Basic Immunology
C. Stephen Foster
Wayne J. Streilein
CELLS OF THE IMMUNE SYSTEM
The cellular components of the immune system include lymphocytes, macrophages, Langerhans cells, neutrophils, eosinophils, basophils and mast cells. Many of these cell types can be further subdivided into subtypes and sub-subsets. For example, lymphocytes include T lymphocytes, B lymphocytes, and non-T, non-B (null) lymphocytes. Each subtype can be further subcategorized, both by functional differences and by differences in cell surface glycoprotein specialization and uniqueness. The latter differentiating aspect of cell types and cell type subsets has been made possible through the development of hybridoma-monoclonal antibody technology (1,2) (Table 3-1).
Lymphocytes
Lymphocytes are mononuclear cells that are round, 7 to 8 μm in diameter, and found in lymphoid tissue (lymph node, spleen, thymus, gut-associated lymphoid tissue, mammary-associated lymphoid tissue, and conjunctiva-associated lymphoid tissue) and in blood. They ordinarily constitute approximately 30% of the total peripheral white blood cell count. The lymphocyte is the premier character in the immune drama; it is the primary recognition unit for foreign material, the principal specific effector cell type in immune reactions, and the cell exclusively responsible for immune memory.
T lymphocytes, or thymus-derived cells, comprise 65% to 80% of the peripheral blood lymphocyte population, 30% to 50% of the splenocyte population, and 70% to 85% of the lymph node cell population. B lymphocytes comprise 5% to 15% of peripheral blood lymphocytes, 20% to 30% of splenocytes, and 10% to 20% of lymph node cells.
T cells possess cell surface receptors for sheep erythrocytes and for the plant-derived mitogens concanavalin A and phytohemagglutinin. They do not possess surface immunoglobulin or surface membrane receptors for the Fc portion of antibody—two notable cell surface differences from B lymphocytes, which do possess these two entities. B cells also exhibit cell surface receptors for the third component of complement, for the Epstein-Barr virus, and for the plant mitogen known as pokeweed mitogen, as well as for the purified protein derivative of Mycobacterium tuberculosis and for lipopolysaccharide.
Null cells are lymphocytes that possess none of the aforementioned cell surface antigens characteristic of T cells or B cells. This cell population is heterogeneous, and some authorities include natural killer (NK) cells among the null cell population, even though the origin of NK cells may be in monocytes/macrophage precursor lines rather than the lymphocyte lineage. Nonetheless, the morphologic characteristics and behaviors of NK cells, along with the ambiguity of their origin, enable their inclusion under the null cell rubric. NK cells are nonadherent (unlike macrophages, they do not stick to the surface of plastic tissue culture dishes) mononuclear cells present in peripheral blood, spleen, and lymph nodes. The most notable function of these cells is the killing of transformed (malignant) cells and virus-infected cells. Because they do this without prior sensitization, they are an important component of the early natural response in the immune system. The cytotoxicity of NK cells is not major histocompatibility complex (MHC)-restricted, a dramatic contrast with cytotoxic T cells. (More about the MHC and the products of those gene loci is provided later.) But they do have recognition structures that detect class I MHC molecules; when these receptors engage class I MHC molecules on target cells, the NK cell fails to trigger cytolysis of that target cell. The large granules present in NK cells (the cells are sometimes called large granular lymphocytes) contain perforin and perhaps other cell membrane-lysing enzymes; it is the enzymes in the granules that are responsible for the lethal-hit cytolysis for which NK cells are famous.
Killer cells are the other notable null cell subpopulation. These cells do have receptors for the Fc portion of immunoglobulin G (IgG) and thus can attach themselves to the Fc portion of IgG molecules. Through this receptor, they are a primary cell responsible for cytolysis in the so-called antibody-dependent, cell-mediated cytotoxicity reaction. These cells probably participate in type II Gell and Coombs hypersensitivity reactions and are involved in immune removal of cellular antigens when the target cell is too large to be phagocytosed.
TABLE 3-1. CLUSTERS OF DIFFERENTIATION (CD) DESIGNATIONS | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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It is clear that both B cells and T cells can be further divided into specialized subsets. B cells, for example, are subdivided into the B cells that synthesize the five separate classes of immunoglobulin (IgG, IgA, IgM, IgD, and IgE). All B cells initially produce IgM specific for an antigenic determinant (epitope) to which it has responded, but some subsequently switch from synthesis of IgM to synthesis of other immunoglobulin classes. The details of the control of antibody synthesis and class switching are discussed later in this chapter. Less known is the fact that functionally distinct subsets of B cells exist, in addition to the different B cells involved in antibody class synthesis. The field of B-cell diversity analysis is embryonic, but it is clear that the exploitation of monoclonal antibody technology will distinguish, with increasingly fine specificity, differences in B-cell subpopulations. It is clear, for example, that a subpopulation of B lymphocytes possess the CD5 glycoprotein on the cell surface plasma membrane (a CD glycoprotein not ordinarily present on B lymphocytes but rather on the cell surfaces of T cells) (3). These cells appear to be associated with autoantibody production (4).
It is also clear now that B cells are functionally important as antigen-presenting cells (APCs) for previously primed or memory (not naive) T cells, a fact that startles most physicians who studied immunology before 1991. T-cell receptors (TCRs) cannot react with native antigen; rather, they respond to processed antigenic determinants of that antigen. APCs phagocytose the antigen, process it, and display denatured, limited peptide sequences of the native antigen on the cell surface of the APC in association with cell surface class II MHC glycoproteins. B cells, as well as classic APCs, such as macrophages and Langerhans cells, can perform this function. The antigen is endocytosed by the B cell and processed in the B-cell endosome (possibly through involvement of cathepsin D) to generate short, denatured peptide fragments, which are then transported to the B-cell surface bound to class II glycoprotein peptides; here, the antigenic peptides are “presented” to CD4 helper T lymphocytes.
Finally, regarding B-cell heterogeneity, it is becoming apparent that some B lymphocytes also have suppressor or regulatory activity. The emerging data on B-cell functional and cell surface heterogeneity will be exciting to follow in the coming years.
Much more widely recognized, of course, is that subsets of T lymphocytes exist. Helper (CD4) T cells “help” in the induction of an immune response, in the generation of an antibody response, and in the generation of other, more specialized components of the immune response. Cytotoxic (CD8) T cells, as the name implies, are involved in cell killing or cytotoxic reactions. Delayed-type hypersensitivity (CD4) T cells are the classic participants in the chronic inflammatory responses characteristic of certain antigens such as mycobacteria. Regulatory T cells (CD8) are responsible for modulating immune responses, thereby preventing uncontrolled, host-damaging inflammatory responses. It is even likely that there are sub-subsets of these T cells. Excellent evidence exists, for example, that there are at least three subsets of regulatory T cells and at least two subsets of helper T cells.
