Blood, lymphoid tissues and haemopoiesis

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CHAPTER 4 Blood, lymphoid tissues and haemopoiesis

In postnatal life blood cells are formed in the bone marrow. Haemopoiesis produces red cells (erythrocytes), and a wide variety of defensive cells (white blood cells, or leukocytes). The latter include neutrophil, eosinophil and basophil granulocytes, B lymphocytes and monocytes. T lymphocytes develop in the thymus from bone marrow-derived progenitors. Platelets are produced in the bone marrow as cellular fragments of megakaryocytes. Only erythrocytes and platelets are generally confined to the blood vascular system, whereas all leukocytes can leave the circulation and enter extravascular tissues. The numbers of cells doing so increases greatly during inflammation caused by local infections and diseases.

The lymphoid tissues are the thymus, lymph nodes, spleen and the lymphoid follicles associated mainly with the alimentary and respiratory tracts. Lymphocytes populate lymphoid tissues and are concerned with various types of immune defence. Lymphoid tissue also contains supportive stromal cells which are non-haemopoietic in origin (e.g. thymic epithelium), non-haemopoietic follicular dendritic cells of lymph nodes and splenic follicles, haemopoietically-derived interdigitating dendritic cells, and macrophages of the mononuclear phagocyte system. Dendritic cells and blood monocyte-derived macrophages are found additionally in most tissues and organs where they function as immunostimulatory antigen-presenting cells (APCs).

CELLS OF PERIPHERAL BLOOD

BLOOD

Blood is an opaque fluid with a viscosity greater than that of water (mean relative viscosity 4.75 at 18°C), and a specific gravity of 1.06 at 15°C. It is bright red when oxygenated, in the systemic arteries, and dark red to purple when deoxygenated, in systemic veins. Blood is a mixture of a clear liquid, plasma, and cellular elements, and consequently the hydrodynamic flow of blood in vessels behaves in a complex manner that is not entirely predictable by simple Newtonian equations.

Plasma

Plasma is a clear, yellowish fluid which contains many substances in solution or suspension: low molecular weight solutes give a mean freezing-point depression of 0.54°C. Plasma contains high concentrations of sodium and chloride ions, potassium, calcium, magnesium, phosphate, bicarbonate, traces of many other ions, glucose, amino acids and vitamins. The colloids include high molecular weight plasma proteins, e.g. clotting factors, particularly prothrombin; immunoglobulins and complement proteins involved in immunological defence; glycoproteins, lipoproteins, polypeptide and steroid hormones and globulins for the transport of hormones and iron. Since most of the metabolic activities of the body are reflected in the plasma composition, its routine chemical analysis is of great diagnostic importance.

The precipitation of the protein fibrin from plasma to form a clot (Fig. 4.1) is initiated by the release of specific materials from damaged cells and blood platelets in the presence of calcium ions. If blood or plasma samples are allowed to stand, they will separate into a clot and a clear yellowish fluid, the serum. Clot formation is prevented by removal of calcium ions, e.g. by addition of citrate, oxalate or various organic calcium chelators (EDTA, EGTA) to the sample. Heparin is also widely used as an anticlotting agent, because it interferes with another part of the complex series of chemical interactions which lead to fibrin clot formation.

ERYTHROCYTES

Erythrocytes (red blood cells, red blood corpuscles [RBC]) account for the largest proportion of blood cells (99% of the total number), with normal values of 4.1–6.0 × 106/μl in adult males and 3.9–5.5 × 106/μl in adult females. Polycythaemia (increased red cell mass) can occur in individuals living at high altitude, or pathologically in conditions resulting in arterial hypoxia. Reduction in red cell mass (anaemia) has many underlying causes but in rare instances can be due to structural defects in erythrocytes (see below).

