PMNs AND ITS ABNORMALITIES

INTRODUCTION
Periodontal diseases involve
A local infection
A host response that may result in connective
tissue alteration.

Leucocytes are critical to periodontal defense, as
most potential periodontal pathogens are
known to be resistant to the antimicrobial
mechanisms of serum.

Control of Local Infection
ACUTE INFLAMMATION
Three primary leucocytes participating in immune
response to periodontal diseases are
NEUTROPHILS , MONOCYTES and LYMPHOCYTES.
The leucocytes infiltrate the gingiva in a temporal
and spatial order, suggesting that the strategy of
host defense against periodontal infections is
similar to that used to combat most other local
infections.(Miyaski KT . THE NEUTROPHIL : MECHANISMS OF CONTROLLING PERIODONTAL BACTERIA. J
Periodontol 62:761, 1991 {55} ) .
CHRONIC INFLAMMATION
It begins with the infiltration of monocytes and
lymphocytes . Unlike neutrophils , monocytes
and lymphocytes primarily infiltrate
connective tissue and develop into tissue
macrophages and activated lymphocytes .
Normal neutrophil function
Neutrophil can crawl at a rate of about 400
micrometer / hour .
This may seem fast but it actually amounts to
only about 40 cell lenghts per hour .
The primary role of the neutrophil is the
destruction of pathogens that threaten tissues
outside of the blood , including the periodontium
For this reason the blood , finding targets , and
killing targets are important phagocyte functions
in the defense of the periodontium .
Normal functions
Adherence
Chemotaxis
Phagocytosis
Metabolic response
Bacteriocidal capacity
Digestion
Secretion



Leukocyte cellular events
Leukocytes leave the vasculature routinely through
the following sequence of events:
Margination and rolling
Adhesion and transmigration
Chemotaxis and activation
They are then free to participate in:
Phagocytosis and degranulation
Leukocyte-induced tissue injury
Leucocytes activation
Margination and Diapedesis
With increased vascular permeability, fluid leaves the
vessel causing leukocytes to settle-out of the central
flow column and marginate along the endothelial
surface
Endothelial cells and leukocytes have complementary
surface adhesion molecules which briefly stick and
release causing the leukocyte to roll along the
endothelium like a tumbleweed until it eventually
comes to a stop as mutual adhesion reaches a peak
Margination and Rolling
Early rolling adhesion mediated by selectin
family:
E-selectin (endothelium), P-selectin (platelets,
endothelium), L-selectin (leukocytes) bind other
surface molecules (i.e.,CD34, Sialyl-Lewis X-
modified GP) that are upregulated on
endothelium by cytokines (TNF, IL-1) at injury sites