Mosmann and Coffman (5) described two types of helper (CD4) T cells with differential cytokine production profiles. TH1 cells secrete interleukin-2 (IL-2) and interferon-γ (IFN-γ) but do not secret IL-4 or IL-5, whereas TH2 cells secrete IL-4, IL-5, IL-10, and IL-13, but not IL-2 or IFN-γ. Furthermore, TH1 cells can by cytolytic and can assist B cells with IgG, IgM, and IgA synthesis but not IgE synthesis. TH2 cells are not cytolytic but can help B cells with IgE synthesis, as well as with IgG, IgM, and IgA production (6). It is becoming clear that TH1 CD4 or TH2 CD4 cells are selected in infection and in autoimmune diseases. Thus, TH1 cells accumulate in the thyroid of patients with autoimmune thyroiditis (7), whereas TH2 cells accumulate in the conjunctiva of patients with vernal conjunctivitis (8). The T cells that respond to M. tuberculosis protein are primarily TH1 cells, whereas those that respond to Toxocara canis antigens are TH2 cells. Romagnani has proposed that TH1 cells are preferentially “selected” as participants in inflammation associated with delayed-type hypersensitivity reactions and low antibody production (as in contact dermatitis or tuberculosis), and TH2 cells are preferentially selected in inflammation associated with persistent antibody production, including allergic responses in which IgE production is prominent (9). Further, it is now clear that these two major CD4 T-lymphocyte subsets regulate each other through their cytokines. Thus, TH2 CD4 lymphocyte cytokines (notably IL-10) inhibit TH1 CD4 lymphocyte proliferation and cytokine secretion, and TH1 CD4 lymphocyte cytokines (notably IFN-γ) inhibit TH2 CD4 lymphocyte proliferation and cytokine production.
Macrophages
The macrophage (“large eater”) and dendritic cells are the preeminent professional APCs. Macrophages are 12 to 15 μm in diameter, the largest of the lymphoid cells. They posses a high density of class II MHC glycoproteins on their cell surfaces, along with receptors for complement components, the Fc portion of immunoglobulin molecules, receptors for fibronectin, IFNs-α, β, and γ, IL-1, tumor necrosis factor (TNF), and macrophage colony-stimulating factor. These cells are widely distributed throughout the various tissues (when found in tissue, they are called histiocytes); the microenvironment of the tissue profoundly influences the extent of the expression of the various cell surface glycoproteins as well as the intracellular metabolic characteristics. It is clear that further compartmentalization of macrophage
subtypes occurs in the spleen. Macrophages that express a high density of class II MHC glycoproteins are present in red pulp, and macrophages with significantly less surface class II MHC glycoprotein expression are in the marginal zone, where intimate contact with B cells exists. It is likely that, just as in the murine system (10), so too in humans, one subclass of macrophage preferentially presents antigen to one particular subset of helper T cells responsible for induction of regulatory T-cell activation, whereas a different subset of macrophage preferentially presents antigen to a different helper T-cell subset responsible for cytotoxic or delayed-type hypersensitivity effector functions.
subtypes occurs in the spleen. Macrophages that express a high density of class II MHC glycoproteins are present in red pulp, and macrophages with significantly less surface class II MHC glycoprotein expression are in the marginal zone, where intimate contact with B cells exists. It is likely that, just as in the murine system (10), so too in humans, one subclass of macrophage preferentially presents antigen to one particular subset of helper T cells responsible for induction of regulatory T-cell activation, whereas a different subset of macrophage preferentially presents antigen to a different helper T-cell subset responsible for cytotoxic or delayed-type hypersensitivity effector functions.
Macrophages also participate more generally in inflammatory reactions. They are members of the natural (early defense) immune system and are incredibly potent in their capacity to synthesize and secrete a variety of powerful biologic molecules, including proteases, collagenase, angiotensin-converting enzyme, lysozyme, IFN-α, IFN-β, IL-6, TNF-α, fibronectin, transforming growth factor (TGF), macrophage colony-stimulating factor, granulocyte colony-stimulating factor, platelet-activating factor, arachidonic acid derivatives (prostaglandins and leukotrienes), and oxygen metabolites (oxygen free radicals, peroxide anion, and hydrogen peroxide). These cells are extremely important, even pivotal, participants in inflammatory reactions and are especially important in chronic inflammation. The epithelioid cell typical of so-called granulomatous inflammatory reactions evolves from the tissue histiocyte, and multinucleated giant cells form through fusion of many epithelioid cells.
Specialized macrophages exist in certain tissues and organs, including the Kupffer cells of the liver, dendritic histiocytes in lymphoid organs, interdigitating reticular cells in lymphoid organs, and Langerhans cells in skin, lymph nodes, conjunctiva, and cornea.
Langerhans Cells
Langerhans cells are particularly important to the ophthalmologist. They probably are the premier APC for the external eye. Derived from bone marrow macrophage precursors, like macrophages, their function is basically identical to that of the macrophage in antigen presentation. They are rich in cell surface class II MHC glycoproteins and have cell surface receptors for the third component of complement and for the Fc portion of IgG. Langerhans cells are abundant in the mucosal epithelium of the mouth, esophagus, vagina, and conjunctiva. They are also abundant at the corneoscleral limbus, less so in the peripheral cornea; they are normally absent from the central third of the cornea (11). If the center of the cornea is provoked through trauma or infection, the peripheral cornea Langerhans cells quickly “stream” into the center of the cornea (12). These CD1+ dendritic cells possess a characteristic racket-shaped cytoplasmic granule on ultrastructural analysis, the Birbeck granule, whose function is unknown.
Polymorphonuclear Leukocytes
Polymorphonuclear leukocytes (PMNs) are part of the natural immune system. They are central to the host defense through phagocytosis, but if they accumulate in excessive numbers, persist, and are activated in an uncontrolled manner, the result may be deleterious to host tissues. As the name suggests, they contain a multilobed nucleus and many granules. PMNs are subcategorized as neutrophils, basophils, or eosinophils, depending on the differential staining of their granules.
Neutrophils
Neutrophils account for more than 90% of circulating granulocytes. They possess surface receptors for the Fc portion of IgG (CD16) and for complement components, including C5a (important in adhesion and phagocytosis). When appropriately stimulated by chemotactic agents (complement components, fibrinolytic and kinin system components, and products from other leukocytes, platelets, and certain bacteria), neutrophils move from blood to tissues through margination (adhesion to receptors or adhesion molecules on vascular endothelial cells) and diapedesis (movement through the capillary wall). Neutrophils release the contents of their primary (azurophilic) granules (lysosomes) and secondary (specific) granules (Table 3-2) into an endocytic vacuole, resulting in (a) phagocytosis of a microorganism or tissue injury, (b) type II antibody-dependent, cell-mediated cytotoxicity, or (c) type III hypersensitivity reactions (immune complex-mediated disease). Secondary granules release collagenase, which mediates collagen degradation. Aside from the products secreted by the granules, neutrophils produce arachidonic acid metabolites (prostaglandins and leukotrienes), as well as oxygen free radical derivatives.
