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.)

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.

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).

Fig. 4.3 Neutrophil (N) and basophil (B) granulocytes within a renal glomerular capillary in a human kidney biopsy. The neutrophil nucleus is more segmented (four lobes are visible) and the granules are smaller and more electron-dense than in the basophil.

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.

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).

Fig. 4.5 Tubular glands in the appendix, showing intraepithelial lymphocytes (arrowed). A lymphocyte in anaphase is indicated (longer arrow).

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.

Antibodies are immunoglobulins, grouped into five classes according to their heavy polypeptide chain. Immunoglobulin G (IgG) forms the bulk of circulating antibodies. Immunoglobulin M (IgM) is normally synthesized early in immune responses. Immunoglobulin A (IgA) is present in breast milk, tears, saliva and other secretions of the alimentary tract, coupled to a secretory piece (a 70 kDa protein) which is synthesized by the epithelial cells and protects the immunoglobulin from proteolytic degradation: IgA thus contributes to mucosal immunity. IgA deficiency is relatively common, particularly in some ethnic groups (reviewed in Woof & Kerr 2006). Immunoglobulin E (IgE) is an antibody which binds to receptors on the surfaces of mast cells, eosinophils and blood basophils; it is found only at low concentrations in the circulation. Immunoglobulin D (IgD) is found together with IgM as a major membrane-bound immunoglobulin on mature, immunocompetent but naïve (prior to antigen exposure) B cells, acting as the cellular receptor for antigen.

When circulating antibodies bind to antigens they form immune complexes. If present in abnormal quantities, these may cause pathological damage to the vascular system and other tissues, either by interfering mechanically with the permeability of the basal lamina (e.g. some types of glomerulonephritis), or by causing local activation of the complement system which generates inflammatory mediators (e.g. C5a), attacks cell membranes and causes vascular disease. In pregnancy, maternal IgG crosses the placenta and confers passive immunity on the fetus. Maternal milk contains secretory immunoglobulins (IgA) which help to combat bacterial and viral organisms in the alimentary tract of the baby during the first few weeks of postnatal life.

T cells

There are a number of sub-sets of T (thymus-derived) lymphocytes, all progeny of haemopoietic stem cells in the bone marrow. They develop and mature in the thymus, and subsequently populate peripheral secondary lymphoid organs, which they constantly leave and re-enter via the circulation. As recirculating cells, their major function is immune surveillance. Their activation and subsequent proliferation and functional maturation is under the control of antigen-presenting cells. T cells undertake a wide variety of cell-mediated defensive functions which are not directly dependent on antibody activity, and which constitute the basis of cellular immunity. T cell responses focus on the destruction of cellular targets such as virus-infected cells, certain bacterial infections, fungi, some protozoal infections, neoplastic cells and the cells of grafts from other individuals (allografts) when the tissue antigens of the donor and recipient are not sufficiently similar. Targets may be killed directly by cytotoxic T cells, or indirectly by accessory cells (e.g. macrophages) which have been recruited and activated by cytokine-secreting helper T cells. A third group, regulatory T cells, acts to regulate or limit immune responses.

Functional groups of T cells are classified according to the molecules they express on their surfaces. The majority of cytokine-secreting helper T cells express CD4, while cytotoxic T cells are characterized by CD8. Regulatory T cells coexpress CD4 and CD25. The CD (cluster of differentiation) prefix provides a standard nomenclature for all cell surface molecules. At present, more than 330 different CD antigens have been designated: each one represents a cell surface molecule that can be identified by specific antibodies. Further details of the classification are beyond the scope of this publication and are given in Male et al (2006).

Structurally, T lymphocytes present different appearances depending on their type and state of activity. When resting, they are typical small lymphocytes and are morphologically indistinguishable from B lymphocytes. When stimulated, they become large (up to 15 μm), moderately basophilic cells, with a partially euchromatic nucleus and numerous free ribosomes, rough and smooth endoplasmic reticulum, a Golgi complex and a few mitochondria, in their cytoplasm. Cytotoxic T cells contain dense lysosome-like vacuoles which function in cytotoxic killing.