Adhesion
Rolling comes to a stop and adhesion results
Other sets of adhesion molecules participate:
Endothelial: ICAM-1, VCAM-1
Leukocyte: LFA-1, Mac-1, VLA-4
(ICAM-1 binds LFA-1/Mac-1, VCAM-1 binds VLA-4)
Ordinarily down-regulated or in an inactive
conformation, but inflammation alters this
Transmigration (diapedesis)
Occurs after firm adhesion within the systemic
venules and pulmonary capillaries via PECAM
1 (CD31)
Must then cross basement membrane
Collagenases
Integrins
Transmigration (diapedesis)
Early in inflammatory response mostly PMNs,
but as cytokine and chemotactic signals
change with progression of inflammatory
response, alteration of endothelial cell
adhesion molecule expression activates other
populations of leukocytes to adhere
(monocytes, lymphocytes, etc)
SELECTINS
Selectins, so called because they are characterized by an
extracellular N-terminal domain related to sugar-binding
mammalian lectins, consist of E-selectin (CD62E, previously known
as ELAM-1), which is confined to endothelium; P-selectin (CD62P,
previously called GMP140 or PADGEM), which is present in
endothelium and platelets; and L-selectin (CD62L, previously known
by many names, including LAM-1), which is expressed on most
leukocyte types.
Selectins bind, through their lectin domain, to sialylated forms of
oligosaccharides (e.g., sialylated Lewis X), which themselves are
covalently bound to various mucin-like glycoproteins (GlyCAM-1,
PSGL-1, ESL-1, and CD34).
The immunoglobulin family molecules include two endothelial
adhesion molecules: ICAM-1 (intercellular adhesion molecule 1) and
VCAM-1 (vascular cell adhesion molecule 1).
INTEGRINS
Integrins are transmembrane heterodimeric
glycoproteins, made up of a and b chains, that
are expressed on many cell types and bind to
ligands on endothelial cells, other leukocytes,
and the extracellular matrix.
The b2 integrins LFA-1 and Mac-1
(CD11a/CD18 and CD11b/CD18) bind to ICAM-
1, and the b1 integrins (such as VLA-4) bind
VCAM-1.
MUCIN
Mucin-like glycoproteins, such as heparan
sulfate, serve as ligands for the leukocyte
adhesion molecule called CD44. These
glycoproteins are found in the extracellular
matrix and on cell surfaces.
Chemotaxis
Leukocytes follow chemical gradient to site of injury
(chemotaxis)
Soluble bacterial products
Complement components (C5a)
Cytokines (chemokine family e.g., IL-8)
LTB
4
(AA metabolite)
Chemotactic agents bind surface receptors inducing
calcium mobilization and assembly of cytoskeletal
contractile elements
Chemotaxis and Activation
Leukocytes:
extend pseudopods with overlying surface
adhesion molecules (integrins) that bind ECM
during chemotaxis
undergo activation:
Prepare AA metabolites from phospholipids
Prepare for degranulation and release of lysosomal
enzymes (oxidative burst)
Regulate leukocyte adhesion molecule affinity as
needed
Phagocytosis:
Derived from the Greek words Eat and cell.
Phagocytosis is carried out by white blood cells:
macrophages, neutrophils, and occasionally
eosinophils.
Neutrophils predominate early in infection.
Wandering macrophages: Originate from
monocytes that leave blood and enter infected
tissue, and develop into phagocytic cells.
Fixed Macrophages (Histiocytes): Located in liver,
nervous system, lungs, lymph nodes, bone marrow,
and several other tissues.
Phagocytic Cells: Macrophages (Monocytes),
Neutrophils, and Eosinophils
(Macrophages)
Stages of Phagocytosis
1. Chemotaxis: Phagocytes are chemically attracted
to site of infection.
2. Adherence: Phagocyte plasma membrane
attaches to surface of pathogen or foreign
material.
Adherence can be inhibited by capsules (S.
pneumoniae) or M protein (S. pyogenes).
Opsonization: Coating process with opsonins that
facilitates attachment.
Opsonins include antibodies and complement proteins.
Phagocytes are Attracted to Site of Infection
by Chemotaxis
Stages of Phagocytosis (Continued)
3. Ingestion: Plasma membrane of phagocytes
extends projections (pseudopods) which engulf the
microbe. Microbe is enclosed in a sac called
phagosome.
4. Digestion: Inside the cell, phagosome fuses with
lysosome to form a phagolysosome.
Lysosomal enzymes kill most bacteria within 30
minutes and include:
Lysozyme: Destroys cell wall peptidoglycan
Lipases and Proteases
RNAses and DNAses
After digestion, residual body with undigestable
material is discharged.
Process of Phagocytosis
Recognition and Atachment
Opsonized by serum complement,
immunoglobulin (C3b, Fc portion of IgG)
Corresponding receptors on leukocytes (FcR,
CR1, 2, 3) leads to binding


Contd.
Triggers an oxidative burst (next slide)
engulfment and formation of vacuole which
fuses with lysosomal granule membrane
(phagolysosome)
Granules discharge within phagolysosome and
extracellularly (degranulation)

Oxidative burst
Reactive oxygen species formed through
oxidative burst that includes:
Increased oxygen consumption
Glycogenolysis
Increased glucose oxidation
Formation of superoxide ion
2O
2
+ NADPH 2O
2
-rad
+ NADP
+
+ H
+