Eosinophils
Eosinophils constitute 3% to 5% of the circulating PMNs. They possess surface receptors for the Fc portion of IgE (low affinity) and IgG (CD16) and for complement components, including C5a, CR1 (CD35), and CR3 (CD11b). Eosinophils play a special role in allergic conditions and parasitoses. They also participate in type III hypersensitivity reactions or immune complex-mediated disease, after attraction to the inflammatory area by products from mast cells (eosinophil chemotactic factor of anaphylaxis), complement, and other cytokines from other inflammatory cells. Eosinophils release the contents of their granules to the outside of the cell after fusion of the intracellular granules with the plasma membrane (degranulation). Table 3-3 shows the known secretory products of eosinophils; the role these products of inflammation play, even in nonallergic diseases (such as Wegener’s granulomatosis), is underappreciated.
TABLE 3-2. NEUTROPHIL GRANULES AND THEIR CONTENTS | |||||||||||||||||||||||||||||||||||||||
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Basophils
Basophils account for les than 0.2% of circulating granulocytes. They possess surface receptors for the Fc portion of IgE (high affinity) and IgG (CD16) and for complement components, including C5a, CR1 (CD35), and CR2 (CD11b). Their role, other than perhaps as tissue mast cells, is unclear.
Mast Cells
The mast cell is indistinguishable from the basophil in many respects, particularly its contents. There are at least two classes of mast cells, based on their neutral protease composition, T-lymphocyte dependence, ultrastructural characteristics, and predominant arachidonic acid metabolites (Table 3-4). Mucosa-associated mast cells (MMC or MC-T) contain primarily tryptase as the major protease (hence, some authors designate these MC-T, or mast cells—tryptase) and prostaglandin D2 as the primary product of arachidonic acid metabolism. MMCs are T-cell dependent for growth and development (specifically IL-3 dependent), and they are located predominantly in mucosal stroma (e.g., gut). MMCs are small and short-lived (<40 days). They contain chondroitin sulfate but not heparin, and their histamine content is modest (Table 3-5). MMCs degranulate in response to antigen-IgE triggering but not to exposure to compound 48/80, and they are not stabilized by disodium cromoglycate. They are formalin sensitive, so formalin fixation of tissue eliminates or greatly reduces our ability to find these cells using staining technique. With special fixation techniques, MMC granules stain with Alcian blue but not with safranin.
TABLE 3-3. GRANULAR CONTENT OF EOSINOPHILS | ||||||||||||||||
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Connective tissue mast cells (CTMCs) contain both tryptase and chymase (so some authors designate them MC-TC), as well as leukotrienes B4, C4, and D4, as the primary products of arachidonic acid metabolism. CTMCs are T-cell independent. They are larger than MMCs and are located principally in skin and at mucosal interfaces with the environment. They contain heparin and large amounts of histamine, and they degranulate in response to compound 48/80 in addition to antigen-IgE interactions. CTMCs are stabilized by disodium cromoglycate. They stain with alkaline Giemsa, toluidine blue, Alcian blue, safranin, and berberine sulfate.
The ultrastructural characteristics of MMCs and CTMCs are also different. Electron microscopy shows that the granules of CTMCs contain scroll-like structures. Mast cells play a special role in allergic reactions—they are the preeminent cell in the allergy drama. However, they also can participate in type II, III, and IV hypersensitivity reactions. Their role in these reactions, aside from notable vascular effects, is not well understood. Non-IgE-mediated mechanisms (e.g., C5a) can trigger mast cells to release histamine, platelet-activating factor, and other biologic molecules when antigen binds to two adjacent IgE molecules on the mast cell surface. Histamine and other vasoactive amines cause increased vascular permeability, allowing immune complexes to become trapped in the vessel wall.
TABLE 3-4. MAST CELL TYPES AND CHARACTERISTICS | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Platelets
Blood platelets, cells well adapted for blood clotting, also are involved in the immune response to injury, which is a reflection of their evolutionary heritage as myeloid (inflammatory) cells. They possess surface receptors for the Fc portion of IgG (CD16) and IgE (low affinity), for class I histocompatibility glycoproteins [human leukocyte antigen (HLA)-A, -B, or -C], and for factor VIII. They also carry molecules such as Gp11b/111a (CDw41), which binds fibrinogen, and Gp1b (CDw42), which binds von Willebrand factor.
After endothelial injury, platelets adhere to and aggregate at the endothelial surface, releasing permeability-increasing molecules from their granules (Table 3-6). Endothelial injury may be caused by type III hypersensitivity. Platelet-activating factor released by mast cells after antigen-IgE antibody complex formation induces platelets to aggregate and release their vasoactive amines. These amines separate endothelial cell tight junctions and allow immune complexes to enter the vessel wall. Once the immune complexes are deposited, they initiate an inflammatory reaction through activation of complement components and neutrophil lysosomal enzyme release.
IMMUNE SYSTEM
Cells of the immune system are derived from primordial stem cell precursors of the bone marrow. They originate in
the blood islands of the yolk sac (13), and populate embryonic liver and bone marrow (14). These stem cells are pluriopotential. Characteristics of the microenvironment in the bone marrow, particularly with respect to a stem cell’s association with other resident cells in the bone marrow, contribute to or are responsible for the different pathways of maturation and differentiation. For example, specific cells in the bone marrow in the endosteal region promote the differentiation of hematopoietic stem cells into B lymphocytes (15). In birds, primordial pluripotential stem cells that migrate to a gland near the cloaca of the chicken known as the bursa of Fabricius (for reasons of probable stimuli in the bone marrow not yet understood) are influenced by the epithelial cells in that gland to differentiate terminally into B lymphocytes (16,17).
the blood islands of the yolk sac (13), and populate embryonic liver and bone marrow (14). These stem cells are pluriopotential. Characteristics of the microenvironment in the bone marrow, particularly with respect to a stem cell’s association with other resident cells in the bone marrow, contribute to or are responsible for the different pathways of maturation and differentiation. For example, specific cells in the bone marrow in the endosteal region promote the differentiation of hematopoietic stem cells into B lymphocytes (15). In birds, primordial pluripotential stem cells that migrate to a gland near the cloaca of the chicken known as the bursa of Fabricius (for reasons of probable stimuli in the bone marrow not yet understood) are influenced by the epithelial cells in that gland to differentiate terminally into B lymphocytes (16,17).
TABLE 3-5. MAST CELL CONTENTS | ||||||||||||||
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T-cell development results from stem cell migration from the bone marrow to the thymus. Thymic hormones (at least 20 have been preliminarily described) produced by the thymic epithelium initiate the complex series of events that results not only in differentiation of the stem cells into T lymphocytes but subdifferentiation of T lymphocytes into their various functional subsets: Helper function, killer function, and suppressor function are acquired while the T cells are still in the thymus. These hormones are also responsible for the induction of cell surface glycoprotein expression on the surfaces of T cells. The cell surface expression of the various glycoproteins changes during T-cell maturation in the thymus. For example, the CD2 glycoprotein is the first that can be identified on the differentiating T cell, but this is eventually joined by CD5; these are both eventually replaced (CD2 completely and CD5 partially) by CD1 glycoprotein, which in turn is lost and replaced by the mature CD3 marker. CD4 and CD8 glycoproteins are acquired before emigration from the thymus by helper and cytotoxic-regulatory T cells, respectively.