Cytotoxic T cells

Cytotoxic T lymphocytes (which express CD8 on their surface) are responsible for the direct cytotoxic killing of target cells (e.g. virus-infected cells): the requirement for direct cell–cell contact ensures the specificity of the response. Recognition of antigen, presented as a peptide fragment on MHC class I molecules, triggers the calcium-dependent release of lytic granules by the T cell. These lysosome-like granules contain perforin which forms a pore in the target cell membrane. They also contain several different serine protease enzymes (granzymes) which enter the target cell via the perforin pore and induce the programmed cell death (apoptosis; p. 24) of the target.

Helper T cells

Helper T cells (which express CD4) are characterized by the secretion of cytokines. Two major populations have been identified according to the range of cytokines produced. Th1 helper T cells typically secrete interleukin (IL)-2, tumour necrosis factor (TNF)-alpha and interferon gamma, while Th2 cells produce cytokines such as IL-4, IL-5 and IL-13. These two CD4-expressing populations are termed ‘helper’ T cells because one aspect of their function is to stimulate the proliferation and maturation of B lymphocytes and cytotoxic T lymphocytes (mediated via cytokines such as IL-4, IL-2 and interferon gamma), thus enabling and enhancing the immune responses mediated by those cells. In addition to Th1 and Th2 cells, other subsets of helper T cells have been described. Notably, Th17 cells (which secrete the cytokine IL-17) have recently been implicated in autoimmune diseases.

However, helper T cells are also important in directing the destruction of pathogens by recruiting accessory cells (e.g. macrophages, neutrophils, eosinophils) to the site of infection and by activating their effector functions. This process is tightly coordinated. For example, Th1 helper T cells secrete cytokines that not only attract and activate macrophages but also provide help for B cells and guide their immunoglobulin production to the subclasses that fix complement. Thus these antibodies opsonize the pathogen target which can then be recognized, ingested and destroyed by the macrophage accessory cells that bear receptors for complement and the Fc region of IgG. These Th1 cells are sometimes referred to as delayed type hypersensitivity T cells. In contrast, Th2 cells secrete cytokines that induce the development and activation of eosinophils, and also induce B cells to switch their immunoglobulins to non-complement fixing classes (e.g. IgE). Pathogens such as parasitic worms can then be coated with IgE antibody and hence recognized and destroyed by the effector functions of the eosinophil accessory cells which bear receptors for the Fc region of IgE.

If helper T-cell activities are non-functional, a state of immunodeficiency results. This means that potentially pathogenic organisms, which are normally kept in check by the immune system, may proliferate and cause overt pathology, e.g. in acquired immune deficiency syndrome (AIDS), where a virus (HIV) specifically infects and kills (predominantly) helper T cells, though some antigen-presenting cells are also killed.

Regulatory T cells

A third population of T cells, ‘regulatory’ T or ‘Treg’ cells has been identified within the last decade (reviewed in O’Garra & Vieira 2004). These CD4⁺, CD25⁺ cells have an immunomodulatory function and can dampen the effector functions of both cytotoxic and helper T cells. Regulatory T cells are produced in the thymus and are an important additional mechanism for maintaining self-tolerance. Treg function is antigen-specific and depends upon direct cell–cell contact. Molecules secreted or induced by Treg cells, such as interleukin (IL)-10 or transforming growth factor (TGF) β, may also play an important role in mediating Treg suppressive effects on the immune system. Similar regulatory T cells can be induced in the periphery and may be important in the induction of oral tolerance to ingested antigens as well as tolerance to tissue specific molecules that are not expressed in the thymus.

Natural killer (NK) cells

Natural killer (NK) cells have functional similarities to cytotoxic T cells. However they lack other typical lymphocyte features, and do not express antigen-specific receptors. They normally form only a small percentage of all lymphocyte-like cells and are usually included in the large granular lymphocyte category. When mature, NK cells have a mildly basophilic cytoplasm. Ultrastructurally, their cytoplasm contains ribosomes, rough endoplasmic reticulum and dense, membrane-bound vesicles 200–500 nm in diameter with crystalline cores. These contain the protein perforin (cytolysin), which is capable of inserting holes in the plasma membranes of target cells, and granzymes (serine proteases), which trigger subsequent target cell death by apoptosis. NK cells are activated to kill target cells by a number of factors. They can recognize and kill antibody coated target cells via a mechanism termed antibody-dependent cell-mediated cytotoxicity (ADCC). They also have receptors that inhibit NK destructive activity when they recognize MHC class I on normal cells. When NK cells detect the loss or downregulation of MHC class I antigens on certain virus-infected cells and some tumour cells, they activate apoptosis-inducing mechanisms which enable them to attack these abnormal cells, albeit relatively non-specifically. For further reading, see Vivier et al 2008.

