Basic science

Structure and function of red and white blood cells

Ted Gordon-Smith

Abstract
Red blood cells (RBCs) carry oxygen bound reversibly to the ferrous Fe++ atoms of the four haem groups of the haemoglobin (Hb) tetramer. In order to transport the Hb around the body in a functional state, the RBC requires a flexible membrane and contents to pass passively through the capillary bed and a source of energy to maintain the internal milieu. ATP is provided by anaerobic glycolysis. Reducing power is provided as NADH and NADPH, via the pentose phosphate pathway. Genetic abnormalities that affect the membrane deformability lead to shape changes and haemolysis; defects in the glycolytic pathway cause non-spheroctic haemolytic anaemia and failure of reducing power to intravascular haemolysis in response to oxidative stress. White blood cells provide the basis for the innate immune system as well as interacting with specific immune processes. They need to pass from the circulation, through the vessel wall into the extravascular tissues in order to carry out these functions. Inherited defects of the migratory process also lead to susceptibility to infection.

cells. Values are usually expressed in absolute numbers (Table 1). As reticulocytes pass through the splenic vessels they lose their organelles and RNA is degraded. About half the reticulocyte population resides in the spleen. Whilst splenectomy increases the number of circulating reticulocytes it does not hinder maturation nor increase the overall RBC count. The function of RBCs is to carry haemoglobin (Hb) around the body in sufficiently high concentration to allow effective transport of oxygen from the lungs to the tissues and the return of carbon dioxide back to the lungs. In order to achieve this, RBCs need a suitably large surface area for rapid gas exchange (the biconcave disc shape), a readily deformable structure to pass through the capillary bed and a source of energy to maintain this structure and provide the internal milieu necessary for functioning haemoglobin. The red cell membrane The RBC membrane (Figure 2)3 is a phospholipid bilayer, stabilized with equimolar amounts of cholesterol. The main phospholipids are phosphatdyl groups linked to ethanolamine, inositol, serine or choline (lecithin) and sphingomyelin. The lipid bilayer provides a stable milieu for the membrane proteins. The proteins determine the shape and flexibility of the RBC. Membrane proteins may be attached to the outer surface of the membrane through integral membrane domains or by the glycophosphatidyl inositol (GPI) anchor. Transmembrane proteins, for example band 3 protein, have both plasma and cytoplasmic domains. External domains include blood groups, binding sites for immune complexes and the external parts of transmembrane channels and signalling proteins. They are mostly heavily glycosylated, which gives the RBC a strong negative surface charge. Two important complement inactivating proteins, decay accelerating factor (CD55) and membrane inhibitor of reactive lysis (CD58), are attached via the GPI anchor. Deficiency of these proteins in paroxysmal nocturnal haemoglobinuria is responsible for the haemolysis in this condition. The main proteins of the cytoskeleton are spectrin, actin, protein 4.1 and ankyrin The two chains of spectrin ( and ) wind round each other to form heterodimers, which are linked end to end by actin, protein 4.1 and other proteins to form a network of tetramers on the inner membrane surface (Figure 2). The spectrin is also linked to transmembrane proteins, band 3 and glycophorin C via ankyrin and actin. Together these proteins give the RBC its biconcave disc shape. Mutations in their genes cause haemolytic anaemias associated with abnormalities of shape,4 the most common being hereditary spherocytosis and elliptocytosis. Red cell metabolism The mature RBC derives its energy from anaerobic respiration via the glycolytic (EmbdenMeyerhof) pathway, in which one molecule of glucose is converted to two of pyruvate, providing energy as ATP and reducing power as NADH. Gene mutations which lead to loss of activity of the enzymes of the pathway lead to shortened RBC survival (non-spherocytic haemolytic anaemias).5 The most common inherited deficiency of this pathway is pyruvate kinase (PK). Since reticulocytes can produce energy through oxidative phosphorylation they can bypass the defect. Splenectomy in PK deficiency greatly alleviates the anaemia.
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Keywords allosteric; cell adhesion; erythrocytes; glycolysis; granulocytes; migration; monocytes; phagocytosis; oxygen carriage; red blood cells

Red blood cells

Values for the numbers of circulating blood cells for normal individuals at different ages are given in Table 1.1,2 Numbers are maintained within fairly close limits under steady conditions but can be increased rapidly and appropriately in response to stress. The value for each cell type is a reflection of the rates of release into and escape from the circulation. Red cells and platelets have a finite life span within the circulation. Granulocytes and monocytes may be marginated on vessel walls and can leave the circulation rapidly in response to inflammatory signals. Red blood cells (RBCs) are released into the circulation as reticulocytes. Reticulocytes are enucleate cells that contain residual RNA. This gives the faintly basophilic appearance to reticulocytes in blood films and provides the reticulate material in supravitally stained preparations (Figure 1). They are about 20% larger than mature RBCs, contain some mitochondria capable of oxidative respiration and make up 12% of the circulating red

Ted Gordon Smith MA FRCP FRCP (Ed) FRCPath FAcadMedSci is Emeritus Professor of Haematology and Honorary Consultant Physician at St Georges Hospital Medical School, London, UK. Competing interests: none declared.