TABLE 3-6. PLATELET GRANULES AND THEIR CONTENTS | ||||||||||||||||||||||||||||||||||||||||
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Monocytes, NK cells, and killer cells evolve from stem cells through influences that are incompletely understood. All three types of cells do arise from a common monocyte precursor and later subdifferentiate under unknown influences.
Lymphoid Traffic
Lymphatic vessels and blood vessels connect the central lymphoid organs (bone marrow, thymus, liver) and the peripheral lymphoid organs (spleen, lymph nodes, gut, bronchial- and conjunctival-associated lymphoid tissues) to one another and to the other organs of the body (18,19) (Table 3-7). Lymphatic vessels drain every organ except the nonconjuntival parts of the eye, internal ear, bone marrow, spleen, and cartilage, and some parts of the central nervous system. The interstitial fluid and cells entering the lymphatic system are propelled (predominantly by skeletal muscle contraction) to regional lymph nodes. Efferent lymphatics draining these regional nodes converge to form large lymph vessels that culminate in the thoracic duct and the right lymphatic duct. The thoracic duct empties into the left subclavian vein, carrying approximately three fourths of the lymph, whereas the right lymphatic duct empties into the right subclavian vein.
One or more homing receptors is present on the surface of all lymphoid cells. These receptors can be regulated, induced, and suppressed. Mature T cells emerging from the thymus cortex toward the medulla are rich either in cell surface or plasma membrane homing receptors, or adhesion molecules or “adhesomes,” which are ligands for various addressins or adhesion molecules at other, remote foci. In the mouse, homing
receptors on the surface of mature T cells have been identified for the lymph node (MEL-14 or L-selectin [leukocyte functional antigen (LFA)-1]) and for Peyer’s patch [leukocyte Peyer’s patch adhesion molecule (LPAM)-1 α4β7 integrin, CD44]. Equivalent homing receptors undoubtedly exist in humans, but work in this area is currently embryonic. A 90-kD glycoprotein designated Hermes-3, however, has been identified as a specific heterotypic recognition unit on lymphocytes (20). The Hermes glycoprotein has been shown to be identical to the CD44 molecule (21). Antibodies to this glycoprotein prevent binding of lymphocytes to mucosal lymph node high-endothelial venules (22,23).
receptors on the surface of mature T cells have been identified for the lymph node (MEL-14 or L-selectin [leukocyte functional antigen (LFA)-1]) and for Peyer’s patch [leukocyte Peyer’s patch adhesion molecule (LPAM)-1 α4β7 integrin, CD44]. Equivalent homing receptors undoubtedly exist in humans, but work in this area is currently embryonic. A 90-kD glycoprotein designated Hermes-3, however, has been identified as a specific heterotypic recognition unit on lymphocytes (20). The Hermes glycoprotein has been shown to be identical to the CD44 molecule (21). Antibodies to this glycoprotein prevent binding of lymphocytes to mucosal lymph node high-endothelial venules (22,23).
TABLE 3-7. LYMPHOID ORGANS | ||||||||
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Immune Response
Professional APCs phagocytose foreign material (antigens), process it through protease endosomal-lysosomal degradation, “package” it with MHC molecules, and transport the peptide-MHC complex to the cell surface. B cells and dendritic cells (including Langerhans cells) also perform this function, but differences in protease types and class II MHC molecules among these APCs may influence the type of T cell activated by an antigen. It is this unit of antigenic peptide determinant and self-MHC glycoproteins, along with the aid of adhesion molecules [intercellular adhesion molecule (ICAM)-1; (CD54) and LFA-3 (CD58)] and costimulatory molecules [B7 (CD80)], that forms the recognition unit for TCRs specific for the antigenic epitope of the foreign material. The TCR is composed of recognition units for the epitope and for the autologous MHC glycoprotein. Endogenous antigens, such as endogenously manufactured viral protein, typically collect in cytoplasm, associate with class I MHC molecules, and are transported to the surface of the APC, where the class I MHC-peptide complex preferentially associates with the TCR of CD8+ cells. As described earlier, exogenous antigens that are phagocytosed typically associate, in the endosomal, endocytic, and exocytic pathways, with class II MHC molecules; this complex preferentially associates with CD4+ TCRs.
The αβ heterodimer of the TCR is associated with CD3 and ζη proteins and (for CD4+ cells) the CD4 molecule, thus forming the TCR complex. Antigen presentation can then occur as the TCR complex interacts with the antigenic determinant/MHC complex on the macrophage, with simultaneous CD28-CD80 interaction or “costimulation.” Macrophage secretion of IL-1 during this cognitive “presentation” phase of the acquired immune response to CD4 T cells completes the requirements for successful antigen presentation to the helper T cell (Fig. 3-1).
The CD3 and ζη proteins are the signal-transducing components of the TCR complex; transmembrane signaling by this pathway results in activation of several phosphotyrosine kinases, including those of the tyk/jak family, and other signal transduction and activation of transcription molecules and phosphorylation of tyrosine residues in the cytoplasmic tails of the CD3 and ζη proteins, leading to the creation of multiple sites that bind proteins (enzymes), like phosphatidylinositol phospholipase C-γ1 (PI-PLC-γ1) with SH2-binding domain. PI-PLC-γ1 in turn is phosphorylated (and thereby activated), and it catalyzes hydrolysis of plasma membrane phosphatidylinositol 4,5-biphosphate into inositol 1,4,5-trisphosphate (IP3), and diaclyglycerol. IP3 then provokes the release of calcium from its endoplasmic reticulum storage sites. The increased intracellular calcium concentration that results from the release from storage in turn results in increased binding of calcium to calmodulin; this then activates the phosphatase, calcineurin. Calcineurin catalyzes the conversion of phosphorylated nuclear factor of activated T cells, cytoplasmic component (NFATc) to free NFATc. This protein (and probably others) then enters the cell nucleus, where gene transcription of cellular protooncogene/transcription factor genes, cytokine receptor genes, and
cytokine genes is then activated and regulated by it (them). For example, NFATc translocates to the nucleus, where it combines with adaptor proteins (AP-1); this complex then binds to the NFAT-binding site of the IL-2 promoter. This, coupled with nuclear factor-kappa B binding by proteins possibly induced by the events stimulated by CD28-CD80 signal transduction, results in IL-2 gene transcription typical of T-cell activation (Fig. 3-1). Thus, this activation phase of the acquired immune response is characterized by lymphocyte proliferation and cytokine production.
cytokine genes is then activated and regulated by it (them). For example, NFATc translocates to the nucleus, where it combines with adaptor proteins (AP-1); this complex then binds to the NFAT-binding site of the IL-2 promoter. This, coupled with nuclear factor-kappa B binding by proteins possibly induced by the events stimulated by CD28-CD80 signal transduction, results in IL-2 gene transcription typical of T-cell activation (Fig. 3-1). Thus, this activation phase of the acquired immune response is characterized by lymphocyte proliferation and cytokine production.
 FIG. 3-1. Signal transduction: intracellular and intranuclear. With antigen-presenting cell presentation of antigen to the T cell [green peptide fragment in the major histocompatibility complex (MHC) class II groove of the macrophage], an extraordinary cascade of events occurs, through the cell membrane, into the cytoplasm, and subsequently into the nucleus, to the level of specific genes on the chromosomes of the nucleus. Specifically, tyrosine-rich phosphorylases catalyze phosphorylation of a series of intracellular proteins, with resultant liberation of calcium stores, and production of the calcineurin-calmodulin complex, which then facilitates the production of nuclear factor of activated T cells, cytoplasmic component capable of being transported through one of the nuclear pores into the nucleus, where interaction then with specific foci on the gene results in induction of gene transcription (in this instance, production of messenger RNA for ultimate synthesis of the protein interleukin-2). (Original drawing by Laurel Cook Lowe.) |
TABLE 3-8. ADHESION MOLECULES | |||||||||||||||||||||||||||||||||||||
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Expression of Immunity
The emigration of hematopoietic cells from the vascular system typically occurs at the region of the postcapillary high-endothelial venule cells. These cells are rich in the constitutive expression of so-called addressins, which are tissue or organ specific endothelial cell molecules involved in lymphocyte homing. These adhesion molecules are lymphocyte-binding molecules for the homing receptors on lymphocytes (Table 3-8). Thus, the mucosal addressin (21) specifically binds to the Hermes 90-kD glycoprotein. In the murine system, a 90-kD glycoprotein (designated MECA-79) is a peripheral lymph node addressin specifically expressed by high-endothelial venules. Along with the constitutive expression of addressins or adhesion molecules, expression of additional adhesion molecules is induced by a panoply of proinflammatory cytokines (Table 3-9). The adhesion molecules give the expression of an immune response its focus, its specifically directed, targeted expression.
Lymphocytes, monocytes, and neutrophils preferentially migrate or “home” to sites of inflammation because of this upregulation of cytokines and the induction of adhesion molecules promoted by them. Thus, L-selectin (CD62L) on the neutrophil cell surface membrane does not adhere to normal vascular endothelium, but ICAM and endothelial leukocyte adhesion molecule (CD62E) expression on the vascular endothelial surface induced by IFN-α, IFN-γ, IL-1, IL-17, or a combination thereof results in low-affinity binding of CD62L, with resultant slowing of neutrophil transit through the vessel, neutrophil “rolling” on the endothelial surface, and (with complement split product and IL-8-driven chemotaxis of increasing numbers of neutrophils) neutrophil margination in the vessels of inflamed tissue. Neutrophil LFA-1 (CD11a, CD18)-activated expression (stimulated by IL-6 and IL-8) then results in stronger adhesion of the neutrophil to endothelial cell ICAM molecules, with resultant neutrophil spreading and diapedesis into the subendothelial spaces and into the surrounding tissue.
Immunologic Memory
The anamnestic capacity of the acquired immune response system is one of its most extraordinary properties. Indeed, it is this remarkable property that was the first to be recognized by the Chinese ancients and (later) by Jenner. We take it as axiomatic that our immunization in childhood with killed or attenuated smallpox and poliovirus provokes not only a primary immune response, but the development of long-lived “memory” cells that immediately produce a rapid, vigorous secondary immune response whenever we might encounter smallpox or poliovirus, thereby resulting in specific antibody- and lymphocyte-mediated killing of the microbe and defending us from the harm the virus would otherwise have done.
Niels Jerne first hypothesized a clonal selection theory to explain at once the specificity and the diversity of the acquired immune response, and Frank Macfarlane Burnet expanded on Jerne’s original hypothesis, clearly predicting the necessary features that would prove the theory; many subsequent studies have done so. Clones are derived from the development of antigen-specific clones of lymphocytes arising from single precursors before and independently of exposure to antigen. Approximately 109 such clones have been estimated to exist in an individual, allowing him or her to respond to all currently known or future antigens. Antigen contact results in preferential activation of the preexisting clone with the cell surface receptors specific for it, with resultant proliferation of the clone and differentiation into effector and memory cells. The secondary or anamnestic immune response is greater and more rapid in onset than is the primary immune response because of the large
number of lymphocytes derived from the original clone of cells stimulated by primary contact with antigen, and because of the longevity of many of the cells (memory cells). The memory cells can survive for very long periods, even decades. They express cell surface proteins not expressed by nonmemory cells (CD45RO). In memory cells, the level of cell surface expression of peripheral lymph node homing receptors is low compared with the population of such receptors on the surfaces of nonmemory cells; in contrast, the population of other adhesion molecules includes CD11a, CD18 (LFA-1), CD44, and HLA molecules. Because of the constitutive expression of the cell surface adhesion molecules, memory T cells rapidly home to sites of inflammation, “looking” for antigen to which they might respond.
number of lymphocytes derived from the original clone of cells stimulated by primary contact with antigen, and because of the longevity of many of the cells (memory cells). The memory cells can survive for very long periods, even decades. They express cell surface proteins not expressed by nonmemory cells (CD45RO). In memory cells, the level of cell surface expression of peripheral lymph node homing receptors is low compared with the population of such receptors on the surfaces of nonmemory cells; in contrast, the population of other adhesion molecules includes CD11a, CD18 (LFA-1), CD44, and HLA molecules. Because of the constitutive expression of the cell surface adhesion molecules, memory T cells rapidly home to sites of inflammation, “looking” for antigen to which they might respond.
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B-LYMPHOCYTE RESPONSES
When an antigen encounters cell surface IgM that has binding specificities for the antigen (e.g., self-antigens), tolerance to the antigen is the typical result if such an encounter precedes emigration of the B cell from the bone marrow.
Once the immature B cell has acquired its “exit visa” (complete surface IgM), it leaves the bone marrow, residing primarily in the peripheral lymphoid organs (and blood), where it further matures to express both IgM and IgD on its cell surface. It is now a mature B cell, responsive to antigen with proliferation and antibody synthesis.
The ability to generate a diverse immune response depends on the assembly of discontinuous genes that encode the antigen-binding sites of immunoglobulin and TCRs during lymphocyte development. Diversity is generated through the recombination of various germline gene segments, the imprecise joining of segments with insertion of additional nucleotides at the junctions, and somatic mutations occurring within the recombining gene segments.
Antibody Diversity
The paradox of an individual possessing a limited number of genes but the capability to generate an almost infinite number of different antibodies remained an enigma to immunologists for a considerable time. The discovery of distinct variable (V) and constant (C) regions in the light (L) and heavy (H) chains of immunoglobulin molecules (Fig. 3-2) raised the
possibility that immunoglobulin genes possess an unusual architecture. In 1965, Dreyer and Bennett proposed that the V and C regions of an immunoglobulin chain are encoded by two separated genes in embryonic (germline) cells (germline gene diversity) (24). According to this model, one of several V genes becomes joined to the C gene during lymphocyte development. In 1976, Hozumi and Tonegawa discovered that V and C regions are encoded by separate, multiple genes far apart in germline DNA that become joined to form a complete immunoglobulin gene active in B lymphocytes (25). Immunoglobulin genes are thus translocated during the differentiation of antibody-producing cells (somatic recombination; Fig. 3-3).
possibility that immunoglobulin genes possess an unusual architecture. In 1965, Dreyer and Bennett proposed that the V and C regions of an immunoglobulin chain are encoded by two separated genes in embryonic (germline) cells (germline gene diversity) (24). According to this model, one of several V genes becomes joined to the C gene during lymphocyte development. In 1976, Hozumi and Tonegawa discovered that V and C regions are encoded by separate, multiple genes far apart in germline DNA that become joined to form a complete immunoglobulin gene active in B lymphocytes (25). Immunoglobulin genes are thus translocated during the differentiation of antibody-producing cells (somatic recombination; Fig. 3-3).
 FIG. 3-2. Structure of immunoglobulin G showing the regions of similar sequence (domains). (From Albert DA, Jakobiec FA. Principles and practice of ophthalmology, 2nd ed. Philadelphia: WB Saunders, 2000:66.) |
Structure and Organization of Immunoglobulin Genes
The V regions of immunoglobulins contain three hypervariable segments that determine antibody specificity (26) (Fig. 3-4). Hypervariable segments of both the L and H chains form the “antigen-binding” site. Hypervariable regions are also called complementarity-determining regions (CDRs). The V regions of L and H chains have several hundred gene segments in germline DNA; the exact number of segments is still being debated but is estimated to range between 250 and 1000 segments.
 FIG. 3-3. Translocation of a V-segment gene to a C gene in the differentiation of an antibody-producing B cell. (From Albert DA, Jakobiec FA. Principles and practice of ophthalmology, 2nd ed. Philadelphia: WB Saunders, 2000:67.) |
 FIG. 3-4. Hypervariable or complementarity-determining regions (CDRs) on the antigen-binding site of the variable regions of immunoglobulin G. (From Albert DA, Jakobiec FA. Principles and practice of ophthalmology, 2nd ed. Philadelphia: WB Saunders, 2000:67.) |
Light-Chain Genes
A complete gene for the V region of an L chain is formed by the splicing of an incomplete V-segment gene with one of several J (joining)-segment genes, which encodes part of the last hypervariable segment (27, 28, 29) (Fig. 3-5). Additional diversity is generated by V and J genes becoming spliced in different joining frames (junctional diversity) (28) (Fig. 3-6). There are at least three frames for the joining of V and J. Two forms of L chain exist: kappa (κ) and lambda (λ). For κλ chains, assume that there are approximately 250 V-segment genes and four J-segment genes. Therefore, a total of 250 × 4 × 3 (for junctional diversity), or 3000, kinds of complete VJ genes can be formed by combinations of V and J.
Heavy-Chain Genes
H-chain V region genes are formed by the somatic recombination of V, an additional segment called D (diversity), and J-segment genes (Fig. 3-7). The third CDR of the heavy chain is encoded mainly by a D segment. Approximately 15 D segments lie between hundreds of VH and at least four JH gene segments. A D segment joins a JH segment; a VH segment then becomes joined to the DJH to form the complete VH gene. To diversify further the third CDR of the heavy chain, extra nucleotides are inserted between V and D, and between D and J (N-region addition) by the action of terminal deoxyribonucleotidyl transferase (30). Introns, which are noncoding intervening sequences, are removed from the primary RNA transcript.
 FIG. 3-5. A V gene is translocated near a J gene in forming a light-chain V region gene. (From Albert DA, Jakobiec FA. Principles and practice of ophthalmology, 2nd ed. Philadelphia: WB Saunders, 2000:67.) |
The site-specific recombination of V, D, and J genes is mediated by enzymes (immunoglobulin recombinase) that recognize conserved nonamer and palindromic heptamer sequences flanking these gene segments (31,32). The nonamer and heptamer sequences are separated by either 12-base pair (bp) or 23-bp spacers (Fig. 3-8). Recombination can occur only between the 12- and 23-bp spacers, not between two 12-bp types or two 23-bp types (called the 12/23 rule of V gene-segment recombination). For example, VH segments and JH segments are flanked by 23-bp types on both their 5′ and 3′ ends. Consequently, they cannot recombine with each other or among themselves. Instead, they recombine with D segments, which are flanked on both 5′ and 3′ ends by recognition sequences of the 12-bp type.
 FIG. 3-6. Imprecision in the site of splicing of a V gene to a J gene (junctional diversity). (From Albert DA, Jakobiec FA. Principles and practice of ophthalmology, 2nd ed. Philadelphia: WB Saunders, 2000:68.) |
Sources of Immunoglobulin Gene Diversity
For 250 VH, 15 DH, and 4 JH gene segments that can be joined in 3 frames, at least 45,000 complete VH genes can be formed. Therefore, more than 108 different specificities can
be generated by combining different V, D, and J gene segments and by combining more than 3000 L chains and 45,000 H chains. If the effects of N-region additional are included, more than 1011 different combinations can be formed. This is large enough to account for the immense range of antibodies that can be synthesized by an individual.
be generated by combining different V, D, and J gene segments and by combining more than 3000 L chains and 45,000 H chains. If the effects of N-region additional are included, more than 1011 different combinations can be formed. This is large enough to account for the immense range of antibodies that can be synthesized by an individual.
 FIG. 3-7. The variable region of the heavy chain is encoded by V-, D-, and J-segment genes. (From Albert DA, Jakobiec FA. Principles and practice of ophthalmology, 2nd ed. Philadelphia: WB Saunders, 2000:68.) |
 FIG. 3-8. Recognition sites for the recombination of V-, D-, and J-segment genes. V and J genes are flanked by sites containing 23-bp spacers, whereas D-segment genes possess 12-bp spacers. Recombination can occur only between sites with different classes of spacers. (From Albert DA, Jakobiec FA. Principles and practice of ophthalmology, 2nd ed. Philadelphia: WB Saunders, 2000:68.) |
Far fewer V genes than Vκ genes encode L chains. However, many more V amino acid sequences are known (33, 34, 35). It is therefore likely that mutations introduced somatically give rise to much of the diversity of λ L chains (somatic hypermutation) (28). Likewise, somatic hypermutation further amplifies the diversity of H chains. To summarize, four sources of diversity are used to form the almost limitless array of antibodies that protect a host from foreign invasion: germline gene diversity, somatic recombination, junctional diversity, and somatic hypermutation.
Regulation of Immunoglobulin Gene Expression
An incomplete V gene becomes paired to a J gene on only one of a pair of homologous chromosomes. Successful rearrangement of one H-chain V region prevents the process from occurring on the other H-chain allele. Only the properly recombined immunoglobulin gene is expressed. Therefore, all of the V regions of immunoglobulins produced by a single lymphocyte are the same. This is called allele exclusion (36,37).
There are five classes of immunoglobulins. An antibody-producing cell first synthesizes IgM and then IgG, IgA, IgE, or IgD of the same specificity. Different classes of antibodies are formed by the translocation of a complete VH (VHDH) gene from the CH gene of one class to that of another (38). Only the constant region of the H chain changes; the variable region of the H chain remains the same (Fig. 3-9). The L chain remains the same in this switch. This step in the differentiation of an antibody-producing cell is called class switching and is mediated by another DNA rearrangement called single-stranded (SS) recombination (39) (Fig. 3-10). This process is regulated by
cytokines produced by helper T cells (28). For example, switching to IgE class immunoglobulin production is provoked by the CD4 TH2 cytokine, IL-4.
cytokines produced by helper T cells (28). For example, switching to IgE class immunoglobulin production is provoked by the CD4 TH2 cytokine, IL-4.
 FIG. 3-9. The VH region is first associated with Cμ and then with another C region to form an H chain of a different class in the synthesis of different classes of immunoglobulins. (From Albert DA, Jakobiec FA. Principles and practice of ophthalmology, 2nd ed. Philadelphia: WB Saunders, 2000:69.) |
 FIG. 3-10. The VHDJH gene moves from its position near Cμ to one near Cγ1 by SS recombination. (From Albert DA, Jakobiec FA. Principles and practice of ophthalmology, 2nd ed. Philadelphia: WB Saunders, 2000:69.) |
Determination of B-Cell Repertoire
V-segment genes can be grouped into families based on their DNA sequence homologies. In general, variable genes sharing greater than 80% nucleotide similarity are defined as a family (40). Currently, there are 11 known VH gene families in the mouse (40, 41, 42, 43) and 6 in humans (44, 45, 46, 47). At least 29 families are known for the V segment of murine L-chain genes (48,49). In fetal pre-B cells, chromosomal position is a major determinant of VH rearrangement frequency, resulting in a nonrandom repertoire that is biased toward use of VH families closest to the JH segments (50, 51, 52, 53, 54, 55). In contrast, random use of VH families based on the number of members in each family occurs in mature B cells without bias toward JH proximal families (54, 55, 56). The preferential VH gene rearrangement frequency seen in pre-B cells presumably becomes normalized when contact of the organism with a foreign antigen selects for the expression of the entire VH gene repertoire. One can speculate that members of VH families preferentially used in the pre-B cell encode antibody specificities that are needed in the early development of the immune system (57).
Immunoglobulin
Immunoglobulins are serum proteins that migrate with the globulin fractions by electrophoresis (25). Although they are glycoproteins, primary functions of the molecules are determined by their polypeptide sequence (26). At one end of the immunoglobulin is the amino terminus, a region that binds a site (epitope) on an antigen with great specificity. At the other end is the carboxyl terminus, a non-antigen-binding region responsible for various functions, including complement fixation and cellular stimulation through binding to cell surface immunoglobulin receptors.
IgG is composed of four polypeptide chains: two identical H chains and two identical L chains. H chains weigh approximately twice as much as L chains. The identical H chains are covalently linked by two disulfide bonds. One L chain is associated with each of the H chains by a disulfide bond and noncovalent forces. The two L chains are not linked. Asparagine residues on the H chains contain carbohydrate groups. The amino terminals of one L chain and its linked H chain compose the region for specific epitope binding. The carboxyl termini of the two H chains constitute the non-antigen-binding region.
Each polypeptide chain, whether L or H, is composed of regions that are called constant (C) or variable (V). A variable region on an L chain is called VL, the constant region of a heavy chain is called CH, and so forth. If the amino acid sequence of multiple L or H chains is compared, the constant regions vary little, whereas the variable regions differ greatly. The L chains are divided approximately equally into a constant (CL) and a variable (VL) region at the carboxyl and amino terminals, respectively. The H chains also contain a similar length of variable region (VH) at the amino terminals, but the constant region (CH) is three times the length of the variable region (VH). The variable regions are responsible for antigen binding, and it is this variability that accounts for the ability to bind to millions of potential and real epitopes (27). Because each antibody molecule has two antigen-binding sites with variable regions, cross-linking of two identical antigens may be performed by an antibody. The constant regions carry out effector functions common to all antibodies of a given class (e.g., IgG) without the requirement of unique binding sites.
The functions of various regions of the immunoglobulin molecule were determined in part by the use of proteolytic enzymes that digest these molecules at specific locations. These enzymes have also been exploited for the development of laboratory reagents. The enzyme papain splits the molecule on the amino terminal side of the disulfide bonds that link the H chains, resulting in three fragments: two identical Fab fragments (each composed of the one entire H chain and a portion of the associated H chain) and one Fc fragment composed of the linked carboxyl terminal ends of the two H chains. In contrast, treatment with the enzyme pepsin results in one molecule composed of two linked Fab fragments know as F(ab′) (25). The Fc fragment is degraded by pepsin treatment.
Within some classes of immunoglobulins, whole molecules may combine with other molecules of the same class to form polymers with additional functional capabilities. J chains facilitate the association of two or more immunoglobulins (Fig. 3-11), most notably IgA and IgM. Secretory component is a polypeptide synthesized by nonmotile epithelium found near mucosal surfaces. This polypeptide may bind noncovalently to IgA molecules, allowing their transport across mucosal surfaces to be elaborated in secretions.
Five immunoglobulin classes are recognized in humans: IgG, IgM, IgA, IgE, and IgD. Some classes are composed of subclasses as well. The class or subclass is determined by the structure of the H-chain constant region (CH) (28). The H chains γ, μ, α, ε, and δ are found in IgG, IgM, IgA, IgE, and IgD, respectively. Four subclasses of IgG and two subclasses of both IgA and IgM exist. The two L chains on any immunoglobulin are identical and, depending on the structures of their constant regions, may be designated κ or λ. The κ chains tend to predominate in human immunoglobulins regardless of the H-chain-determined class. Whether an immunoglobulin is composed of two κ or two λ chains does not determine its functional capabilities. H-chain-determined class does not dictate important capacities (29).
 FIG. 3-11. Schematic diagram of polymeric human immunoglobulins. (From Albert DA, Jakobiec FA. Principles and practice of ophthalmology, 2nd ed. Philadelphia: WB Saunders, 2000:70.) |
Immunoglobulin G
The most abundant of the human classes in serum, IgG constitutes approximately three fourths of the total serum immunoglobulins. Respectively, IgG1 and IgG2 make up approximately 60% and 20% of the total IgG. IgG3 and IgG4 are relatively minor components. IgG is the primary immunoglobulin providing immune protection in the extravascular compartments of the body. IgG is able to fix complement in the serum, an important function in inducing inflammation and controlling infection. IgG3 and IgG1 are most adept at complement fixation. IgG is the only immunoglobulin class to cross the placenta, an important aspect in fetal defense. Through their Fc portions, IgG molecules bind Fc receptors found on a host of inflammatory cells. Such binding activates cells such as macrophages and NK cells, enhancing cytotoxic activities important in the immune response.
Immunoglobulin M
Less abundant in the serum than IgG, IgM typically exists as a permanent form, stabilized by J chains, theoretically allowing the biding of 10 epitopes. (In vivo, this is usually limited by steric considerations.) IgM appears early in the immune response to antigen and is especially efficient at initiating agglutination, complement fixation, and cytolosis. IgM probably preceded IgG in the evolution of the immune response and is the most important antibody class in defending the circulation.
Immunoglobulin A
IgA is found in secretions of mucosal surfaces as well as in the serum. In secretions, it exists as a dimer coupled by J chain and stabilized by secretory component. IgA protects mucosal surfaces from infection but may also be responsible for immunologic surveillance at the site of first contact with antigen. IgA secretion is hardy, able to withstand the ravages of proteolytic degradation.
Immunoglobulin D
IgD is present in minute amounts in the serum and is the least stable of the immunoglobulins. Its function is not known, but it probably serves as a differentiation marker. IgD is found on the surfaces of B lymphocytes (along with IgM) and may have a role in class switching and tolerance.
Immunoglobulin E
IgE is notable for its ability to bind to mast cells; when cross-linked by antigen, it causes a variety of changes in the mast cell, including release of granular contents and membrane-derived mediators. Although IgE is recognized as a component of the allergic response, its role in protective immunity is speculative.
Immunoglobulin Intraclass Differences
Differences among the immunoglobulin classes are known as isotypes because all normal individuals in a species possess all of the classes. Allotype refers to antigenic structures on immunoglobulins that may differ from one individual to another within a species. Idiotype refers to differences among individual antibody molecules in a given individual, and is determined by the variable domain. Just as the variable domain allows for antibodies to recognize many antigens (epitopes), these differences also allow individual antibodies to be recognized on the basis of idiotype. In fact, antibodies directed against antibodies exist and are called anti-idiotypic antibodies. These anti-idiotypic antibodies are crucial to the regulation of the antibody response and constitute the basis for Jerne’s idiotype network.
Complement
The complement system functions in the immune response by allowing animals to recognize foreign substances
and defend themselves against infection (46). The pathways of complement activation are complex (47) (Fig. 3-12). Activation begins with the formation of antigen-antibody complexes and the ensuing generation of peptides that leads to a cascade of proteolytic events. The particle that activates the system accumulates a protein complex on its surface that often leads to cellular destruction through disruption of membranes.
and defend themselves against infection (46). The pathways of complement activation are complex (47) (Fig. 3-12). Activation begins with the formation of antigen-antibody complexes and the ensuing generation of peptides that leads to a cascade of proteolytic events. The particle that activates the system accumulates a protein complex on its surface that often leads to cellular destruction through disruption of membranes.
 FIG. 3-12. Simplified schematic of steps in classic and alternate complement cascades. (From Albert DA, Jakobiec FA. Principles and practice of ophthalmology, 2nd ed. Philadelphia: WB Saunders, 2000:72.) |
Two independent pathways of complement activation are known. The classic pathway is initiated by IgG- and IgM-containing immune complexes. The alternative pathway is activated by aggravated IgA or complex polysaccharides from microbial cell walls (49). One component, C3, is crucial to both pathways and in its proactive form can be found circulating in plasma in large concentrations. Deficiency or absence of C3 results in increased susceptibility to infection (50). Cleavage of C3 may result in at least seven products (lettered a through g), each with biologic properties related to cellular activation and immune and nonimmune responses (51). C3a, for instance, causes the release of histamine from mast cells, neutrophil enzyme release, smooth muscle contraction, suppressor T-cell induction, and secretion of macrophage IL-1, prostaglandin, and leukotriene (52). C3e enhances vascular permeability. C3b binds to target cell surfaces and allows opsonization of biologic particles.
The alternative pathway probably is a first line of defense because, unlike the classic pathway, it may neutralize foreign material in the absence of antibody. The initiating enzyme of this pathway, factor D, circulates in an active form and may protect bystander cells from inadvertent destruction after activation of the pathway.
The final step of both pathways is membrane damage leading to cytolysis. Both pathways require the assembly of five precursor proteins to effect this damage: C5, C6, C7, C8, and C9. The mechanism of complement-mediated cell lysis is similar to that of cell-mediated cytotoxicity (as with NK cells). Membrane lesions result from insertion of tubular complexes into the membranes, leading to uptake of water with ion exchange disruption and eventual osmotic lysis.
The complement system interfaces with a variety of immune responses, as outlined earlier, and with the intrinsic coagulation pathways (53). Complement activity is usually measured by assessing the ability of serum to lyse sensitized sheep red blood cells (54). Values are expressed as 50% hemolytic complement units per millimeter. The function of an individual component may be studied by supplying excess quantities of all other components in a sheep red blood cell lysis assay (55). Components are quantitated by radical diffusion or immunoassay. Complement may be demonstrated in tissue sections by immunofluorescence or enzymatic techniques.
Complement plays a role in a number of human diseases. Complement-mediated cell lysis is the final common pathologic event in type II hypersensitivity reactions. Deficiencies of complement exist in recurrent gonococcal and meningococcal infections, hereditary angioedema, and others (50).
B-Cell Response to Antigen
Primary Response
Naive B cells respond to protein antigen in much the same way that T cells do, through the help of APCs and helper T cells. An APC (usually a macrophage or dendritic cell) processes the antigen and presents it to an antigen-specific helper (CD4) T cell, usually in the T-cell-rich zones of the required lymph node. The T cell is thus activated, expresses the membrane protein gp39, secretes cytokines (e.g., IL-2 and IL-6), and binds to similarly activated antigen-specific B cells (activated by the binding cross-linking of antigen to surface IgMand IgD-binding sites). The T-cell/B-cell proliferation and a cascade of intracellular protein phosphorylation events, together with T-cell cytokine signals, results in production of IgM L and H chains with paratopes specific to the antigen epitopes that initiated this primary B-cell response. The proliferating B cells form germinal centers in the lymph node follicles, and somatic hypermutation of the IgM genes in some of these cells results in the evolution of a collection of B cells in the germinal center with surface IgM of even higher antigen-binding affinity. This phenomenon is called affinity
maturation of the primary antibody response. Those cells with the greatest antigen-binding affinity survive as this primary B-cell response subsides, persisting as long-lived memory cells responsible for the classic distinguishing characteristics of secondary humoral immune response.
maturation of the primary antibody response. Those cells with the greatest antigen-binding affinity survive as this primary B-cell response subsides, persisting as long-lived memory cells responsible for the classic distinguishing characteristics of secondary humoral immune response.
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