Platelets

Blood platelets, also known as thrombocytes, are relatively small (2–4 μm across) irregular or oval discs present in large numbers (200,000–400,000/μl) in blood. In freshly harvested blood samples they readily adhere to each other and to all available surfaces, unless the blood is treated with citrate or other substances which reduce the availability of calcium ions. Platelets are anucleate cell fragments, derived from megakaryocytes in the bone marrow. They are surrounded by a plasma membrane with a thick glycoprotein coat, which is responsible for their adhesive properties. A band of 10 microtubules lies around the perimeter of the platelet beneath the plasma membrane: the microtubules are associated with actin filaments, myosin and other proteins related to cell contraction. The cytoplasm also contains mitochondria, glycogen, scant smooth endoplasmic reticulum, tubular invaginations of the plasma membrane, and three major types of membrane-bound vesicle, designated alpha, delta and lambda granules.

Alpha granules are the largest, and have diameters of up to 500 nm. They contain platelet-derived growth factor (PDGF), fibrinogen and other substances. Delta granules are smaller (up to 300 nm), and contain 5-hydroxytryptamine (serotonin) which has been endocytosed from the blood plasma. Lambda granules are the smallest (up to 250 nm) and contain lysosomal enzymes.

Platelets play an important role in haemostasis. When a blood vessel is damaged, platelets become activated, evert their membrane invaginations to form lamellipodia and filopodia, and aggregate at the site of injury, plugging the wound. They adhere to each other (agglutination), and to other tissues. Adhesion is a function of the thick platelet coat and is promoted by the release of ADP and calcium ions from the platelets in response to vessel injury. The contents of released alpha granules, together with factors released from the damaged tissues, initiate a complex sequence of chemical reactions in the blood plasma, which leads to the precipitation of insoluble fibrin filaments in a three-dimensional meshwork, the fibrin clot (Fig. 4.1). More platelets attach to the clot, inserting extensions of their surfaces, filopodia, deep into the spaces between the fibrin filaments, to which they adhere strongly. The platelets then contract (clot retraction) by actin–myosin interactions within their cytoplasm, and this concentrates the fibrin clot and pulls the walls of the blood vessel together, which limits any further leakage of blood. After repair of the vessel wall, which may be promoted by the mitogenic activity of PDGF, the clot is dissolved by enzymes such as plasmin. Plasmin is formed by plasminogen activators in the plasma, probably assisted by lysosomal enzymes derived from the lambda granules of platelets. Platelets typically circulate for 10 days before they are removed, mainly by splenic macrophages.

Microstructure

Lymph nodes (Fig. 4.8) are small, oval or kidney-shaped bodies, 0.1–2.5 cm long, lying along the course of the lymphatic vessels. Each usually has a slight indentation on one side, the hilum, through which blood vessels enter and leave and the efferent lymphatic vessel leaves. Several afferent lymphatic vessels enter the capsule around the periphery. Lymph nodes have a highly cellular cortex and a medulla which contains a network of minute lymphatic channels (sinuses) through which lymph from the afferent lymphatics is filtered, to be collected at the hilum by the efferent lymphatic. The cortex is absent at the hilum, where the medulla reaches the surface.

Fig. 4.8 Lymph node (human) sectioned mainly through cortical tissue, showing lymphoid follicles (F), some with germinal centres (G), and connective tissue trabeculae (T). Also shown is the subcapsular sinus (short arrow) and hilum (long arrow) with blood vessels.

The capsule is composed mainly of collagen fibres, elastin fibres (especially in the deeper layers), and a few fibroblasts. From the capsule, trabeculae of dense connective tissue extend radially into the interior of the node. They are continuous with a network of fine type III collagen (reticulin) fibrils which supports the lymphoid tissue. At the hilum, dense fibrous tissue may extend into the medulla, surrounding the efferent lymphatic vessel.