Each erythrocyte is a biconcave disc (Fig. 4.1, Fig. 4.2) with a mean diameter in dried smear preparations of 7.1 μm; in fresh preparations the mean diameter is 7.8 μm, decreasing slightly with age. Mature erythrocytes lack nuclei. They are pale red by transmitted light, with paler centres because of their biconcave shape. The properties of their cell coat cause them to adhere to one another by their rims to form loose piles of cells (rouleaux). In normal blood, a few cells assume a shrunken star-like, crenated form: this shape can be reproduced by placing normal biconcave erythrocytes in a hypertonic solution, which causes osmotic shrinkage. In hypotonic solutions erythrocytes take up water and become spherical; they may eventually lyse to release their haemoglobin (haemolysis), leaving red cell ghosts.

Erythrocytes have a limiting plasma membrane which encloses mainly a single protein, haemoglobin, as a 33% solution. The plasma membrane of erythrocytes is 60% lipid and glycolipid, and 40% protein and glycoprotein. More than 15 classes of protein are present, including two major types. Glycophorins A and B (each with a molecular mass of approximately 50 kDa) span the membrane, and their negatively charged carbohydrate chains project from the outer surface of the cell. Their sialic acid groups confer most of the fixed charge on the cell surface. A second transmembrane macromolecule, band 3 protein, forms an important anion channel, exchanging bicarbonate for chloride ions across the membrane and allowing the release of CO2 in the lungs. The ABO blood group antigens are all membrane glycolipids.

The shape of the erythrocyte is largely determined by the filamentous protein dimer, spectrin, a name which reflects its original isolation from red cell ghosts. Spectrin dimers associate as tetramers through their head regions, and are attached to the cytoplasmic domain of the anion carrier, band 3 protein, via ankyrin. Other proteins, including tropomyosin, tropomodulin and short actin filaments form junctional complexes which link spectrin to glycophorin transmembrane proteins, forming a stabilizing cytoskeletal network. This gives the membrane great flexibility: red cells are deformable but regain their biconcave shape and dimensions after passing through the smallest capillaries, which are 4 μm in diameter. Erythrocyte membrane flexibility also contributes to the normally low viscosity of blood. Defects in the cytoskeleton occur in autosomal dominant disorders (some cases of elliptocytosis result from mutations in spectrin and of spherocytosis from ankyrin dysfunction) which result in abnormalities of red cell shape, membrane fragility, premature destruction of erythrocytes in the spleen and haemolytic anaemia.

Fetal erythrocytes up to the fourth month of gestation differ markedly from those of adults, in that they are larger, are nucleated and contain a different type of haemoglobin (HbF). After this time they are progressively replaced by the adult type of cell.

Haemoglobin

Haemoglobin (Hb) is a globular protein with a molecular mass of 67 kDa. It consists of globulin molecules bound to haem, an iron-containing porphyrin group. The oxygen-binding power of haemoglobin is provided by the iron atoms of the haem groups, and these are maintained in the ferrous (Fe++) state by the presence of glutathione within the erythrocyte. The haemoglobin molecule is a tetramer, made up of four subunits, each a coiled polypeptide chain holding a single haem group. In normal blood, five types of polypeptide chain can occur, namely; α, β and two β-like polypeptides, γ and δ. A third, β-like η chain is restricted to early fetal development. Each haemoglobin molecule contains two α-chains and two others, so that several combinations, and hence a number of different types of haemoglobin molecule, are possible. For example, haemoglobin A (HbA), which is the major adult class, contains 2α- and 2β-chains; a variant, HbA2 with 2α and 2δ chains, accounts for only 2% of adult haemoglobin. Haemoglobin F (HbF), found in fetal and early postnatal life, consists of 2α- and 2γ-chains. Adult red cells normally contain less than 1% of HbF.

In the pathological genetic condition thalassaemia, only one type of chain is expressed normally, the mutant chain being absent or present at much reduced levels. Thus, a molecule may contain 4 α-chains (β-thalassaemia) or, more commonly, 4 β-chains (α-thalassaemia) where individuals affected carry haemoglobin H (HbH). In haemoglobin S (HbS) of sickle-cell disease, a point mutation in the β-chain gene (valine substituted for glutamine) causes a major alteration in the behaviour of the red cell and its oxygen-carrying capacity.

Lifespan

Erythrocytes last between 100 and 120 days before being destroyed. As erythrocytes age they become increasingly fragile, and their surface charges decrease as their content of negatively charged membrane glycoproteins diminishes. The lipid content of their membranes also reduces. Aged erythrocytes are eventually ingested by the macrophages of the spleen and liver sinusoids, usually without prior lysis, and are hydrolysed in phagocytic vacuoles where the haemoglobin is split into its globulin and porphyrin moieties. Globulin is further degraded to amino acids which pass into the general amino acid pool. Iron is removed from the porphyrin ring and either transported in the circulation bound to transferrin and used in the synthesis of new haemoglobin in the bone marrow, or stored in the liver as ferritin or haemosiderin. The remainder of the haem group is converted in the liver to bilirubin and excreted in the bile.

The recognition of effete erythrocytes by macrophages appears to depend in part on the exposure of normally inaccessible parts of membrane proteins, enabling autoantibodies to these erythrocyte senescence antigens to bind to them and flag them for macrophage removal. Red cells are destroyed at the rate of 5 × 1011 cells a day and are normally replaced from the bone marrow (see Fig. 4.12) at the same rate.

Blood groups

Over 300 red cell antigens are recognizable with specific antisera. They can interact with naturally occurring or induced antibodies in the plasma of recipients of an unmatched transfusion, causing agglutination and lysis of the erythrocytes. Erythrocytes of a single individual can carry several different types of antigen, each type belonging to an antigenic system in which a number of alternative antigens are possible in different persons. So far, 19 major groups have been identified. They vary in their distribution frequencies between different populations, and include the ABO, Rhesus, MNS, Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Cartwright, Colton, Sid, Scianna, Yt, Auberger, Ii, Xg, Indian and Dombrock systems. Clinically, the ABO and Rhesus groups are of most importance.

In the ABO system, two allelic genes are inherited in simple Mendelian fashion. Thus the genome may be homozygous and carry the AA complement, the blood group being A, or the BB complement which gives blood group B, or it may carry neither (OO), producing blood group O. In the heterozygous condition the following combinations can occur: AB (blood group AB), AO (blood group A) and BO (blood group B).

Individuals with group AB blood lack antibodies to both A and B antigens, and so can be transfused with blood of any group: they are termed universal recipients. Conversely, those with group O, universal donors, can give blood to any recipient, since anti-A and anti-B antibodies in the donated blood are diluted to insignificant levels. Normally, however, blood is only transfused between persons with precisely corresponding groups, because anomalous antibodies of the ABO system are occasionally found in blood and may cause agglutination or lysis. The anti-ABO agglutinins, unlike those of the Rhesus system, belong to the immunoglobulin M (IgM) class and do not cross the placenta during pregnancy.

The Rhesus antigen system is determined by three sets of alleles, namely Cc, Dd and Ee: the most important clinically is Dd. Inheritance of the Rh factor also obeys simple Mendelian laws and it is therefore possible for a Rhesus-negative mother to bear a Rhesus-positive child. Fetal Rh antigens can, under these circumstances, stimulate the production of anti-Rh antibodies by the mother: since these belong to the immunoglobulin G (IgG) class of antibodies they are able to cross the placental barrier (generally late in the last trimester) and cause agglutination of fetal erythrocytes. In the first such pregnancy little damage usually occurs because anti-Rh antibodies are present only at low levels, but in subsequent Rh-positive pregnancies massive destruction of fetal red cells (haemolytic disease of the newborn) may result, causing fetal or neonatal death. Sensitization of the maternal immune system can also result from abortion or miscarriage, or even occasionally amniocentesis, which may introduce fetal antigens into the maternal circulation. Treatment is by exchange transfusion of the neonate or, prophylactically, by giving Rh-immune (anti-D) serum to the mother after the first Rh-positive pregnancy, which destroys the fetal Rh antigen in her circulation before sensitization can occur.

Leukocytes also bear highly polymorphic antigens encoded by allelic gene variants. These belong to the group of major histocompatibility complex (MHC) antigens, also termed human leukocyte antigens (HLA) in man. HLA Class I antigens are expressed by all nucleated cells. Class II antigen expression is more restricted outside the immune system, but is inducible on many parenchymal cell types e.g. after exposure to interferon. HLA Class I and II antigens play important roles in cell–cell interactions in the immune system, particularly in the presentation of antigens to T lymphocytes by APCs.

LEUKOCYTES

Leukocytes (white blood cells) belong to at least five different categories (see Fig. 4.12), and are distinguishable by their size, nuclear shape and cytoplasmic inclusions. In practice, leukocytes are often divided into two main groups, namely those with prominent stainable cytoplasmic granules, the granulocytes, and those without.

Granulocytes

This group consists of eosinophil granulocytes, with granules which bind acidic dyes such as eosin; basophil granulocytes, with granules which bind basic dyes strongly; and neutrophil granulocytes, with granules which stain only weakly with either type of dye. Granulocytes (Fig. 4.3) all possess irregular or multilobed nuclei and belong to the myeloid series of blood cells (p. 77 see Fig. 4.12).

Neutrophil granulocytes

Neutrophil granulocytes (neutrophils), are also referred to as polymorphonuclear leukocytes (polymorphs) because of their irregularly segmented (multilobed) nuclei. They form the largest proportion of the white blood cells (40–75% in adults, with a normal count of 2500–7500/μl) and have a diameter of 12–14 μm. The cells may be spherical in the circulation, but they can flatten and become actively motile within the extracellular matrix of connective tissues.

The numerous cytoplasmic granules are heterogeneous in size, shape and content, but all are membrane-bound and contain hydrolytic and other enzymes. Two major types can be distinguished according to their developmental origin and contents. Non-specific or primary (azurophilic) granules are formed early in neutrophil maturation. They are relatively large (0.5 μm) spheroidal lysosomes containing myeloperoxidase, acid phosphatase, elastase and several other enzymes. Specific or secondary granules are formed later, and occur in a wide range of shapes including spheres, ellipsoids and rods. These contain strong bacteriocidal components including alkaline phosphatase, lactoferrin and collagenase, none of which are found in primary granules. Conversely, secondary granules lack peroxidase and acid phosphatase. Some enzymes, e.g. lysozyme, are present in both types of granule.

In mature neutrophils the nucleus is characteristically multilobed with up to six (usually three or four) segments joined by narrow nuclear strands: this is known as the segmented stage. Less mature cells have fewer lobes. The earliest to be released under normal conditions are juveniles (band or stab cells) in which the nucleus is an unsegmented crescent or band. In certain clinical conditions, even earlier stages in neutrophil formation, when cells display indented or rounded nuclei (metamyelocytes or myelocytes) may be released from the bone marrow. In mature cells the edges of the nuclear lobes are often irregular. In females 3% of the nuclei of neutrophils show a conspicuous ‘drumstick’ formation which represents the sex chromatin of the inactive X chromosome (Barr body). Neutrophil cytoplasm contains few mitochondria but abundant cytoskeletal elements, including actin filaments, microtubules and their associated proteins, all characteristic of highly motile cells.

Neutrophils are important in the defence of the body against microorganisms. They can phagocytose microbes and small particles in the circulation and, after extravasation, they carry out similar activities in other tissues. They function effectively in relatively anaerobic conditions, relying largely on glycolytic metabolism, and they fulfil an important role in the acute inflammatory phase of tissue injury, responding to chemotaxins released by damaged tissue. Phagocytosis of cellular debris or invading microorganisms is followed by fusion of the phagocytic vacuole, first with specific granules, whose pH is reduced to 5.0 by active transport of protons, then with non-specific (primary) granules, which complete the process of bacterial killing and digestion. Actively phagocytic neutrophils are able to reduce oxygen enzymatically to form reactive oxygen species including superoxide radicals and hydrogen peroxide, which enhance bacterial destruction.

Phagocytosis is greatly facilitated by circulating antibodies to molecules such as bacterial antigens which the body has previously encountered. Antibodies coat the antigenic target and bind the plasma complement protein, C1, to their non-variable Fc regions. This activates the complement cascade, which involves some 20 plasma proteins synthesized mainly in the liver, and completes the process of opsonization. The complement cascade involves the sequential cleavage of the complement proteins into a large fragment, which generally binds to the antigenic surface, and a small bioactive fragment which is released. The final step is the recognition of complement by receptors on the surfaces of neutrophils (and macrophages), which promotes phagocytosis of the organism.

Neutrophils are short-lived; they spend some 6–7 hours circulating in the blood and a few days in connective tissues. The number of circulating neutrophils varies, and often rises during episodes of acute bacterial infection. They die after carrying out their phagocytic role: dead neutrophils, bacteria, tissue debris (including tissue damaged by neutrophil enzymes and toxins) and interstitial fluid form the characteristic, greenish-yellow pus of infected tissue. The colour is derived from the natural colour of neutrophil myeloperoxidase.

Granules may also be inappropriately released from neutrophils. Their enzymes are implicated in various pathological conditions, e.g. rheumatoid arthritis, where tissue destruction and chronic inflammation occur.

Eosinophil granulocytes

Eosinophil granulocytes (eosinophils) are similar in size (12–15 μm), shape and motile capacity to neutrophils, but are present only in small numbers in normal blood (100–400/μl). The nucleus has two prominent lobes connected by a thin strand of chromatin. Their cytoplasmic specific granules are uniformly large (0.5 μm) and give the living cell a slightly yellowish colour. The cytoplasm is packed with granules which are spherical or ellipsoid and membrane-bound. The core of each granule is composed of a lattice of major basic protein, which is responsible for its strong eosinophilic staining properties. The surrounding matrix contains several lysosomal enzymes including acid phosphatase, ribonuclease, phospholipase and a myeloperoxidase unique to eosinophils.

Like other leukocytes, eosinophils are motile. When suitably stimulated, they are able to pass into the extravascular tissues from the circulation. They are typical minor constituents of the dermis, and of the connective tissue components of the bronchial tree, alimentary tract, uterus and vagina. The total lifespan of these cells is a few days, of which some 10 hours is spent in the circulation, and the remainder in the extravascular tissues.

Eosinophil numbers rise (eosinophilia) in worm infestations and also in certain allergic disorders, and it is thought that they evolved as a primary defence against parasitic attack. They have surface receptors for IgE which bind to IgE-antigen complexes, triggering phagocytosis and release of granule contents. However, they are only weakly phagocytic and their most important function is the destruction of parasites too large to phagocytose. This anti-parasitic effect is mediated via toxic molecules released from their granules (e.g. eosinophil cationic protein and major basic protein). They also release histaminase, which limits the inflammatory consequences of mast cell degranulation. High local concentrations of eosinophils, e.g. in bronchial asthma and in cutaneous contact sensitivity and allergic eczema, can cause tissue destruction as a consequence of the release of molecules such as collagenase from their granules.

Basophil granulocytes

Slightly smaller than other granulocytes, basophil granulocytes are 10–14 μm in diameter, and form only 0.5–1% of the total leukocyte population of normal blood, with a count of 25–200/μl. Their distinguishing feature is the presence of large, conspicuous basophilic granules. The nucleus is somewhat irregular or bilobed, and is usually obscured in stained blood smears by the similar colour of the basophilic granules. The granules are membrane-bound vesicles which display a variety of crystalline, lamellar and granular inclusions: they contain heparin, histamine and several other inflammatory agents, and closely resemble those of tissue mast cells (see p. 36). Both basophils and mast cells have high affinity membrane receptors for IgE and are therefore coated with IgE antibody. If this binds to its antigen it triggers degranulation of the cells, producing vasodilation, increased vascular permeability, chemotactic stimuli for other granulocytes, and the symptoms of immediate hypersensitivity, e.g. in allergic rhinitis (hay fever). Despite these similarities, basophils and mast cells develop as separate lineages in the myeloid series, from haemopoietic stem cells in the bone marrow. Evidence from experimental animal models suggests that they are closely related (see Fig. 4.12) but studies on mast cell disorders in humans indicate that their lineages diverge from a more distant ancestral progenitor (Kocabas et al 2005).