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Normal values at different ages

Unit Haemoglobin Red blood cells Packed cell volume Mean corpuscular volume Mean corpuscular haemoglobin Mean corpuscular haemoglobin concentration Reticulocytes White blood cells Neutrophils Eosinophils Basophils Monocytes Lymphocytes T (CD8+) T(CD4+) B cells NK cells Platelets g/dL 1012/L % fl pg g/dL 109/L 109/dL 109/dL 109/dL 109/dL 109/dL 109/dL 109/dL 109/dL 109/dL 109/dL 109/dL At birth 1524 3.76.5 4775 100125 3137 3236 120400 930 2.714.4 0.00.8 0.01.9 2.07.25 Infant 913 35 2838 8498 3036 3035 2060 517 1.06.0 0.091.1 0.41.2 3.311.5 Child 1115 45 3243 7090 2530 3336 30100 615 1.08.5 0.041.04 0.151.28 2.34.6 0.81.5 1.02.0 0.51.5 170450 Adult M: 13.517.5, F: 11.515.5 M: 4.56.5, F: 3.95.6 M: 4052, F: 3648 8095 2734 2035 30100 4.011.0 2.0*7.5 <0.45 <0.1 0.20.8 1.53.5 0.330.67 0.41.8 0.050.4 0.20.4 150450

150450

210650

Figures are derived from several sources,1,2 which should be consulted for more detailed results for specific ages. They are given here to emphasize the shifts which occur during growth and development. People of African ethnicity may constitutionally have neutrophil counts down to 1.0109/litre.

Table 1

The glycolytic pathway also links to the production of 2,3di-phosphoglycerate (2,3-DPG), a molecule essential for the effective carriage of oxygen by haemoglobin (see below). The RBC needs reducing power not only to prevent the oxidation of Hb to methaemoglobin, mainly provided by NADH, but also as NADPH to prevent oxidative damage to the red cell membrane. The RBC is subject to attack by reactive oxygen species in its circulation through the blood and the potentially harmful presence of iron in the Hb. Protection comes through reduced glutathione, maintained by the production of NADPH. Inherited mutations of gene on the X chromosome for the enzyme which catalyses the

reaction for NADPH, glucose-6-phosphate dehydrogenase, leads to various intravascular haemolytic syndromes, including favism and oxidative drug-induced haemolysis. The main pathways of metabolism in the mature RBC are represented in Figure 3, which indicates the main active products. Haemoglobin and the carriage of oxygen Hb is a tetramer of two and two non- globin chains, each of the chains enclosing one of the four haem groups which carry oxygen in the complete tetramer (Figure 4). The genes are on chromosome 6, the non- on chromosome 11. In the foetus and infant the non- chain of the main Hb is (HbF; 22), and in the adult it is (HbA; 22). The minor HbA2 (22) comprises less than 3.0% in adults but is useful in the diagnosis of thalassaemia trait. The pocket which contains the haem group prevents its oxidation to methaem, which cannot combine with O2. Mutations in the globin genes which affect the haem pocket lead to methaemoglobinaemia or instability of the Hb molecule.6 Hb has two slightly different tertiary structures depending on whether or not O2 is bound:7 In the absence of O2 on the haem group it has a tense (T) structure with low affinity for O2. When O2 attaches to a haem it initiates a structural change in globin so that the tetramer becomes more avid for further O2 molecules (high affinity, relaxed (R) form). This allosteric transformation accounts for the sigmoid O2 dissociation curve. Various ligands, including hydrogen ions (pH) and 2,3-DPG, affect the position of the curve and hence the readiness of the O2 to dissociate at different O2 tensions (p O2). 2,3-DPG, which is increased in PK deficiency, decreases the affinity of Hb for O2 so that it is given up more readily (Figure 3d) and
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a Normal (see pages 143-148). RBC with area of central pallor the result of the biconcave disc shape. b Reticulocytes stained with supravital stain showing reticulate precipitation of RNA. Figure 1