The fine reticulin bundles branch and interconnect to form a very dense network in the cortex: there are fewer fibres in the germinal centres of follicles (see below). They provide attachment for various cells, mostly dendritic cells, macrophages and lymphocytes. Reticulin and the associated proteoglycan matrix are produced by fibroblasts, a few of which are associated with the fibre network.

Lymphatic and vascular supply

Lymph nodes are permeated by channels through which lymph percolates after its entry from the afferent vessels. Macrophages line the channels or migrate along the reticulin which crosses them, and so lymph is exposed to their phagocytic activities, as well as to B and T lymphocytes which lie within the various regions of a node. Afferent lymphatic vessels enter at many points on the periphery, branch to form a dense intracapsular plexus, and then open into the subcapsular sinus, a cavity which is peripheral to the whole cortex except at the hilum (Fig. 4.7). Numerous radial cortical sinuses lead from the subcapsular sinus to the medulla, where they coalesce as larger medullary sinuses. The latter become confluent at the hilum with the efferent vessel which drains the node. All of these spaces are lined by a continuous endothelium and traversed by fine reticular fibres, which support sinus macrophages.

Arteries and veins serving lymph nodes pass through the hilum, giving off straight branches which traverse the medulla, and sending out minor branches as they do so. In the cortex, arteries form dense arcades of arterioles and capillaries in numerous anastomosing loops, eventually returning to highly branched venules and veins. Capillaries are especially profuse around the follicles, which contain fewer vessels. Postcapillary HEV are abundant in the paracortical zones. They form an important site of blood-borne lymphocyte extravasation into lymphoid tissue, apparently by migration through labile endothelial tight junctions. The density of the capillary beds increases greatly when lymphocytes multiply in response to antigenic stimulation. Veins leave a node through its principal trabeculae and capsule, and drain them and the surrounding connective tissue.

Cells and cellular zones of lymph nodes

Although most of the cells in a lymph node are B and T lymphocytes, their distribution is not homogeneous. In the cortex, cells are densely packed and in the outer cortical area they form lymphoid follicles or nodules (Fig. 4.8), which are populated mainly by B cells and specialized follicular dendritic cells (FDC) (see Fig. 4.15). The number, degree of isolation and staining characteristics of follicles vary according to their state of antigenic stimulation. A primary follicle is uniformly populated by small, quiescent lymphocytes, whereas a secondary follicle has a germinal centre (Fig. 4.9) which is composed mainly of antigen-stimulated B cells which are larger, less deeply staining and more rapidly dividing than those at its periphery.

Fig. 4.15 Follicular dendritic cells (brown) in a germinal centre of the human palatine tonsil (immunoperoxidase labelled).

(By courtesy of Dr Marta Perry, UMDS, London.)

Fig. 4.9 A germinal centre in a human tonsil lymphoid follicle, immunolabelled to show CD 38-positive B cells in the germinal centre (red), IgD-positive naïve B cells (green) in the mantle zone and activated, transferrin receptor (CD 71)-positive cells of various lineages (blue).

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

The role of the germinal centre is to provide a microenvironment which allows the affinity maturation of the B cell response, so that as the immune response progresses the affinity or strength with which antibodies bind their antigen also increases. There are several zones in the germinal centre where this is allowed to happen. In the ‘dark zone’ the B cells (centroblasts) undergo rapid proliferation which is associated with hypermutation of their antibody molecules. They then move into the light zone (as centrocytes), where they can interact with the FDCs which carry intact unprocessed antigen on their surface. The centrocytes compete for binding to the antigen; those whose antibody has the highest affinity survive and the rest die. T cells are also present, helping the survival of the B cells and inducing class switching. Macrophages in the germinal centre phagocytose apoptotic lymphocytes (e.g. those B cells which die as part of the process of affinity maturation), and consequently macrophage cytoplasm becomes filled with engulfed lipid and nuclear debris.

The mantle zone (Fig. 4.9) is produced as surrounding cells are marginalized by the rapidly growing germinal centre. It is populated by cells similar to those found in primary follicles, mainly quiescent B cells with condensed heterochromatic nuclei and little cytoplasm (hence the deeply basophilic staining of this region in routine preparations; Fig. 4.10), a few helper T cells, FDCs and macrophages. After numerous mitotic divisions the selected B cells give rise to small lymphocytes, some of which become memory B cells and leave the lymph node to join the recirculating pool, while others leave to mature as antibody-secreting plasma cells either in the lymph node medulla or in peripheral tissues.

