. Sontheimer RD: Cutaneous features of classic dermatomyositis and
amyopathic dermatomyositis. Curr Opin Rheumatol 11:475-482,
1999.
30. Hill CL, Zhang Y, Sigurgeirsson B, et al: Frequency of specific cancer
types in dermatomyositis and polymyositis: A population-based study.
Lancet 357:96-100, 2001.
31. Buchbinder R, Forbes A, Hall S, et al: Incidence of malignant disease
in biopsy-proven inflammatory myopathy: A population-based cohort
study. Ann Intern Med 134:1087-1095, 2001.
32. Ang GC, Werth VP: Combination antimalarials in the treatment of
cutaneous dermatomyositis: A retrospective study. Arch Dermatol
141:855-859, 2005.
33. Dalakas MC: Controlled studies with high-dose intravenous immuno-
globulin in the treatment of dermatomyositis, inclusion body myositis,
and polymyositis. Neurology 51:S37-S45, 1998.
34. Peterson LS, Nelson AM, Su WP, et al: The epidemiology of morphea
(localized scleroderma) in Olmsted County 1960-1993. J Rheumatol
24:73-80, 1997.
35. Elst EF, Van Suijlekom-Smit LW, Oranje AP: Treatment of linear
scleroderma with oral 1,25-dihydroxyvitamin D3 (calcitriol) in seven
children. Pediatr Dermatol 16:53-58, 1999.
36. Kreuter A, Hyun J, Stucker M, et al: A randomized controlled study of
low-dose UVA1, medium-dose UVA1, and narrowband UVB photo-
therapy in the treatment of localized scleroderma. J Am Acad Derma-
tol 54:440-447, 2006.
37. Brenner M, Herzinger T, Berking C, et al: Phototherapy and photo-
chemotherapy of sclerosing skin diseases. Photodermatol Photoimmu-
nol Photomed 21:157-165, 2005.
 PART 6  |  DIFFERENTIAL DIAGNOSIS OF REGIONAL MUSCULOSKELETAL PAIN
38. Masi AT, Subcommittee for Scleroderma Criteria of the American
Rheumatism Association Diagnostic and Therapeutic Criteria Com-
mittee: Preliminary criteria for the classification of systemic sclerosis
(scleroderma). Arthritis Rheum 5:581-590, 1980.
39. Zandman-Goddard G, Tweezer-Zaks N, Shoenfeld Y: New therapeutic
strategies for systemic sclerosis—a critical analysis of the literature.
Clin Dev Immunol 12:165-173, 2005.
40. DeMarco PJ, Weisman MH, Seibold JR, et al: Predictors and out-
comes of scleroderma renal crisis: The high-dose versus low-dose D-
penicillamine in early diffuse systemic sclerosis trial. Arthritis Rheum
46:2983-2989, 2002.
41. Tashkin DP, Elashoff R, Clements PJ, et al: Cyclophosphamide versus
placebo in scleroderma lung disease. N Engl J Med 354:2655-2666,
2006.
42. Chung L, Fiorentino D: Digital ulcers in patients with systemic sclero-
sis. Autoimmun Rev 5:125-128, 2006.
43. Korn JH, Mayes M, Matucci Cerinic M, et al: Digital ulcers in systemic
sclerosis: Prevention by treatment with bosentan, an oral endothelin
receptor antagonist. Arthritis Rheum 50:3985-3993, 2004.
44. Mayeno AN, Lin F, Foote CS, et al: Characterization of “peak E,” a
novel amino acid associated with eosinophilia-myalgia syndrome. Sci-
ence 250:1707-1708, 1990.
45. Falanga V, Medsger TA Jr, Reichlin M, et al: Linear scleroderma:
Clinical spectrum, prognosis, and laboratory abnormalities. Ann
Intern Med 104:849-857, 1986.
46. Chan JK, Fletcher CD, Hicklin GA, et al: Glomeruloid hemangioma:
A distinctive cutaneous lesion of multicentric Castleman’s disease
associated with POEMS syndrome. Am J Surg Pathol 14:1036-1046,
1990.
47. Dispenzieri A, Gertz MA: Treatment options for POEMS syndrome.
Exp Opin Pharmacother 6:945-953, 2005.
48. Dispenzieri A, Moreno-Aspitia A, Suare GA, et al: Peripheral blood
stem cell transplantation in 16 patients with POEMS syndrome, and a
review of the literature. Blood 104:3400-3407, 2004.
49. Kreuter A, Altmeyer P: High-dose dexamethasone in scleromyxedema:
Report of 2 additional cases. J Am Acad Dermatol 53:739-740, 2004.
