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.

Fig. 4.1 Erythrocytes enmeshed in filaments of fibrin in a clot.

(By courtesy of Michael Crowder.)

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.

Fig. 4.2 A human heart muscle biopsy specimen, showing an erythrocyte within a capillary. The erythrocyte biconcave disc is typically electron dense and almost fills the capillary lumen.

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.

Fig. 4.12 The origins and lineage relationships of haemopoietically-derived cells of the immune system. Mature cells and selected progenitors (all human) are illustrated (magnifications vary). The dendritic cell was cultured from peripheral blood, immunolabelled to show HLA-DR and photographed using Nomarski optics. The megakaryocyte and erythroblast are from a bone marrow smear, stained with MGG; the remaining cells illustrated are from peripheral blood smears (Wright’s stain), sections of connective tissue (plasma cell, mast cell), bone (osteoclast) and lung alveolus (macrophage). Platelets (one is arrowed) are subcellular fragments of bone marrow megakaryocytes. Note that circulating small lymphocytes cannot be classified further with routine staining methods. For further explanation of cellular structure and staining properties, see the text.

(by courtesy of Dr Cécile Chalouni, Ludwig Institute for Cancer Research, Yale University School of Medicine, USA)

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.

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

Monocytes

Monocytes are the largest of the leukocytes (15–20 μm in diameter), but they form only a small proportion of the total population (2–8% with a count of 100–700/μl of blood). The nucleus, which is euchromatic, is relatively large and irregular, often with a characteristic indentation on one side. The cytoplasm is pale-staining, particulate and typically vacuolated. Near the nuclear indentation it contains a prominent Golgi complex and vesicles. Monocytes are actively phagocytic cells, and contain numerous lysosomes. Phagocytosis is triggered by recognition of opsonized material, as described for neutrophils. Monocytes are highly motile, and possess a well-developed cytoskeleton.

Monocytes express Class II MHC antigens and share other similarities to tissue macrophages and dendritic cells. Most monocytes are thought to be in transit via the bloodstream from the bone marrow to the peripheral tissues where they give rise to macrophages and dendritic cells; different monocyte subsets may target inflamed tissues. Like other leukocytes, they pass into extravascular sites through the walls of capillaries and venules.

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.

Fig. 4.4 A small, resting lymphocyte in human peripheral blood. The nuclear/cytoplasmic ratio is high and the cytoplasm contains few organelles, indicative of its quiescent state.

Fig. 4.6 A mature B cell (plasma cell) in human connective tissue. The abundant rough endoplasmic reticulum is typical of a cell actively synthesizing secretory protein, in this case immunoglobulin. The cell to the left is a fibroblast.

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).