Fig. 4.10 Germinal centre in a follicle of mucosa-associated lymphoid tissue (MALT) in the mucosa and submucosa of the appendix. The bases of tubular glands of the mucosal epithelium are seen in the upper field.

The deep cortex or paracortex lies between the cortical follicles and the medulla, and is populated mainly by T cells, which are not organized into follicles. Both CD4 and CD8 T cell subsets are present. The paracortex also contains interdigitating dendritic cells. These dendritic cells include Langerhans cells from the skin and other squamous epithelia which have migrated as veiled cells via the afferent lymphatics into the draining lymph nodes (see Fig. 4.14). Their role is to present processed antigen to T cells. The region expands greatly in T cell-dominated immune responses, when its cells are stimulated to proliferate and disperse to peripheral sites.

Fig. 4.14 Dendritic cells in the skin and lymphoid tissues. Their migratory routes are shown: from blood-borne, marrow-derived precursors, to immature dendritic cells (Langerhans cells) in skin, and then to migrating veiled cells in afferent lymphatic vessels and interdigitating dendritic cells in lymph nodes. An example of each cell in the sequence (arrowed) is shown in red.

In the medulla, lymphocytes are much less densely packed, forming irregular, branching medullary cords between which the reticulin network is easily seen. Other cells include macrophages, which are more numerous in the medulla than in the cortex, plasma cells and a few granulocytes.

Follicle-associated epithelium

The epithelium covering mucosa-associated lymphoid tissue, which varies in type according to its location, is unusual in possessing cells which are involved in sampling antigens and passing them to the underlying tissues. The main function of B lymphocytes in MALT is to produce IgA for secretion into the lumen of the tracts which they line. They are exposed to antigens present in the lumen because the epithelium samples and transfers these antigens to antigen-presenting cells in the underlying tissues. Appropriate clones of T and B cells in local lymphoid tissues are then activated and amplified prior to their exit via the lymphatics. In the small and large intestine these specialized epithelial cells have characteristic short microvilli on their luminal surface and are known as microfold (M) cells. In the palatine tonsils they include modified stratified squamous reticulated epithelial cells.

Thus, many of the lymphocytes migrating between cells in the basal regions of epithelia (Fig. 4.5) are effector cytotoxic and helper T cells that have already been selected in lymphoid nodules and are engaged in immune responses. Similar cells, and activated IgA-producing B cells and plasma cells, are also scattered throughout the entire mucosal lamina propria.

Yellow marrow

Yellow marrow consists of a framework of connective tissue which supports numerous blood vessels and cells, most of which are adipocytes. A small population of typical red marrow cells persists and may be reactivated when the demand for blood cells becomes sufficiently great.

Red marrow

Red marrow is found throughout the skeleton in the fetus and during the first years of life. After about the fifth year the red marrow, which represents actively haemopoietic tissue, is gradually replaced in the long bones by yellow marrow. The replacement starts earlier, and is generally more advanced, in the more distal bones. By 20–25 years of age, red marrow persists only in the vertebrae, sternum, ribs, clavicles, scapulae, pelvis, cranial bones and in the proximal ends of the femur and humerus.

Red bone marrow consists of a network of loose connective tissue, the stroma, which supports clusters of haemopoietic cells (haemopoietic cords or islands) and a rich vascular supply in which large, thin-walled sinusoids are the main feature (Fig. 4.11). The vascular supply is derived from the nutrient artery to the bone, which ramifies in the bone marrow, and terminates in thin-walled arterioles from which the sinusoids arise. These, in turn, drain into disproportionately large veins. Lymphatic vessels are absent from bone marrow. The stroma contains a variable amount of fat, depending on age, site and the haematological status of the body, and small patches of lymphoid tissue are also present. Marrow thus consists of vascular and extravascular compartments, both enclosed within a bony framework from which they are separated by a thin layer of endosteal cells (p. 91).