(NADPH oxidase)
O
2
+ 2H
+
H
2
O
2
(dismutase)
Reactive oxygen species
Hydrogen peroxide alone insufficient
MPO (azurophilic granules) converts hydrogen
peroxide to HOCl
-
(in presence of Cl
-
), an
oxidant/antimicrobial agent
Therefore, PMNs can kill by halogenation, or
lipid/protein peroxidation
Degradation and Clean-up
Reactive end-products only active within
phagolysosome
Hydrogen peroxide broken down to water and
oxygen by catalase
Dead microorganisms degraded by lysosomal
acid hydrolases
Leucocytes abnormalities
Disorders of Neutrophil Numbers
Definition
Neutropenia
Less than 1500/ml
Neutrophila
Greater than 7700/ml
Acquired
Or
Inherited
35
J. Levine
Definition of Neutrophilia - too many
Normal ANC is 1500-7700/ml
Neutrophilia: abnormally high ANC
Shift to the left: d release of precursors
from the bone marrow
not necessarily associated with
neutrophilia
36
NEUTROPHILIA
Often accompanied by other reactive changes, e.g. atypical lymphocytes.
Varied aetiology, e.g. excercise, emotional (anger, stress), inflammation, infection,
chemicals or drugs (glucocorticoids, epinephrine, G-CSF), tumours, smoking.
Left shift results from increased granulopoiesis.
Leukaemoid reactions must be distinguished from CML.
Morphological changes include Dhle bodies (aggregates of polyribosomes), toxic
granulation (persistence of primary granules) and cytoplasmic vacuolation.
Neutrophilia
Chronic Stimulation
Excess cytokine stimulates
proliferative pool

Causes:
Infection
Down's Syndrome
Pregnancy/Eclampsia
Chemotherapy recovery
Myeloproliferative disorders
Marrow metastases
Acute shift from
marginating to
circulating pool
measured WBC, not total
WBC
Causes:
Steroid treatment
Exercise
Epinephrine
Hypoxia
Seizures
Other stress
38
Example: exercise induced neutrophilia
39
Neutropenia: too few
Neutropenia
Definition: ANC < 1500/l
ANC 500-1000 increased risk of infection from
exposure
ANC < 500: increased risk of infection from host
organisms
African-Americans: lower normal neutrophil
counts (1000-1200)
40
NEUTROPENIA (<1.5 10
9
/L)
Drug-induced, e.g. cancer chemotherapy, chloramphenicol.
Immune neutropenia (antibodies to surface antigens) associated with
autoimmune disorders (SLE or RA).
Myelodysplastic syndromes (MDS).
Infections; HIV, increased utilization, increased adherence to damaged
endothelium.
Chronic idiopathic neutropenia; possibly a mixture of congenital and
acquired disorders.
Congenital neutropenia, e.g. Kostmann's syndrome (very rare).
Cyclic neutropenia (three week intervals), due to abnormal regulation of
haemopoietic stem cells.
Diagnosis: BM helps to discriminate between peripheral destruction and
decreased production.
Management: Remove underlying cause. Granulocyte transfusion has risk of
alloimmunisation and CMV.
Acquired Causes of Neutropenia
Decreased
Production
Increased
Destruction
Shift to
Marginating Pool
Bone marrow

Peripheral
circulation
Move from the
circulating pool to
attach along the
vessel wall
Medication:
Chemotherapy
Antibiotics, etc