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The red blood cell membrane

Phospholipid Cholesterol

Sialoprotein (blood group ABH) Band 3

Glycophorins

Vertical interactions

4 .9
2.1 2.1
4.1

4.1 Artin

-spectrin Horizontal interactions

Spectrin-spectrin interaction to form tetramer

Spectrin interacts with other proteins, some of which are transmembrane proteins, and with the lipid bilayer. Oligosaccharides on the cytoplasmic domains of proteins determine various blood group antigens. Some proteins of the cytoskeleton are identified by numbers corresponding to their position on electrophoresis. Figure 2

the effects of anaemia are mitigated. Sickle cell Hb also has a low O2 affinity so patients tolerate apparently low Hb levels well. Ageing and destruction of RBCs RBCs have a life span in the circulation of 120 days. Senescent RBCs are phagocytosed by macrophages of the reticuloendothelial system, mainly in the marrow, liver and spleen. Iron from the haem group is available for erythropoiesis after transfer to the marrow bound to transferrin.

Outline of the metabolic pathways in the mature red blood cell

Glycolysis Glucose Pentose Glutathione phosphate cycle pathway

The porphyrin ring is converted through a series of reactions to bilirubin. Bilirubin is not conjugated to a glucuronide until it has been processed in the hepatocyte. Unconjugated bilirubin does not pass through the kidney. The hyperbilirubinaemia of haemolysis is not accompanied by bilirubin in the urine (acholuric jaundice). RBCs are enucleate and contain no RNA so are unable to synthesize proteins. The activity of some enzymes decreases with the age of the RBC and the consequent decline in energy production may account for increased rigidity of ageing cells. Rearrangement of the lipid distribution in the cell membrane with the replacement of the choline phospholipids lecithin and sphingomyelin by phosphatidyl-serine from the inner layer may also be a consequence that leads to phagocytosis and apoptosis.8

White blood cells

White blood cells (WBCs), which circulate in the blood, fulfil most of their functions outside the circulation. To achieve this they have systems which respond to specific stimuli and which enable them to enter and traffic through the extravascular milieu. WBCs derived from the common myeloid progenitor cell, granulocytes and monocytes mainly provide a first-line defence against microbial invasion through phagocytosis and killing, whereas lymphocytes, derived from the common lymphoid precursor, provide specific cellular and humoral immune responses. Granulocytes Granulocytes are recognized by the presence of specific granules within their cytoplasm and their multilobed nuclei (Figure 5). They have the capacity to change shape and migrate, via the vessel wall,9 through tissues in response to chemotactic signals from infection and inflammation. The granules mediate the specific killing and signalling functions of the cells.10
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NADPH MetHb Reduction NADH Reducing power

GSH

Hb O2 affinity Rapoport luebering shunt

2,3DPG ATP Metabolic energy Antioxidant

Pyruvate/lactate

Figure 3

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Basic science

Functioning of haemoglobin
a O2 b

F8 Haem Fe
++

Haem E7 Globin SH Globin

Tyr HC1 F9

d
FG2 CD2 C3 F8 CD1 E7 E11 CD7 D1 E5 G15 B5 A16 B1 G19 AB1 E1

G1 F1 H16 E18 EF3 NA1 NA2 H5 A1 H1 EF1 E20 G5

1 2

H H

H 2,3,DPG H

GH4

A 1 2

a The haem group sits in the pocket stabilised by amino acids of the globin chains. b The haem is connected to the globin chain by histidines in the pocket which allows reversible binding of O2 to the Fe++ atom of haem. c The Hb molecule consists of helices supporting the haem group. d The tetrameric heterodimer undergoes allosteric conformational changes during oxygenation and deoxygenation.7 2,3 DPG occupies the central space of the deoxygenated form.

Figure 4

Neutrophils Neutrophils circulate in the blood for 610 hours. A considerable fraction of the total intravascular population is marginated on vessel walls at any one time; the major mass is in the extravascular system, where they eventually undergo apoptosis. Circulating neutrophils are increased following a number of stimuli. Exercise causes demargination and hence an increase in the circulating count. Inflammation leads to a rapid increase in neutrophil numbers through the action of a number of cytokines, including IL-1 and granulocyte colony stimulating factor (G-CSF). The primary role of neutrophils is to engulf and kill bacteria encountered in the extravascular space and take part in the inflammatory response. The process may be divided into migration, chemotaxis, ingestion, degranulation and oxidative burst activity.10 Migration across the vascular endothelium requires engagement of adhesion molecules on the neutrophils and endothelial cells (Figure 6). There are two steps: Rolling along the endothelium is mediated by reversible association of molecules of the selectin family (E-selectin, L-selectin).
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Activation and adhesion inflammation activates the adhesion system whereby the neutrophil attaches to the endothelium, changes shape and passes between two endothelial cells to begin the migration towards the site of inflammation. The process is regulated by adhesion molecules of the leucocyte integrins (LFA- 1 family), imunnoglobulin superfamily (ICAM-3) and the cytoadhesins (leucocyte response integrin). Chemotaxis: agents produced by activation complement (C5a), breakdown of bacterial call walls (N-formyl peptides) and cytokine-stimulated endothelial cells (leukotriene B4, IL-8), produced at the site of inflammation, provide a concentration gradient to which the neutrophil reacts by moving towards the higher concentration. Ingestion: neutrophils engulf microorganisms into the phagosome. This may occur before there is an immune response but is greatly enhanced by antibody and complement binding to the microorganism. Receptors on the neutrophil stimulate the phagocytosis and killing.

