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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).

  

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.

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

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).

  

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.

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