Haemopoietic stem cells

Within the adult marrow there is a very small number (0.05% of haemopoietic cells) of self-renewing, pluripotent stem cells which are capable of giving rise to all blood cell types, including lymphocytes (Fig. 4.12). Although they cannot be identified morphologically in the marrow, they can be recognized in aspirates by the expression of specific cell surface marker proteins (e.g. CD34). It is thought that haemopoietic stem cells occupy specific environmental niches in the marrow associated with the endosteum of trabecular bone or with sinusoidal endothelium and that their microenvironment is important in homeostasis, the balance between self-renewal and differentiation. Stem cells can also be found (at lower concentrations) in the peripheral blood, particularly after treatment with appropriate cytokines.

Progressively more lineage-restricted committed progenitor cells develop from these ancestors (see Laiosa et al 2006 for a recent review) to produce the various cell types found in peripheral blood. The committed progenitor cells are often termed colony-forming units (CFU) of the lineage(s), e.g. CFU-GM cells give rise, after proliferation, to neutrophil granulocytes, monocytes and certain dendritic cells, whereas CFU-E produce only erythrocytes. Each cell type undergoes a period of maturation in the marrow, often accompanied by several structural changes, before release into the general circulation. In some lineages, e.g. the erythroid series, the final stages of maturation take place in the circulation, whereas in the monocytic lineage, they occur after the cells have left the circulation and entered peripheral tissues where they differentiate into macrophages and some dendritic cells.

To generate a complete set of blood cells from a single pluripotent cell may take some months. The later progenitor cells form mature cells of their particular lineages more quickly. However, because they are not self-renewing, grafts of these later cells eventually fail because the cells they produce all ultimately die. This is of considerable importance in bone marrow replacement therapy. The presence of pluripotent stem cells in the donor marrow is essential for success: only 5% of the normal number are needed to repopulate the marrow. Following replacement therapy, T lymphocytes reconstitute more slowly than the other haemopoietic lineages, reflecting the progressive reduction in size of the thymus with age (chronic involution).

Lymphocytes

Lymphocytes are a heterogeneous group of cells which may share a common ancestral lymphoid progenitor cell, distinct from the myeloid progenitor cell which gives rise to all of the cell types described above. The first identifiable progenitor cell is the lymphoblast, which divides several times to form prolymphocytes: both cells are characterized by a high nuclear to cytoplasmic ratio. B cells undergo differentiation to their specific lineage subset entirely within the bone marrow and migrate to peripheral or secondary lymphoid tissues as naïve B cells, ready to respond to antigen. However, T cells require the specialized thymic microenvironment for their development. During fetal and early postnatal life, and subsequently at lower levels throughout life, progenitor cells migrate to the thymus where they undergo a process of differentiation and selection as T cells, before leaving to populate secondary lymphoid tissues.

Erythrocytes

Erythrocytes and granulocytes belong to the myeloid lineage. The earliest identifiable erythroid progenitor cells are capable of rapid bursts of cell division to form numerous daughter cells; they have thus been named burst-forming units of the erythroid line (BFU-E). They give rise to the CFU-E, which, with their immediate progeny, are sensitive to the hormone erythropoietin. This hormone, produced in the kidney, induces further differentiation along the erythroid line.

The first readily identifiable cell of the erythroid series is the proerythroblast, which is a large (about 20 μm) cell with a large euchromatic nucleus and a moderately basophilic cytoplasm. It also responds to erythropoietin. The proerythroblast contains small amounts of ferritin and bears some of the protein spectrin on its plasma membrane. Proerythroblasts proliferate to produce smaller (12–16 μm) basophilic erythroblasts, rich in ribosomes, in which haemoglobin-RNA synthesis begins. The cytoplasm becomes partially, and then uniformly, eosinophilic (the polychromatic erythroblast and orthochromatic erythroblast respectively). These cells are only 8–10 μm in diameter and contain very little cytoplasmic RNA. The nucleus becomes pyknotic (dense, deep-staining, shrunken) and is finally extruded from the cell, leaving an anucleate reticulocyte, which enters a sinusoid. Its reticular staining pattern, visible using special stains, results from residual cytoplasmic RNA which is usually lost within 24 hours of entering the peripheral blood circulation. Reticulocyte numbers in peripheral blood are therefore a good indicator of the rate of red cell production. The whole process of erythropoiesis takes 5–9 days.