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Circulating WBCs, a Neutrophil, b eosinophil, c basophil, d monocyte, e lymphocytes. Figure 5

Degranulation: primary granules contain a variety of bactericidal enzymes, including myeloperoxidase and lysozyme. Secondary granules contain lactoferrin, which chelates iron and is essential for bacterial growth. The granules are released into the phagosome.11 Oxidative burst: degranulation activates a switch to oxidative respiration and the production of high concentrations of reactive oxygen species (including free radical O2, OH radicals and H2O2), which are directly toxic to the microorganisms. Once the killing process is complete the neutrophil is lysed and forms pus, the greenish colour of which is due to myeloperoxidase (Figure 6).

Eosinophils The majority of eosinophils (Figure 5B) are in the extravascular tissue space, particularly in the subepithelium of areas exposed to the external environment (tracheobronchial tree, gastrointestinal tract). Their main function is protection against parasitic organisms. They are weakly phagocytic and act mainly by degranulating on to the surface of the parasite. The granules have a high content of basic proteins, including major basic protein (MBP) and an eosinophil specific peroxidise. Eosinophil numbers are increased in allergic reactions (e.g. asthma) as well as in response to parasitic infection.12 The main mediators are IL-5, IL-3 and GM-CSF. Inappropriate increase in numbers

Neutrophil migration
G-CSF, IL-1 Blood Rolling LFA-1,VLA-5 ICAM-3,LRI Activation and adhesion Transendothelial migration

E-Selectin L-Selectin

Vessel wall

Endothelial cells

LFA-1,HCAM

Tissue

Migration towards inflammatory site Phagocytosis killing Cell death (pus) Phagosomes Degranulation Oxidative burst Chemotaxis C5a, N-formyl peptides Leukotriene-B4, IL-8. Inflammation

Figure 6

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may lead to the hypereosinophilic syndrome (fever, weight loss, anorexia, thromboembolic episodes, heart failure, splenomegaly, skin lesions and central nervous system disease). Basophils Basophils (Figure 5C) and the related mast cells are also associated with allergic and parasitic disease.13 They have a high affinity IgE receptor: when this binds to antigen, degranulation occurs with release of histamine. They also synthesise and release leukotrieneD4, the slow acting substance of anaphylaxis. The main results are vasodilatation, mucus secretion and smooth muscle contraction (type 1 hypersensitivity reaction). Mast cells release eosinophil chemotactic factors. The incoming eosinophils release histaminases, which limit mast cell mediated hypersensitivity reactions. Monocytes and macrophages When monocytes (Figure 5D) leave the circulation they reside in the tissues for a considerable time, where they differentiate into macrophages. These are the main phagocytic cells but also have a major antigen-presenting role. Monocytes increase in many chronic infective and inflammatory states. Phagocytic role: Macrophages ingest and kill particular microorganisms, including Mycobacterium, Listeria, Candida and other fungal species; functions that are enhanced by IFN.13 Activated monocytes express tissue factor (see pages 133-136) and can initiate coagulation. Diffuse intravascular coagulation may be an inappropriate response in some infections. Macrophage cells engulf foreign proteins by endocytosis and fuse the endosome with a lysosome where the protein is degraded to short peptides, which are presented on the cell surface in class II histocompatiblity molecules. The class II presentation is recognized by CD4+ T cells. As well as foreign proteins macrophages engulf and degrade senescent cells and, in the lungs, inhaled particulate matter.

are mobile cells. Mutations which interfere with the integrity of their systems lead to predictable clinical syndromes: haemolysis in disorders of the red cell membrane, metabolism or haemoglobin, and susceptibility to infection when systems of adhesion, migration and killing are deficient in WBCs.

Summary
Blood cells have specialized functions which depend on structural and metabolic characteristics. As well as their actions they

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