Autoimmune
diseases
(Rheumatoid
arthritis, SLE, etc)
Severe infection
Endotoxin release
Hemodialysis
Cardiopulmonary
bypass
42
Increased Destruction
Anti-neutrophil
antibody
Neutrophil-Antibody
Complex
Uptake and
destruction of
neutrophil by the
RE system
43
Shift to Marginating Pool
Circulating
Marginating
Circulating
Marginating
Severe infection / Endotoxin release
Hemodialysis
Cardiopulmonary bypass
44
Evaluation of Neutropenia
If visit prompted by a fever and ANC is
low, treat promptly for infection
Suspect medication: major cause of
neutropenia
If no culprits, bone marrow exam for:
Malignancy
Infiltration by non-marrow cells
Arrest of cell growth
Myeloproliferative disorder
45
Cyclic Neutropenia
21 day cycle
autosomal dominant
fever, mouth ulcers
Treatment G-CSF
usually improves after
puberty
46
Congenital Neutropenia
Maturation arrest
frequent infections,
often serious
mouth sores
may lose teeth or
develop severe gum
infections
Increased risk of
leukemia
Tx: G-CSF, BMT
47
Role of Neutrophil
Responds to chemotactic factors released from damaged
tissue
Rolls and attaches to the endothelial cell wall
protein and carbohydrate interactions (selectins and their ligands).
Becomes activated by chemotactic factors
Tightly adheres through the integrin family of proteins.
Migrates across the endothelial cell wall.
Phagocytizes organisms so that they are contained within a
vesicle or phagosome.
Releases granule products and reduced oxygen species (e.g.
hydrogen peroxide and superoxide) to kill organisms
48
Function of the Circulating Neutrophil
Chemoattractant
Attachment/rolling
Activation
Adhesion
Migration
Phagocytosis
49
Disruption of Neutrophil Function
Steps where defects in structural components
of neutrophils results in impaired ability to
fight infection
Recruitment from the circulation
Adhesion and subsequent migration
Defective production in active oxygen metabolites
Deficiency in granules
50
Defect in Attachment/Rolling
Attachment/rolling
Sialyl Lewis
X
Selectins
Cell surface molecules expressing Sialyl Lewis X
interact with selectin proteins on the cell
surface of endothelial cells
LAD-2 Impaired expression of sialyl LewisX -
Neutrophils do not attach and are not recruited to the site of inflammation
Chemoattractant
51
Defect in Adhesion
Chemoattractant
Adhesion
Integrins on the surface of
neutrophils mediate tight adhesion
to the endothelial cell wall.
Cells then migrate.
Migration
Integrin
LAD-1 results from a defect in leukocyte integrins.
Decreased to absent expression on the cell surface.
Cells can not adhere and subsequently cannot migrate.
52
Clinical manifestations: LAD
53
Source Undetermined (Both Images)
Phagocytosis
Chemoattractant
Bacteria are engulfed and contained in a phagosome.
Contents of the granules are released.
Oxygen metabolites (superoxide and H
2
O
2
) kill bacteria
CGD: NADPH-Oxidase-defective
Cannot produce active oxygen species
Chediak-Higashi Syndrome: Defect in granule formation
54
Chediak-Higashi Syndrome
55
Chediak-Higashi Syndrome
Oculocutaneous
albinism
Photophobia
Sun sensitivity
Neuropathy
Infections, esp Staph
aureus

TX: BMT
56
Chronic granulomatous disease (CGD)
57
Chronic granulomatous disease: CGD
Catalase positive organisms
Staph aureus
Serratia marcescens
Burkholderia cepacia
Fungal
Skin, lungs, bones, abscesses
Granuloma formation from chronic infection

58
Myeloperoxidase deficiency
One of the more common disorders
1: 4000
Decreased production of hypochlorous acid
(HOCl)
Killing takes longer than normal
Clinically silent for most people
59
Diseases with Neutrophil Defects
Disease Step Molecular Defect
LAD-2 Rolling Sialyl Lewis X
Carbohydrate

LAD-1 Adhesion
Phagocytosis
Integrin
expression

Chediak-
Higashi
Syndrome

Migration
Degranulation

Vacuolar sorting
protein (large
granules interfere
with traversing
endothelial wall)




60
Diseases with Neutrophil Defects
61
LEUKOCYTE ADHESION DEFICIENCY
TYPE I :
LAD 1 is an autosomal disorder (localized to
chromosome 21q22.3) characterized by the
inability of individuals to express beta2
subunit (CD18) common to leukocytes
integrins LFA-1 , Mac 1 and p 150/95 .
Periodontal disease is related to if one or two
defective alleles are present .
TYPE II
There appears to be a selectin ligand
deficiency i.e., the leukocytes do not express
sialo-Lewis x or gp150-Lewis x , referred to as
leucocyte adhesion deficiency, type II , in
which neutrophil rolling doesnot increase in
response to inflammation .
Individuals with this deficiency suffer from
recurrent bacterial infections, neutrophilia
and severe early onset bacteria .
Localized juvenile periodontitis
Neutrophils with LJP exhibit a selective decrease in
ability to kill A. actinomycetamcomitans, despite
normal phagocytosis, oxygen production, and secretion
of specific granule components.
Neutrophils from individuals with the classic form of
LJP are characterized by a decrease in chemotactic
responses to a variety of chemotactic factors, including
C5a, FMLP and leukotriene B
4.

The neutrophil dysfunction is associated with a
functional decrease in chemotaxin receptors on the
PMN surface.