Granulocytes

Granulocyte formation involves major changes in nuclear morphology and cytoplasmic contents which are best known for the neutrophil. Initially, myeloid progenitor cells transform into large (10–20 μm) myeloblasts which are similar in general size and appearance to proerythroblasts. These proliferative cells have large euchromatic nuclei and lack cytoplasmic granules. They differentiate into slightly larger promyelocytes, in which the first group of specific proteins is synthesized in the rough endoplasmic reticulum and Golgi apparatus. The proteins are stored in large (0.3 μm) primary (non-specific) granules, which are large lysosomes containing acid phosphatase. Smaller secondary (specific) granules are formed in the smaller myelocyte, which is the last proliferative stage. The nucleus is typically flattened or slightly indented on one side in myelocytes.

In the next, metamyelocyte, stage, the cell size (10–15 μm) decreases, the nucleus becomes heterochromatic and horse-shoe shaped, and protein synthesis almost stops. As the neutrophil is released, the nucleus becomes first heavily indented (the juvenile stab or band form), and subsequently segmented into up to six lobes, characteristic of the mature neutrophil. The whole process usually takes 7 days to complete, of which 3 days are spent proliferating, and 4 days maturing. Neutrophils may then be stored in the marrow for a further 4 days, depending on demand, before their final release into the circulation.

Eosinophils and basophils pass through a similar sequence but their nuclei do not become as irregular as that of the neutrophil. It is thought that these cells each arise from distinct colony-forming units, which are separate from the CFU-GM.

Monocytes

Monocytes are formed in the bone marrow. Monocytes and neutrophils appear to be closely related cells: together with some of the antigen-presenting dendritic cells, they arise from a shared progenitor, the colony-forming unit for granulocytes and macrophages (CFU-GM). Different colony-stimulating factors (CSF) act on the common progenitor to direct its subsequent differentiation pathway. Monocyte progenitors pass through a proliferative monoblast stage (14 μm) and then form differentiating promonocytes, which are slightly smaller cells in which production of small lysosomes begins. After further divisions, monocytes (up to 20 μm) are released into the general circulation. Most migrate into perivascular and extravascular sites, which they then populate as macrophages, while others may give rise to certain dendritic cells, including Langerhans cells.

Platelets

Platelets arise in a unique manner by the shedding of thousands of cytoplasmic fragments from the tips of processes of megakaryocytes in the bone marrow. The first detectable cell of this line is the highly basophilic megakaryoblast (15–50 μm), followed by a promegakaryocyte stage (20–80 μm), in which synthesis of granules begins. Finally, the fully differentiated megakaryocyte, a giant cell (35–160 μm) with a large, dense, polyploid, multilobed nucleus, appears. Once differentiation is initiated from the CFU-Meg, DNA replicates without cytoplasmic division (endoreduplication), and the chromosomes are retained within a single polyploid nucleus which may contain up to 256n chromosomes (where n is the haploid complement present in gametes). Megakaryocyte lineage characteristics and disorders are reviewed in Sun et al (2006).

The cytoplasm contains fine basophilic granules and becomes partitioned into proplatelets by invaginations of the plasma membrane. These are seen ultrastructurally as a network of tubular profiles which coalesce to form cytoplasmic islands 3–4 μm in diameter. Individual platelets are shed into the circulation from a long, narrow process of megakaryocyte cytoplasm which is protruded through an aperture in the sinusoidal endothelium.

Phagocytosis

The uptake of particulate material and microorganisms is carried out by macrophages in many tissues and organs. When present in general connective tissue, they ingest and kill invading microorganisms and remove debris that has been produced as a consequence of tissue damage. They recognize, engulf and rapidly ingest apoptotic cells in all situations: the mechanism of apoptotic cell uptake does not activate the phagocyte for antigen presentation, and so the process is immunologically silent. In the lung, alveolar macrophages constantly patrol the respiratory surfaces, to which they migrate from pulmonary connective tissue (Fig. 4.13). They engulf inhaled particles including bacteria, surfactant and debris and many enter the sputum (hence their alternative names, dust cells or, in cardiac disease, heart failure cells, which are full of extravasated erythrocytes). They perform similar scavenger functions in the pleural and peritoneal cavities. In lymph nodes, macrophages line the walls of sinuses and remove particulate matter from lymph as it percolates through them. In the spleen and liver, macrophages are involved in particle removal and in the detection and destruction of aged or damaged erythrocytes. They begin the degradation of haemoglobin for recycling iron and amino acids.

Fig. 4.13 Alveolar macrophages (dust cells, arrows) containing ingested carbon particles, in alveoli and interalveolar septa of the human lung.

Macrophages bear surface receptors for the Fc portions of antibodies and for the C3 component of complement. Phagocytic activity is greatly increased when the target has been coated in antibody (opsonized) or complement, or both. Once phagocytosis has occurred, the vacuole bearing the ingested particle fuses with endosomal vesicles which contain a wide range of lysosomal enzymes, including many hydrolases, and oxidative systems capable of rapid bacteriocidal action. These activities are much enhanced when macrophages are stimulated (activated macrophages) by cytokines, e.g. interferon (IFN)-γ, which are secreted by other cells of the immune system, especially T lymphocytes.

Close antibody-mediated binding may initiate the release of lysosomal enzymes onto the surfaces of the cellular targets to which the macrophages bind. This process of cytotoxicity is also used by other cells, including neutrophils and eosinophils, particularly if the targets are too large to be phagocytosed (e.g. nematode worm parasites).

Secretory activities

Activated macrophages can synthesize and secrete various bioactive substances, e.g. IL-1, which stimulate the proliferation and maturation of other lymphocytes, greatly amplifying the reaction of the immune system to foreign antigens. They also synthesize tumour necrosis factor (TNF)-α, which is able to kill small numbers of neoplastic cells. TNF-α depresses the anabolic activities of many cells in the body, and may be a major factor mediating cachexia (wasting) which typically accompanies more advanced cancers. Other macrophage products include plasminogen activator, which promotes clot removal; various lysosomal enzymes; several complement and clotting factors; and lysozyme (an antibacterial protein). In pathogenesis, these substances may be released inappropriately and damage healthy tissues, e.g. in rheumatoid arthritis and various other inflammatory conditions.

Langerhans cells

Langerhans cells (Fig. 4.14) are one of the most well-studied types of immature dendritic cell (reviewed in Berger et al 2006). They are present throughout the epidermis of skin, where they were first described, but are most clearly identifiable in the stratum spinosum (see Ch. 7). They have an irregular nucleus and a clear cytoplasm, and contain characteristic elongated membranous vesicles (Birbeck granules). Langerhans cells endocytose and process antigens, undergoing a process of maturation from antigen-capturing to antigen-presenting cells which express high levels of MHC class I and II molecules, co-stimulatory molecules and adhesion molecules. They migrate to lymph nodes to activate T lymphocytes.

Interdigitating dendritic cells

Immature dendritic cells are found all over the body, including peripheral blood, and function in antigen-processing and immune surveillance. Mature dendritic cells are present in T cell-rich areas of secondary lymphoid tissue (paracortical areas of lymph nodes, interfollicular areas of MALT, periarteriolar sheaths of splenic white pulp), where they are frequently referred to as interdigitating dendritic cells. Within the secondary lymphoid tissues, they are involved in the presentation to T lymphocytes of antigens associated with either MHC class I (CD8 T cells) or MHC class II (CD4 T cells) molecules. Naïve T cells can only respond to antigen presented by dendritic cells. The T cells are stimulated not only by recognition of the antigen-MHC complex by the TcR, but also by interaction with co-stimulatory molecules expressed by the dendritic cells, and by cytokines secreted by the cells. These cytokines not only help activate the T cell but can also direct the nature of the T-cell response (e.g. Th1 or Th2). Appropriate T cells are thus activated to proliferate and are primed for carrying out their immunological functions. Once primed, T cells can then be stimulated by any APC, including macrophages and B cells.

Follicular dendritic cells

Follicular dendritic cells, FDCs (Fig. 4.15), are a non-migratory population of cells found in the follicles of secondary lymphoid tissues, where they attract and interact with B cells. Unlike other dendritic cells, FDCs are not haemopoietic in origin, but are probably derived from the stromal cells of lymphoid tissues. They are unable to endocytose and process antigen, and they lack MHC class II molecules. However, Fc receptors and complement receptors CD21 and CD35 on FDCs allow the cells to bind immune complexes to their surface for subsequent presentation, as unprocessed antigen, to germinal centre B cells. Interactions between B cells, CD4 helper T cells and FDCs in the germinal centres are important in the selection of high affinity B cells and their maturation to either plasma cells or memory B lymphocytes.