Mononuclear leukocytes

Lymphocytes

Lymphocytes (Fig. 4.4, see Fig. 4.6, see Fig. 4.12) are the second most numerous type of leukocyte in adulthood, forming 20–30% of the total population (1500–2700/μl of blood). In young children they are the most numerous blood leukocyte. Most circulating lymphocytes are small, 6–8 μm in diameter; a few are medium-sized and have an increased cytoplasmic volume, often in response to antigenic stimulation. Occasionally, cells up to 16 μm are seen in peripheral blood. Lymphocytes, like other leukocytes, are found in extravascular tissues (including lymphoid tissue); however, they are the only white blood cells which return to the circulation. The lifespan of lymphocytes ranges from a few days (short-lived) to many years (long-lived). Long-lived lymphocytes play a significant role in the maintenance of immunological memory.

Blood lymphocytes are a heterogeneous collection mainly of B and T cells and consist of different subsets and different stages of activity and maturity. About 85% of all circulating lymphocytes in normal blood are T cells. Primary immunodeficiency diseases can result from molecular defects in T and B lymphocytes (reviewed in Cunningham-Rundles & Ponda 2005). Included with the lymphocytes, but probably a separate lineage subset, are the natural killer (NK) cells. NK cells most closely resemble large T cells morphologically.

Small lymphocytes (both B and T cells) contain a rounded, densely staining nucleus which is surrounded by a very narrow rim of cytoplasm, barely visible in the light microscope. In the electron microscope (Fig. 4.4), few cytoplasmic organelles can be seen apart from a small number of mitochondria, single ribosomes, sparse profiles of endoplasmic reticulum and occasional lysosomes: these features indicate a low metabolic rate and a quiescent phenotype. However, these cells become motile when they contact solid surfaces, and can pass between endothelial cells to exit from, or re-enter, the vascular system. They migrate extensively within various tissues, including epithelia (Fig. 4.5).

Larger lymphocytes include T and B cells which are functionally activated or proliferating after stimulation by antigen, and NK cells. They contain a nucleus, which is at least in part euchromatic, a basophilic cytoplasm, which may appear granular, and numerous polyribosome clusters, consistent with active protein synthesis. The ultrastructural appearance of these cells varies according to their class and is described below.

B cells

B cells and the plasma cells that develop from them synthesize and secrete antibodies which can specifically recognize and neutralize foreign (non-self) macromolecules (antigens), and can prime various non-lymphocytic cells (e.g. neutrophils, macrophages and dendritic cells) to phagocytose pathogens. B cells differentiate from haemopoietic stem cells in the bone marrow. After deletion of autoreactive cells, the selected B lymphocytes then leave the bone marrow and migrate to peripheral lymphoid sites (e.g. lymph nodes). Here, following stimulation by antigen, they undergo further proliferation and selection, forming germinal centres in the lymphoid tissues. Following this, some B cells differentiate into large basophilic (RNA-rich) plasma cells, either within or outside the lymphoid tissues. Plasma cells produce antibodies in their extensive rough endoplasmic reticulum (Fig. 4.6) and secrete them into the adjacent tissues. They have a prominent pale-staining Golgi complex adjacent to an eccentrically-placed nucleus, typically with peripheral blocks of condensed heterochromatin resembling the numerals of a clock (clock-faced nucleus) (see Fig. 4.12). Other germinal centre B cells develop into long-lived memory cells capable of responding to their specific antigens not only with a more rapid and higher antibody output, but also with an increased antibody affinity compared with the primary response.

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