50. Sansbury JC, Cocuroccia B, Jorizzo JL, et al: Treatment of recalcitrant
scleromyxedema with thalidomide in 3 patients. J Am Acad Dermatol
51:126-131, 2004.
51. Donato ML, Feasel AM, Weber DM, et al: Scleromyxedema: Role of
high-dose melphalan with autologous stem cell transplantation. Blood
107:463-466, 2006.
52. Cowper SE, Su L, Robin H, et al: Nephrogenic fibrosing dermopathy.
Am J Dermatopathol 23:383-393, 2001.
53. Kucher C, Xu X, Pasha T, et al: Histopathologic comparison of neph-
rogenic fibrosing dermopathy and scleromyxedema. J Cutan Pathol
32:484-490, 2005.
54. Mendoza FA, Artlett CM, Sandorfi N, et al: Description of 12 cases of
nephrogenic fibrosing dermopathy and review of the literature. Semin
Arthritis Rhe
701
64. Lanham JG, Elkon KB, Pusey CD, et al: Systemic vasculitis with
asthma and eosinophilia: A clinical approach to the Churg-Strauss
syndrome. Medicine 63:65-81, 1984.
65. Khan-Sabir SM, Werth VP: Infectious diseases that affect the skin
and joint. In Sontheimer RD, Provost TT (eds): Cutaneous Mani-
festations of Rheumatic Diseases, 2nd ed. Philadelphia, Lippincott
Williams & Wilkins, 2004, pp 242-264.
66. Fujiwara H, Fujiwara K, Hashimoto K, et al: Detection of Borrelia burg-
dorferi DNA (B garinii or B afzelii) in morphea and lichen sclerosus
et atrophicus tissues of German and Japanese but not of US patients.
Arch Dermatol 133:41-44, 1997.
67. Steere A: Lyme disease. N Engl J Med 345:115-125, 2001.
68. Asbrink E, Hovmark A: Successful cultivation of spirochetes from
skin lesions of patients with erythema chronicum migrans afzelius and
acrodermatitis chronica atrophicans. Acta Pathol Microbiol Immunol
Scand B Microbiol 93:161-163, 1985.
69. Aberer E, Breier F, Stanek G, et al: Success and failure in the treatment
of acrodermatitis chronica atrophicans. Infection 24:85-87, 1996.
70. Woolf AD: Clinical manifestations of human parvovirus B19 in adults.
Arch Intern Med 149:1153-1156, 1989.
71. Psychos DN, Voulgari PV, Skopouli FN, et al: Erythema nodosum:
The underlying conditions. Clin Rheumatol 19:212-216, 2000.
72. White JW Jr, Winkelmann RK: Weber-Christian panniculitis: A review
of 30 cases with this diagnosis. J Am Acad Dermatol 39:56-62, 1998.
73. Nakane S, Kawabe Y, Eguchi K, et al: A case of cytophagic histiocytic
panniculitis: Successful treatment of recurrent attacks with steroid pulse
therapy and oral cyclosporin A. Clin Rheumatol 16:417-421, 1997.
74. Panush RS, Yonker RA, Dlesk A, et al: Weber-Christian disease:
Analysis of 15 cases and review of the literature. Medicine
64:181-191, 1985.
75. Salhany KE, Macon WR, Choi JK, et al: Subcutaneous panniculitis-
like T-cell lymphoma: Clinicopathologic, immunophenotypic, and
genotypic analysis of alpha/beta and gamma/delta subtypes. Am J Surg
Pathol 22:881-893, 1998.
76. Peters MS, Su WP: Panniculitis. Dermatol Clin 10:37-57, 1992.
77. Enk AH, Knop J: Treatment of relapsing idiopathic nodular pannicu-
litis (Pfeifer-Weber-Christian disease) with mycophenolate mofetil.
J Am Acad Dermatol 39:508-509, 1998.
78. Calderon P, Anzilotti M, Phelps R: Thalidomide in dermatology: New
indications for an old drug. Int J Dermatol 36:881-887, 1997.
79. Saadoun D, Deslandre CJ, Allanore Y, et al: Sustained response to
infliximab in 2 patients wtih refractory relapsing polychondritis.
J Rheumatol 30:1394-1395, 2003.
80. Rajkumar SV, Dispenzieri A, Kyle RA: Monoclonal gammopathy
of undetermined significance, Waldenstrom macroglobulinemia, AL
amyloidosis, and related plasma cell disorders: Diagnosis and treat-
ment. Mayo Clin Proc 81:693-703, 2006.
81. Mana J, Marcoval J, Graells J, et al: Cutaneous involvement in
sarcoidosis: Relationship to systemic disease. Arch Dermatol 133: