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emerged. Despite this long history, new discoveries continue to be made and this superficially simple system continues to surprise. In this chapter I will provide a concise but up-to-date summary of the complement system, focussing on the activation of the system, its regulation, its biologically active products and its involvement in disease. Complement plays a central role in innate immune defense. The primary purpose of complement is to kill invading microorganisms but it is also important in the mediation of inflammatory reactions and the clearance of immune complexes. Complement, via interactions with other components of the immune system, also plays a role in the development of immunity. The complement system consists of a group of eleven soluble plasma proteins which interact with one another in two distinct enzymatic activation cascades (the classical and alternative pathways) and in the non-enzymatic assembly of a cytolytic complex (the membrane attack pathway) (Figure 1). Control of these enzymatic cascades is essential to prevent the rapid consumption of complement in response to trivial stimuli and is provided by ten or more plasma and membranebound inhibitory proteins which act at multiple stages of the system to regulate activation (Table 1). In the first section of this chapter I will describe the activation of the system, the components of the activation and membrane attack pathways and their interactions. In the second section I will discuss the control of the pathway and the regulatory proteins involved. In the third section I will outline the physiological importance of complement, its role in host defence and the biological activities of the products of activation. In the final section I will focus on pathological processes involving complement.

ACTIVATION OF COMPLEMENT The Classical Activation Pathway Activation of CI

The classical pathway is so called because it was the first activation pathway to be recognized. The pathway is initiated by the binding of CI to an activator (Hughes-Jones, 1986; Loos, 1988). Antibody bound to antigen is the best known activator of the classical pathway but many other substances can also bind CI and initiate the classical pathway and these non-antibody activators may be of considerable physiological and pathological significance (Taylor, 1993). CI is a large heterooligomeric complex (molecular weight approx. 800 kD) consisting of a single molecule of Clq and two molecules each of Clr and Cls (Ziccardi, 1983; Arlaud et al., 1990). These components are held together noncovalently in a Ca^^-dependent complex (Figure 2). Clq is itself composed of six copies of each of three distinct polypeptide chains (A, B, and C). One copy each of the A, B and

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Bound C1

Figure 2. Structure and activation of C I . The fluid-phase CI complex, consisting of one molecule of C1 q and two molecules each of CI r and C1 s, binds via at least two of its six 'heads' to immunoglobulin. (1) binding causes conformational changes in C l q , resulting in activation; (2) this causes conformational changes and activation of the first CI r molecule which (3) cleaves and activates the second CI r which in turn (4) cleaves and activates CI s. Reproduced from Morgan (1990). Complement: Clinical aspects and relevance to disease. Academic Press, London, with permission.

C chains are wound around each other in a triple helix containing an extended, collagen-like tail and a large globular head (Reid and Porter, 1981). In the intact Clq molecule the collagen tails of the six triple helices are tightly associated along the amino terminal half but then diverge to form six connecting strands, each bearing a globular head. This complicated molecule is encoded by three closely linked genes present on chromosome Iq in the order A-C-B (Sellar et al., 1991). Clq binds via its globular heads to the Fc portion of IgG. Activation requires the binding of multiple heads of a C1 q molecule by aggregates of IgG. This multivalent interaction greatly increases the strength of binding and triggers conformational changes within Clq which induce activation. IgM is a multivalent molecule and can thus activate Clq efficiently and without the need for aggregate formation. Among the human IgG subclasses the ranking of efficiency for activation of CI is IgG3 > IgGl > IgG2; IgG4 does not activate CI. Clr and C Is are homologous, single chain molecules of molecular weight 80 kD which, in the presence of Ca^"^, form an elongated Clr2Cls2 complex which sits

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between the globular heads of Clq (Figure 2) (Reid, 1986; Sim and Reid, 1991). They are encoded by closely linked genes on chromosome 12. Conformational changes within Clq upon binding antibody allow the auto-activation of the proenzyme Clr, a process which involves cleavage at a single site within the molecule, thereby revealing the active site. Clr then activates Cls in the complex, again by cleaving at a single site in the molecule (Sim and Reid, 1991). Of the non-antibody activators of CI, mannose binding protein (MBP) is of particular importance. MBP is a high molecular weight serum lectin made up of many copies of a single 32 kD chain (Taylor et al., 1989). Its role in vivo is to bind mannose and N-acetyl glucosamine residues in bacterial cell walls. Once bound, MBP can develop the capacity to activate the classical pathway of complement either by binding Clr and Cls to form a MBP-Clr2-Cls2 complex or by binding a novel protein termed MBP-associated serine protease or MASP (Matsuhita and Fujita, 1992). MBP thus provides a rapid, antibody-independent means of activating the classical pathway on bacteria and deficiency is associated with defective bacterial opsonization and repeated infections (Turner, 1991). Other lectins have also been shown to activate complement, leading to the proposal that they represent a distinct complement activation pathway (Holmskov et al., 1994). Binding and Cleavage of C4 The next component in the classical pathway is C4, a large plasma protein (200 kD) which consists of three disulfide-bonded chains (a, (3 and y) (Shreiber and Muller-Eberhard, 1974; Janatova and Tack, 1981). C4 is synthesized as a singlechain precursor which is processed to give the three-chain molecule prior to secretion. The gene for C4 is in the class III region of the major histocompatibility complex (MHC) on the short arm of chromosome 6. In fact, C4 is encoded by two closely linked genes which give rise to the two isotypic variants, C4A and C4B (Campbell et al., 1990). These variants differ by only six amino acids but this small difference causes significant changes in function, C4A binding preferentially to amino groups after cleavage and C4B to hydroxyl groups (see below). C4B is also more efficient in propagating continued activation of complement. C1 s cleaves fluid-phase C4 at a single site near the amino-terminus of the a chain, releasing a small fragment, C4a and in the process exposing a reactive thiolester group in the a chain of the large fragment, C4b. Exposure of this reactive group bestows upon C4b the capacity to bind to membranes or other surfaces (Law et al., 1980; Law and Dodds, 1990). The thiolester group forms covalent amide or ester bonds with exposed amino or hydroxyl groups respectively on the activating surface (Figure 3). The thiolester is extremely short-lived due to its susceptibility to inactivation by hydrolysis. As a consequence, C4b binding to surfaces is a very inefficient process, most of the C4b formed decaying in the fluid phase and C4b binding being restricted to the immediate vicinity of the activating CI complex.

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Binding and Activation of C2

The next component of the classical pathway is C2, a single chain plasma protein of molecular weight 102 kD which, like C4, is encoded by a gene in the class III region of the MHC on chromosome 6. The gene for C2 is closely linked to that for factor B, its functional homologue in the altemative pathway (see below). Membranebound C4b expresses a binding site which, in the presence of Mg^^ ions, can bind C2 near its amino terminus and present it for cleavage by an adjacent CI complex to yield a 30 kD amino-terminal fragment, C2b, and a 70 kD carboxy-terminal fragment, C2a (Nagasawa and Stroud, 1977). The C2b fragment may be released or remain loosely attached to C4a but is not required for enzymatic activity. The C2a fragment binds to C4b through a newly exposed site near its new amino terminus to form the C4b2a complex, the next enzyme in the classical pathway. Binding and Cleavage of C3

The penultimate component of the classical pathway, C3, is the most abundant of the complement components (1-2 mg/ml in serum). It is a large (185 kD) molecule composed of two chains (a, 110 kD and P, 75 kD) held together by disulfide bonds (Lambris, 1988). Like C4, with which it shares many structural features, C3 is synthesized as a single chain precursor molecule and cleaved intracellularly prior to secretion. The gene for C3 is on chromosome 19. C2a in the C4b2a complex cleaves C3 in the fluid phase at a single site in the a chain, releasing a small fragment from the amino-terminus (C3a, 9 kD) and exposing in the large fragment, C3b, a thioester group and binding sites for several complement receptors and regulatory proteins (see below). The labile thioester group confers upon C3b the capacity to bind covalently to the activating surface as described above for C4b (Figure 3). Binding is inefficient and the bulk of the C3b formed decays in the fluid phase to form C3bi ('i' = inactive). Only C3b bound to the activating surface in close proximity to the C4b2a complex takes any further part in activation. Binding and Cleavage of C5

The final component of the classical pathway, and also the first component of the membrane attack pathway, is C5. C5 is a two-chain plasma protein of 190 kD molecular weight, encoded on chromosome 9, which is structurally related to C3 and C4. Like these molecules it is synthesized as a single-chain precursor and cleaved prior to secretion, however, C5 does not contain a thioester group and thus cannot itself bind covalently to surfaces (Law and Dodds, 1990). C3b attached to the membrane binds C5 and presents it for cleavage by C2a in an adjacent C4b2a complex. The C4b2a complex can only cleave C5 bound to C3b and hence the C5 cleaving enzyme (C5 convertase) of the classical pathway is termed C4b2a3b. Cleavage occurs at a single site in the a chain of C5, releasing a small amino-

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terminal fragment, C5a (approx. 10 kD), and exposing on the larger fragment, C5b, a labile hydrophobic surface binding site and a site for binding C6, neither of which are expressed by uncleaved C5 (Hughes-Jones, 1986; Loos, 1988). The Alternative Activation Pathway Initiation of the Alternative Pathway

The alternative pathway provides a rapid, antibody independent route for complement activation and amplification on invading microorganisms and other foreign surfaces. C3 is the key component but two other proteins, unique to the alternative pathway, are also required. Factor B is a single-chain 93 kD plasma protein, structurally and functionally homologous with C2 which, once activated, can cleave C3 at a site identical to that used by the C4b2a enzyme. C2 and factor B have 40% protein sequence identity and are encoded by closely linked genes in the MHC on chromosome 6. Activation of factor B is accomplished by factor D, a 26 kD serine protease enzyme present in plasma in its active form, which cleaves factor B at a single site, releasing a 30 kD fragment, Ba, and exposing a serine protease domain on the large (60 kD) fragment, Bb (Gotze, 1986). Factor D can only cleave factor B bound to C3b. Initiation of the alternative pathway on a surface therefore requires that C3b be deposited in a conformation which allows factor B to bind and be cleaved by factor D. How then is the initiating C3b formed? In many circumstances some degree of classical pathway activation may have occurred, depositing sufficient C3b to trigger the alternative pathway. Nevertheless, it appears that the alternative pathway can be initiated independent of the classical pathway. In biological fluids C3 is continuously hydrolysed at a slow rate to form a metastable C3 (H2O) molecule which, in the presence of Mg^"^ ions, can bind factor B in solution and render it susceptible to cleavage by factor D. Bb in the fluid phase C3 convertase thus formed (C3(H20)Bb) cleaves C3, releasing C3a and exposing the thioester group in C3b which can then bind to adjacent surfaces (Lachmann and Hughes-Jones, 1984; Law and Dodds, 1990). As a result of this "tickover" phenomenon C3b is continuously deposited in small amounts on all cells in the body. Amplification on Activator Surfaces

Deposition of C3b on host cells does not result in continued activation because the surface features do not favor a binding of factor B (non-activator surfaces) and bound C3b decays, a process catalyzed by fluid-phase and membrane inhibitors (see below). In contrast, the surface features of many microorganisms and foreign cells favor amplification (activator surfaces). C3b binds factor B (Mg^'^-dependent) and presents it for cleavage by fluid-phase factor D, thus forming the alternative pathway C3 convertase C3bBb which cleaves more C3. Activating surfaces thus rapidly become coated with C3b molecules, each one of which can itself recruit

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factor B to form more convertases and further amplify activation. The precise nature of the surface features which determine whether activation will occur are still not clear; however, surface carbohydrates, particularly sialic acid, appear to be important (Pangbum and Muller-Eberhard, 1984; Pangburn, 1986). Cleavage of C5 As in the classical pathway, bound C3b acts as a receptor for C5, enabling the cleavage of C5 by Bb in an adjacent C3bBb complex. The site of cleavage is identical to that caused by C2a in the classical pathway convertase (C4b2a3b). C5a is released and labile membrane- and C6-binding sites are exposed on C5b as described above for the classical pathway. The Membrane Attack Pathway

Cleavage of C5 by the C5 convertase of either the classical or alternative pathways is the final enzymatic step in the complement cascade. The membrane attack pathway involves the non-covalent association of C5b with the four terminal complement components, all of which are hydrophilic plasma proteins, to form an amphipathic membrane-inserted complex. While still attached to C3b in the convertase C5b binds C6, a large (120 kD) single chain plasma protein. Binding of C6 stabilizes the membrane binding site in C5b and exposes a site for C7, a 110 kD single chain plasma protein which is homologous to C6 (see below). Attachment of C7 causes conformational changes in the complex which result in its release from the convertase to the fluid phase and the transient expression of a hydrophobic membrane binding site. If the C5b67 complex encounters a membrane during the brief lifetime of this site, it binds tightly. However, the complex does not penetrate deeply into the membrane and does not disturb the integrity of the lipid bilayer. In addition to the natural decay of the membrane binding site, attachment of C5b67 is further limited by the tendency of the complex to aggregate and by the presence of multiple fluid-phase inhibitors (see below). Deposition of C5b67 is thus limited to the target cell. A small proportion of complexes may attach to the membranes of closely apposed host cells and cause damage or lysis, so-called innocent bystander lysis (Podack and Muller-Eberhard, 1978). The penultimate component of the membrane attack pathway, C8, is a complex molecule made up of three chains, a, p and y (molecular weights 65 kD, 65 kD and 22 kD respectively), encoded by separate genes, a and p closely linked on chromosome 1, y on chromosome 9. The a and p chains are homologous with each other and with C6 and C7 whereas C8y displays no homology. The a and y chains are covalently linked whereas the P chain is non-covalently associated in the complex. The P chain in C8 binds C7 in the C5b67 complex and the resulting complex, C5b-8, becomes more deeply buried in the membrane and forms small pores, causing the cell to become slightly leaky.

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The final component in the pathway, C9, is a single chain plasma protein (molecular weight 69 kD) which is homologous with C6, C7, C8a, and C8(3. The first C9 molecule to enter the C5b-8 complex binds to C8a and undergoes a major conformational change from a globular, hydrophilic form to an elongated, amphipathic form which traverses the membrane and exacerbates membrane leakiness. Unfolding of C9 also exposes binding sites which enable additional C9 molecules to bind, unfold and insert in the membrane. The pore thus grows with the recruitment of additional C9 molecules, individual complexes (membrane attack complexes, MACs) containing anything up to 18 C9 molecules. MACs containing multiple C9 molecules can be visualized in the electron microscope as ring-like structures enclosing a 10 nm pore. Evidence that the pore is formed mainly from C9 molecules is provided by the observation that C9 incubated in vitro in the presence of Zn^"^ ions forms identical ring structures (Podack, 1988; Esser, 1990). The exact mechanism by which the MAC causes lysis is still the subject of debate between those who state that the MAC ring surrounds a rigid, transmembrane pore and those who consider that the MAC induces areas of lipid perturbation (leaky patches) in the membrane (Bhakdi and Tranum-Jensen, 1991; Esser, 1991). Whatever the exact mechanism, the MAC forms functional pores in cell membranes through which ions and small molecules pass, bringing about osmotic lysis of the cell. This strategy of pore formation as a means of killing targets is widely used in nature, notably by bacterial toxins and by mammalian lymphocytes (Peitsch and Tschopp, 1991). The MAC component proteins C6, C7, C8a, C8p and C9 are all highly homologous and have clearly arisen from a common ancestor. C6 and CI are closely linked on chromosome 5 with C9 more distant on the same chromosome (Rogne et al., 1991). C8a and C8p are closely linked on chromosome I. These duplication events have probably arisen to increase the efficiency of targeting and of cytolysis by the MAC.

CONTROL OF THE COMPLEMENT SYSTEM As a consequence of the proteolytic cascade nature of the complement system, small initiating events could result in catastrophic activation of the system. In order to prevent this each part of the complement system is tightly controlled. This control is mediated in part by the inherent instability of the activation pathway enzymes but is also provided by a number offluid-phaseand membrane-associated inhibitors (Figure 4). Control in the Classical Pathway The first step of the classical pathway is regulated by CI-inhibitor (Clinh), a 90 kD plasma protein, a member of the serine protease inhibitor (Serpin) family, which binds to CI and prevents its activation. Clinh also binds activated CI, physically removing Clr and Cls and thus causing disruption of the multimolecular CI

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complex (Davis, 1988). CI inh is the only serum protease inhibitor capable of inhibiting activated C1. Control of the later steps of the classical pathw^ay primarily involves Factor I, a two-chain (55 kD and 42 kD) plasma serine protease, together with a number of fluid-phase and membrane proteins which act as cofactors for factor I and/or accelerate the dissociation of the classical pathway C3- and C5-convertases (C4b2a and C4b2a3b). From the fluid-phase two proteins are involved, C4b-binding protein (C4bp), a large, multimeric plasma protein (6 or 7 a chains, each of approx. 75 kD, and zero or 1 (3 chain) and Factor H, a single-chain 150 kD plasma protein. C4bp binds C4b in the C4b2a complex and accelerates the dissociation (decay) of the enzyme into its component molecules (Gigli et al., 1979: Reid et al., 1986). It also acts as a cofactor for cleavage of C4b in the complex by factor I, a process which inactivates the enzyme complex (see below). The (3 chain confers upon C4bp the capacity to bind Protein S, a cofactor involved in the coagulation system (Hessing, 1991). Factor H binds C3b in the C4b2a3b complex and causes inactivation of the enzyme complex both by accelerating dissociation and by acting as a cofactor for factor I cleavage of C3b (Vik et al., 1990). On the membrane, at least three proteins help regulate the classical pathway convertases. Decay accelerating factor (DAF) is a 65 kD single-chain protein

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tethered to the membrane by a glycosyl phosphatidylinositol (GPI) anchor. DAF binds to and causes dissociation of the enzyme complexes C4b2a and C4b2a3b on the cell membrane. Membrane cofactor protein (MCP) is a 60 kD transmembrane protein which, like DAF, binds to the C4b2a and C4b2a3b enzyme complexes but, instead of causing dissociation, acts as a cofactor for the cleavage of C4b and C3b in the complexes by factor I, thus inactivating the enzyme. Complement receptor 1 (CRl) is a large (approx. 200 kD) transmembrane protein which inactivates the C4b2a and C4b2a3b enzymes both by causing dissociation (like DAF) and by acting as a cofactor for cleavage by factor I (like MCP). DAF, MCP, CRl, factor H and C4bp all share important structural features and all are tightly linked in a region known as the Regulators of Complement Activation (RCA) cluster on the short arm of chromosome 1. These important molecules are thoroughly described in several recent reviews (Reid et al., 1986; Hourcade et al., 1989; Liszewski et al., 1991; Morgan and Meri, 1994). Factor I, in the presence of an appropriate cofactor (C4bp, factor H, MCP or CR1), can enzymatically cleave both C4b and C3b and hence inactivate the classical pathway convertases. The cleavage events are similar. In the case of C3b, Factor I sequentially cleaves at two sites in the a-chain, releasing a small fragment (C3f, 2 kD) and yielding iC3b ('i' = inactive). A third factor I cleavage in the a chain releases a large fragment (C3c, 140 kD), leaving the C3dg fragment (40 kD) attached to the membrane. Factor H, C4bp, MCP and CRl all act as cofactors for the first two cleavages (factor H only for C3b and C4bp only for C4b) but only CR 1 has good cofactor activity for the third factor I cleavage. Further cleavage of C3dg by serum proteases may occur to yield the cell-bound fragment C3d (Figure 3). Control in the Alternative Pathway Control in the alternative pathway involves many of the same membrane and fluid-phase regulatory proteins described above for the classical pathway. Factor I again assumes a pivotal role. From the fluid phase, factor H binds C3b in the C3bBb and C3bBbC3b complexes and inactivates the enzyme both by causing dissociation and by acting as a cofactor for factor I-mediated cleavage of C3b. On the membrane, control is mediated by DAF, MCP and CRl. DAF and CRl cause dissociation of Bb from the C3bBb and C3bBbC3b complexes, and MCP and CRl act as cofactors for factor I-mediated cleavage of C3b in these complexes. A unique feature of the alternative pathway is the existence of a protein which stabilizes the C3 and C5 cleaving enzymes. Properdin (P) is a large, multimeric (2,4 or more subunits each of 56 kD) plasma protein which binds to C3b in the complexes and inhibits the spontaneous and accelerated (Factor H, DAF, CRl) decay (Smith et al., 1984; Pangbum, 1989).

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Control in the Membrane Attack Pathway Like the activation pathways, the membrane attack pathway is tightly regulated by fluid phase and membrane inhibitors. The fluid-phase C5b-7 complex is intrinsically unstable, its hydrophobic membrane binding site being susceptible to inactivation by hydrolysis. Binding of C5b-7 to membranes is further restricted by several serum proteins. The most studied of these is S protein (vitronectin), an 80 kD single-chain protein, a single molecule of which binds tightly to the C5b-7 complex and prevents its binding to membranes. C8 and C9 bind but the resultant SC5b-9 complex is inactive (Podack et al., 1984). The fluid-phase C5b-7 complex can also be inactivated by binding to a recently described serum protein, clusterin, which consists of two disulfide-bonded 40 kD chains (Jenne and Tschopp, 1992). Clusterin is a muki-fiinctional protein which is a serum lipoprotein, is present in high concentration in seminal plasma and also appears to be a marker for cell death. C5b-7 is also inactivated by binding other lipoproteins and, perhaps most important of all, by binding C8 in the fluid phase. With the exception of C8, all these fluid-phase inhibitors have other functions and it is likely that their MAC inhibiting activity is a rather non-specific by-product of their affinity for hydrophobic molecules. Their importance in vivo as complement inhibitors is unproven. On the membrane, two inhibitors have been described, homologous restriction factor (HRF), a 70 kD GPI-anchored protein the significance of which is unproven (Zalman, 1992), and CD59 antigen (CD59), a 20 kD GPI-anchored molecule which is the major MAC inhibitor on most cells. CD59 binds to C8 in the C5b-8 complex and blocks incorporation of C9, thereby preventing formation of the lytic MAC (Lachmann, 1991; Davies and Lachmann, 1993). CD59 is encoded on chromosome 11, and shows no structural similarities with any of the other complement inhibitors.

PHYSIOLOGICAL ROLES OF COMPLEMENT The physiological importance of complement is most dramatically demonstrated in individuals deficient in components of the system (see below). The main problems encountered by such individuals are recurrent and severe bacterial infections and immune complex disease, vividly demonstrating that the principal roles of complement are to mediate killing of invading bacteria and to solubilize immune complexes. Complement also plays an important physiological role as an initiator or enhancer of inflammation at sites of infection or injury and in recent years it has become evident that complement also makes an important contribution to the induction of antibody responses. These roles are all mediated by the products of complement activation and are described below.

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B.PAUL MORGAN Bacterial Killing by Complement

Most bacteria, with the important exception of the Gram negative organisms belonging to the genus Neisseria, are resistant to the lytic effects of the MAC, a consequence of their thick cell walls and capsules. Nevertheless, complement contributes to bacterial killing by attracting phagocytic cells to the site of infection and coating the bacteria in readiness for phagocytosis. The surfaces of most bacteria activate complement via the alternative pathway and rapidly become coated with complement fragments, particularly C3b and its breakdown product iC3b (Taylor, 1983). This process, known as opsonization, enables phagocytic cells to recognize bacteria by binding to fragments of C3 through specific receptors, complement receptors 1 and 3 (CRl and CR3) on the phagocyte membrane. The bacterium can then be efficiently internalized, killed and digested within the cell (Shreiber, 1984; Ross, 1986). Opsonization can also be achieved by bacterium-bound antibody interacting with Fc receptors on phagocytes but this process requires preexisting antibacterial antibody. Complement activation at the site of infection also attracts phagocytic cells and activates them for efficient killing. These activities are mediated by the small fragments released from C3, C4, and C5 during the activation process. These fragments, C4a, C3a, and C5a, are all very similar in structure and have similar biological effects although their potencies differ, C5a being the most active, C3a of intermediate activity and C4a the least active. All bind to receptors on mast cells and cause degranulation with the consequent release of vasoactive amines which mediate vasodilatation and increased vascular permeability (anaphylaxis). Removal of the carboxy-terminal arginine residue by a plasma carboxypeptidase N (anaphylatoxin inactivator) completely abrogates anaphylactic activity of these fragments, thus restricting their activity to the site of activation (Hugh, 1981). C5a and, to a lesser extent, C3a also attract phagocytic cells to the site of activation (chemotaxis). This activity is mediated through specific receptors on phagocytes. Binding of C5a stimulates cell adhesion to and migration through vessel walls and the concentration gradient of C5a guides the cells towards the inflammatory site. In addition, C5a also causes activation of phagocytic cells, priming them for bacterial killing. Importantly, C5a retains some chemotactic activity even after loss of the carboxy-terminal arginine (C5adesArg) and is thus able to signal chemotaxis at some distance from the activation site (Till, 1986). It has recently been demonstrated that cells other than phagocytes, including hepatocytes and brain cells, express receptors for C5a and perhaps also C3a (Haviland et al., 1995; Gasque et al., 1995). The physiological relevance of these findings have yet to be elucidated. Immune Complex Solubilization by Complement

Immune complexes consist of aggregates of antibody and antigen which form in the plasma under physiological and pathological conditions. The complexes effi-

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ciently activate the classical pathway of complement and hence become coated with complement fragments. Coating with complement serves two main purposes; it masks antigenic sites, limiting further growth of the immune complex, and it provides a ligand for immune complexes to attach to cells bearing complement receptors (opsonization). These events are essential for efficient immune complex solubilization and clearance. Erythrocytes are a central component of the immune complex clearance system. CRl on the erythrocyte surface binds C3b on the immune complex, effectively removing it from the circulation. Erythrocyte-bound complexes are then carried to the liver and spleen where transfer to tissue phagocytes occurs with subsequent digestion (Pascual and Schifferli, 1992). Individuals with deficiencies of early components of the classical pathway, particularly those with C1 deficiency, are unable to solubilize immune complexes efficiently and as a consequence have severe immune complex disease. Cell Activation by Complement

Several of the products of complement cause priming of phagocytic cells for more efficient bactericidal activity at the site of inflammation. C5a binds specific receptors on the phagocyte surface and enhances the capacity of the cell to mount an oxidase response and to produce enzymes. The MAC, which forms pores in membranes and can thus bring about lysis of some target cells, is also important as a cell activator in vivo. Metabolically active nucleated cells at the inflammatory site are resistant to lysis by the MAC and are activated to release proinflammatory molecules, thus enhancing the inflammatory response. These important nonlytic effects of the MAC are discussed in several recent reviews (Morgan, 1989; 1992). Complement and the Immune Response

Several of the complement components and fragments generated during complement activation influence the cellular immune response. Most work has focussed on C3 and its fragments (Pepys, 1974). C3 and the fragments C3b and iC3b have been reported to enhance the mitogen-induced proliferative response of B and T lymphocytes, probably acting through complement receptors (CRl, CR2, and CR3) on phagocytic cells and lymphocytes (Kinoshita, 1993). A novel mechanism for the potentiation of immune responses by fragments of C3 has recently been revealed by Fearon and co-workers (Fearon and Carter 1995). C3 fragments bound to antigen act as a bridge between B cell membrane immunoglobulin and a signaling complex containing CD 19, CR2 (to which C3 fragments bind) and TAPA-1. The resulting close association of membrane immunoglobulin with this complex greatly enhances the B cell response to antigen. In vivo, fragments of C3 play an important role in the trapping of antigen-containing immune complexes in lymph nodes. Follicular dendritic cells express complement receptors which bind C3 fragments on the complexes. Antigen in the

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trapped complexes can then be presented to B cells. Individuals deficient in C3 trap antigen very poorly and as a consequence do not mount a good antibody response (Klaus and Humphrey, 1986; Erdei et al., 1991). The anaphylatoxin C3a has been shown to inhibit lymphocyte activation induced by mitogen or antigen. These effects are probably mediated through C3a receptors on T lymphocytes. C3adesArg (C3a which has lost its carboxy-terminal arginine) has no inhibitory effect. Other fragments of C3 have also been demonstrated to influence the cellular immune system in vitro although the physiological relevance of these findings is unclear. C5a indirectly enhances antibody responses by stimulating cytokine release from macrophages. Importantly, C5adesArg, which is much more abundant and stable than C5a in vivo, retains this activity. As a consequence, the stimulatory effect of C5a and C5adesArg is likely to outweigh the inhibitory effect of C3a and the net result is that complement activation causes enhanced humoral immune responses (Weiler, 1987).

PATHOLOGY OF COMPLEMENT Complement Deficiencies Deficiencies of almost every complement protein and regulator have been described and more detailed accounts of the various complement deficiencies can be found in several recent reviews (Morgan and Walport, 1991; Colten and Rosen, 1992;Figueroaetal., 1993). Deficiencies of components of the classical pathway (C1, C4, or C2) are particularly associated with an increased susceptibility to immune complex disease, a consequence of the failure of immune complex solubilizafion. The role of complement in immune complex solubilization is detailed on page 186. The frequency and severity of disease is greatest with Clq deficiency, closely followed by total C4 deficiency, each giving rise to a severe immune complex disease which closely resembles systemic lupus erythematosus (SLE). Subtotal deficiency of C4 is common, due to the extremely high frequency of null alleles at both the C4A and C4B loci, but total C4 deficiency is very rare. Deficiency of C2 is the commonest homozygous complement deficiency in Caucasoids but causes much less severe disease because deposition of the early components, C1 and C4, provides some solubilization of immune complexes. C3 is an essential component of both activation pathways and is vital for efficient opsonization of bacteria. Deficiency thus causes a marked propensity to bacterial infections. Factors I and H are essential for fluid phase regulation of complement activation and deficiency of either causes uncontrolled activation with a consequent secondary deficiency of C3 which is often severe and predisposes to bacterial infections. Deficiencies of components of the alternative pathway are rare and do not predispose to immune complex disease or pyogenic infections. A few individuals

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deficient in factor D have been described all of whom have presented with recurrent Neisseria infections, usually meningococcal meningitis. Properdin deficiency is the commonest disorder of the alternative pathway. It is inherited in an X-linked manner and also predisposes to infection with Neisseria. Deficiencies of terminal pathway components (C5, C6, C7, C8, or C9) also cause susceptibility to infection with Neisseria. Deficiency of C6 is the second most common complement deficiency among Caucasians and is frequently associated with meningococcal meningitis and systemic infection with the meningococcus. C9 deficiency, rare in Caucasians, is by far the most frequent complement deficiency in Japan with an incidence approaching 1 in 1000. CI inhibitor (Clinh) is the sole regulator of activated CI. Partial (heterozygous) deficiency of Clinh is relatively common and is associated with the disease hereditary angioedema (HAE) (Kerr & Yeung-Laiwah, 1987; Davis, 1988). HAE is thus inherited in an autosomal dominant manner. Minor trauma initiates complement activation in skin or mucous membranes and the low tissue levels of Clinh are quickly exhausted. Complement activation (and other enzymatic events usually inhibited by Clinh) then proceeds in an uncontrolled manner, resulting in tissue inflammation. The individual presents with recurrent episodes of edema in skin and mucous membranes. Although most episodes of edema are of little clinical consequence, edema of the gut wall may present with acute abdominal symptoms, and laryngeal or tracheal edema can cause acute airways obstruction. Deficiencies of cell membrane regulators or receptors may also cause disease. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hemolytic disorder caused by an absence of the GPI-anchored complement regulatory molecules DAF and CD59 on an expanding clone of blood cells. Affected erythrocytes and platelets become susceptible to damage by complement and hence hemolysis and thrombotic episodes occur (Rosse, 1990). The molecular basis of this fascinating disorder has recently been revealed. Mutations in the gene for one of the key enzymes, termed PIG-A, in the GPI anchor biosynthesis pathway have been found in every case so far examined. All cells in the affected clone are deficient in all GPI-linked molecules (Takahashi et al., 1993). Deficiency of erythrocyte CRl has been reported in association with systemic lupus erythematosus (SLE) but this association is probably a consequence rather than cause of the disease. Complement in Autoimmune Diseases

The complement system contributes to tissue damage in a large number of autoimmune diseases. Autoantibodies and immune complexes in the affected organs activate complement and exacerbate inflammation. In many autoimmune diseases, from the organ specific (e.g., autoimmune thyroid disease) to the disseminated (e.g., systemic lupus erythematosus), complement deposition can be detected in the affected tissues and products of complement activation are found in the plasma (Laurell, 1986; Rauterberg, 1986; Whaley, 1989). In all these autoimmune

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diseases, complement is just one of several factors which contribute to pathogenesis. Nevertheless, modulation of complement activation may be of therapeutic benefit in these conditions (see below). Evasion of Complement Killing by Microorganisms

Many microorganisms have developed mechanisms for avoiding complement mediated damage and a few have even taken advantage of complement activation to enhance their infectivity (Fishelson, 1994). Complement is activated on the surfaces of most microorganisms, with deposition of fragments of C3 and other components. This process of opsonization, by rendering the microorganisms more susceptible to uptake by phagocytes, is among the most important roles of the complement system. However, some microorganisms have subverted this process to their own advantage (Cooper, 1991; Frank, 1992). Organisms which infect cells must find a way of entering the cell and it is here that complement activation may act to enhance infectivity. The strategy is dramatically illustrated by the human retroviruses HIV and HTLV-1. HIV activates complement efficiently through the classical pathway but is resistant to lysis (Ebenblicher et al., 1991). The virus thus becomes coated with complement fragments which can bind complement receptors CRl and CR2 on cells and facilitate uptake into the cell. In this way, complement activation enhances uptake of HIV into CD4-positive T cells (the main receptor being CD4 itself) and may also enable the virus to infect CD4-negative cells, including monocytes, macrophages and B lymphocytes (Haeffner-Cavaillon et al., 1992). Other viruses directly utilize host complement receptors or regulators as receptors to gain entry into the cell. The Epstein-Barr virus binds to CR2 (McClure, 1992). Recently it has been reported that MCP is the receptor for the measles virus (Naniche et al., 1993) and the group A streptococcus (Okada et al., 1995), and that DAP is a receptor for some strains of Echo virus (Bergelson et al., 1994). Many pathogenic microorganisms are highly resistant to complement killing and this complement resistance correlates closely with virulence. It has recently been shown that in organisms as diverse as vaccinia viruses and schistosome worms, complement resistance is achieved by the expression of complement inhibitor molecules which mimic the effects of the human complement inhibitors (Parizade et al., 1991; Isaacs et al., 1992). In many cases it is clear that the microorganism has acquired its complement regulators from the human host—a particularly spectacular piece of molecular piracy. Iatrogenic Activation of Complement

Many materials used in the tubing and membrane components of extrocorporeal circuits are potentially complement activating and exposure of blood to these materials in renal hemodialysis, cardiopulmonary bypass or plasmapheresis is hazardous (Mollnes et al., 1991). In hemodialysis blood is in contact with a large

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surface area of dialysis membrane, commonly made of cuprophane, a complex polysaccharide material which activates complement via the alternative pathway. Release of active fragments, particularly C3a and C5a, causes activation of neutrophils and monocytes which aggregate and become deposited in the lung. Release of toxic molecules from these cells causes the lung damage which is a common complication of long-term dialysis. Activation of complement may also occur in transplanted organs. When a poorly matched organ is used for transplant it is rejected within minutes due to complement activation in the graft, the phenomenon of hyperacute rejection. Complementmediated hyperacute rejection is an almost universal outcome of cross-species transplants and is thus the major barrier to the use of animal organs for human recipients (xenotransplantation) (White, 1992). Attempts are now underway to produce transgenic animals (pigs) expressing human complement regulatory molecules in the hope that the organs from these animals will be less complement activating, enabling them to be used for human recipients. Control of Complement Activation In Vivo Despite extensive research, safe, non-toxic pharmacological agents capable of efficiently inhibiting complement activation have not been found. Those agents which do inhibit complement efficiently in vivo, for example CVF, are toxic and not suitable for therapy. Many substances, including antiinflammatory agents, cause some inhibition of complement but are of low potency. A promising new approach to the inhibition of complement activation in vivo involves the use of molecular biological techniques to produce soluble, recombinant forms of the natural regulatory molecules (Fearon, 1992). Soluble recombinant CRl has been shown to inhibit complement activation and pathology in several animal models of human diseases and has recently begun clinical trials in man. The use of recombinant forms of CRl and other complement regulatory molecules in therapy is likely to be an area of considerable interest and importance in the next few years.

SUMMARY The complement system consists of a group of plasma proteins which interact with one another in a sequential manner to enhance inflammation and to help kill bacteria and other foreign organisms. The two distinct activation pathways, one triggered through antibody-antigen complexes (the classical pathway), the other triggered by foreign surfaces (the alternative pathway), are both proteolytic cascades with considerable potential for the amplification of complement activation. The activation pathways converge on a common membrane attack pathway during which the component proteins complex together and insert as a pore into cell membranes, thus bringing about cell lysis. During activation several biologically active fragments are produced from the complement proteins which are important in defence against

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infection. The system is tightly controlled by a battery of regulatory molecules in plasma and on cell membranes which act to minimize damage to self cells. Overactivation of the complement system can exacerbate tissue damage in many diseases, including autoimmune disease and infection. Inherited deficiencies of components of the complement system graphically reveal the biological importance of complement. Although the precise consequences depend on the component involved, most individuals with complement deficiencies are prone to bacterial infections and to immune complex diseases. The recent development of specific complement inhibitors opens the possibility of preventing activation in the numerous diseases in which complement contributes to tissue injury. REFERENCES Arlaud, G.J., Thielens, N.M., & Illy, C. (1990). Arrangement of the CI complex of complement. Biochem. Soc. Trans. 18, 1148-1151. Bergelson, J.M., Chan, M., Solomon, K.R., St. John, N.F., Lin, H., & Finberg, R.W. (1994). Decay accelerating factor (CD55), a glycosylphosphatidylinositol-anchored complement regulatory protein, is a receptor for several echoviruses. Proc. Natl. Acad. Sci. USA 91, 6245-6248. Bhakdi, S., & Tranum-Jensen, J. (1991). Complement lysis: A hole is a hole. Immunol. Today 12, 318-320. Buchner, H. (1889). Ueber die nahere Natur der bakterientodtenden substanz im blutserum. Zentralbl Bacteriol. 5, 817-823. Campbell, R.D., Dunham, I., Kendall, E., & Sargent, C.A. (1990). Polymorphism of the human complement component C4. Exp. Clin. Immunogenet. 7, 69-84. Colten, H.R., & Rosen, F. (1992). Complement deficiencies. Ann. Rev. Immunol. 10, 809-834. Cooper, N.R. (1991). Complement evasion strategies of microorganisms. Immunol. Today 12,327—331. Davis III, A.E. (1988). CI inhibitor deficiency and hereditary angioneurotic edema. Ann. Rev. Immunol. 6, 595-628. Davies, A., & Lachmann, P.J. (1993). Membrane defence against complement lysis: The structure and biological properties of CD59. Immunol. Res. 12, 258-275. Ebenblicher C.E., Thielens N.M., Vomhagen R., Marschang P., Arlaud G.J., & Dierich M.P. (1991). Human immunodeficiency virus Type 1 activates the classical pathway of complement by direct CI binding through specific sites in the transmembrane glycoprotein gp41. J. Exp. Med. 174, 1417-1424. Erdei, A., Fust, G., & Gergely, J. (1991). The role of C3 in the immune response. Immunol. Today 12, 332-337. Esser, A.F. (1990). The membrane attack pathway of complement. Year Immunol. 6, 229-244. Esser, A.F. (1991). Big MAC attack: Complement proteins cause leaky patches. Immunol. Today 12, 316-317. Fearon, D.T. (1991). Anti-inflammatory and immunosuppressive effects of recombinant soluble complement receptors. Clin. Exp. Immunol. 12(suppl.l), 43-46. Fearon, D.T., & Carter, R.H. (1995). The CD19/CR2/TAPA-1 complex of B lymphocytes: linking natural to acquired immunity. Ann. Rev. Immunol. 13, 127-149. Ferrata, A. (1907). Die univerksamkeit der komplex haemolysise in salzfreien losungen und ihre ursache. Berlin Klin. Wochenschr. 44, 366-369. Figueroa, J., Andreoni, J., & Densen, P. (1993). Infectious diseases associated with complement deficiencies. Immunol. Res. 12, 295-311.

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Fishelson, Z. (1994). Complement-related proteins in microorganisms. Springer Semin. Immunopathol. 15,345-368. Frank, M.M. (1992). The mechanism by which microorganisms avoid complement attack. Curr. Opin. Immunol. 4, 14-19. Gasque, P., Chan, P., Fontaine, M., Ischenko, A., Lamacz, M., Gotze, O., & Morgan, B.P. (1995). Identification and characterisation of the complement C5a anaphylatoxin receptor on human astrocytes. J. Immunol. 155, 4882-4889. Gigli, I., Fujita, T., & Nussenzweig V. (1979). Modulation of the classical pathway C3 convertase by plasma proteins C4 binding protein and C3b inactivator. Proc. Natl. Acad. Sci. USA 76, 6596-6600. Gotze, O. (1986). The alternative pathway of activation. In: The Complement System (Rother, K., & Till, G.O., Eds.), pp. 154-168. Springer, Berlin. Haeffner-Cavaillon, N., Desgranges, C , & Kazatchkine, M.D. (1992). The role of complement in enhancing infection of target cells with human immunodeficiency virus. In: Progress in Immunology VIII (Gergely, J. et al., Eds.), pp. 509-516. Springer, Hungary. Haviland, D.L., McCoy, R.L., Whitehead, W.T., Akama, H., Molmeti, E.P., Brown, A., Haviland, J.C., Parks, W.C., Perlmutter, D.H., & Wetsel, R.A. (1995). Cellular expression of the C5a anaphylatoxin receptor (C5aR): demonstration of C5aR expression on non-myeloid cells of the liver and lung. J. Immunol. 154, 1861-1869. Hessing, M. (1991). The interaction between complement component C4b-binding protein and the vitamin K-dependent protein S forms a link between blood coagulation and the complement system. Biochem J. 277, 581-592. Holmskov, U., Malhotra, R., Sim, R.B., & Jesenius, J.C. (1994). CoUectins: Collagenous C-type lectins of the innate immune defense system. Immunol. Today 15, 67-74. Hourcade, D., Holers, V.M., & Atkinson, J.P. (1989). The regulators of complement gene cluster. Adv Immunol. 45, 381-416. Hughes-Jones, N.C. (1986). The classical pathway. In: Immunobiology of the Complement System (Ross, G.D., Ed.), pp. 2 1 ^ 4 . Academic Press, New York. Hugli, T.E. (1981). The structural basis for anaphylatoxic and chemotactic functions of C3a, C4a and C5a. Crit. Rev. Immunol. 1, 321-366. Isaacs, S., Kotwal, G.J., & Moss, B. (1992). Vaccinia virus complement control protein prevents antibody-dependent complement enhanced neutralization of infectivity and contributes to virulence. Proc. Natl. Acad. Sci. USA 89, 628-632. Janatova, J., & Tack, B.F. (1981). Fourth component of human complement: Studies of an amine-sensitive site comprised of a thiol component. Biochemistry 20, 2394-2402. Jenne, D., & Tschopp, J. (1992). Clusterin: The intriguing guises of a widely expressed glycoprotein. Trends Biochem. Sci. 17, 154-159. Kerr, M.A., & Yeung-Laiwah A.C. (1987). Hereditary angioedema. In: Complement in Health and Disease (Whaley, K., Ed.), pp. 53-786. MTP Press, Lancaster. Kinoshita, T. (1993). Complement receptors and regulation of humoral immune response. Complement Today 1,46-55. Klaus, C.G.B., & Humphrey J.H. (1986). A re-evaluation of the role of C3 in B-cell activation. Immunol. Today 7, 163-165. Lachmann, P.J. (1991). The control of homologous lysis. Immunol. Today 12, 312-316. Lachmann, P.J., & Hughes-Jones, N.C. (1984). Initiation of complement activation. Springer Semin Immunopathol. 7, 143-162. Lambris, J.D. (1988). The multifunctional role of C3, the third component of complement. Immunol. Today 9, 387-393. Laurell, A-B. (1986). Complement determinations in clinical diagnosis. In: The Complement System (Rother, K., & Till, G.O., Eds.), pp. 272-287. Springer, Berlin.

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Law, S.K., & Dodds, A.W. (1990). C3, C4 and C5: the thiolester site. Biochem. Soc. Trans. 18, 1155-1159. Law, S.K., Litchenberg, N.A., & Levine, R.P. (1980). Covalent binding and hemolytic activity of complement proteins. Proc. Natl. Acad. Sci. USA 77, 71294-71298. Liszewski, M.K., Post, T.W., & Atkinson, J.P. (1991). Membrane cofactor protein (MCP or CD46): Newest member of the regulators of complement activation gene cluster. Ann. Rev. Immunol. 9, 431-455. Loos, M. (1988). "Classical" pathway of activation. In: The Complement System (Rother, K., & Till, G.O. Eds.), pp. 136-154. Springer, Berlin. Matsuhita, M. & Fujita, T. (1992). Activation of the classical complement pathway by mannose-binding protein in association with a novel Cls-like serine protease. J. Exp. Med. 176, 1497-1502. Mayer, M.M. (1984). Complement: Historical perspectives and some current issues. Complement. 1, 2-26. McClure, J.E. (1992). Cellular receptor for the Epstein-Barr virus. Prog. Med. Virol. 39, 116-138. Mollnes, T.E., Videm, V., Riesenfeld, J., Garred, P., Svennevig, J.L., Fosse, E., Hogasen, K., & Harboe, M. (1991). Complement activation and bioincompatibility: The terminal complement complex for evaluation and surface modification with heparin for improvement of biomaterials. Clin. Exp. Immunol. 86(suppl.l), 21-26. Morgan, B.P. (1989). Complement membrane attack on nucleated cells: Resistance, recovery and non-lethal effects. Biochem. J. 164, 1-14. Morgan, B.P. (1992). Effects of the membrane attack complex of complement on nucleated cells. In: Current Topics in Microbiology and Immunology Vol. 178. Membrane defenses against attack by complement and perforins (Parker, C.J., Ed.), pp. 115-140. Springer-Verlag, Berlin. Morgan, B.P., & Meri, S. (1994). Membrane regulators of complement. Springer Semin. Immunopathol. 15,36^396. Morgan, B.P., & Walport, M.J. (1991). Complement deficiency and disease. Immunol. Today 12, 301-306. Nagasawa, S., & Stroud, R.M. (1977). Cleavage of C2 by Cls into the antigenically distinct fragments C2a and C2b: Demonstration of binding of C2b to C4b. Proc. Natl. Acad. Sci. USA 74,299^-3001. Naniche, D., Varior Krishnan, G., Cervoni, F., Wild, T.F., Rossi, B., Rabourdin-Combe, C , & Gerlier, D. (1993). Human membrane cofactor protein (CD46) acts as a cellular receptor for measles virus. J. Virol. 67, 6025-6032. Okada, N., Liszewski, M.K., Atkinson, J.P., & Caparon, M. (1995). Membrane cofactor protein (CD46) is a keratinocyte receptor for the M protein of the group A streptococcus. Proc. Natl. Acad. Sci. USA 92, 2489-2493. Pangbum, M.K. (1986). The alternative pathway. In: Immunobiology of the Complement System (Ross, G.D., Ed.), pp. 45-62. Academic Press, New York. Pangbum, M.K. (1989). Analysis of the natural polymeric forms of human properdin and their functions in complement activation. J. Immunol. 142, 202-207. Pangbum, M.K., & MuUer-Eberhard H.J. (1984). The alternative pathway of complement. Springer Semin. Immunopathol. 7, 163—192. Parizade, M., Amon, R., Lachmann, P.J., & Fishelson, Z. (1994). Functional and antigenic similarities between a 94-kD protein of Schistosoma mansoni (SCIP-1) and human CD59. J. Exp. Med. 179, 1625-1636. Pascual, M., & Schifferli, J.A. (1992). The binding of immune complexes by the erythrocyte complement receptor 1 (CRl). Immunopharmacol. 24, 101-106. Peitsch, M.C., & Tschopp, J. (1991). Assembly of macromolecular pores by immune defense systems. Curr. Opin. Cell Biol. 3, 710-716. Pepys, M.B. (1974). Role of complement in induction of antibody production in vivo. Effect of cobra venom factor and other C3 reactive reagents on thymus-dependent and thymus-independent antibody responses. J. Exp. Med. 140, 126-145.

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Podack, E.R. (1988). Assembly and structure of the membrane attack complex (MAC) of complement. In: Cytolytic Lymphocytes and Complement (Podack, E.R., Ed.), pp. 173-184. CRC Press, Boca Raton. Podack, E.R., & Muller-Eberhard, H.J. (1978). Binding of desoxycholate, phosphatidylcholine vesicles, lipoprotein and of the S-protein to complexes of terminal complement components. J. Immunol. 121,1025-1030. Podack, E.R., Preissner, K.T., & Muller-Eberhard, H.J. (1984). Inhibition of C9 polymerisation within the SC5b-9 complex of complement by S-protein. Acta Path. Microbiol. Immunol. Scand. Sec. C, (Suppl 284). 92, 89^96. Rauterberg, E.W. (1986). Demonstration of complement deposits in tissues. In: The Complement System (Rother, K., & Till, CO., Eds.), pp. 287-326. Springer, Berlin. Reid, K.B.M. (1986). Activation and control of the complement system. Essays Biochem. 22, 27-68. Reid, K.B.M., & Porter, R.R. (1981). The proteolytic activation system of complement. Ann. Rev. Biochem. 50, 433-465. Reid, K.B.M., Bentley, D.R., Campbell, R.D., Chung, L.P., Sim, R.B., Kristensen, T., & Tack, B.F. (1986). Immunol. Today 7, 230-234. Rogne, S., Mykelbost, O., Giving, J.H., Kyrkjebo, H.T., Jonassen, R., Olaisen, B., & Gedde-Dahl, T. (1991). The gene for human complement component C9 is on chromosome 5. J. Med. Genet. 28, 587-590. Ross, G.D. (1986). Opsonization and membrane complement receptors. In: Immunobiology of the Complement System (Ross, G.D., Ed.), pp. 87-114. Academic Press, New York. Rosse, W.F. (1990). Phosphatidylinositol-1 inked proteins and paroxysmal nocturnal hemoglobinuria. Blood. 75, 1595-1601. Schreiber, R.D. (1984). The chemistry and biology of complement receptors. Springer Semin. Immunopathol. 7,221-249. Schreiber, R.D., & Muller-Eberhard, H.J. (1974). Fourth component of human complement: Description of a three polypeptide chain structure. J. Exp. Med. 140, 1324—1335. Sellar, G.C., Blake, D.J., & Reid, K.B. (1991). Characterization of the genes encoding the A-, B- and C- chains of human complement subcomponent C1 q. The complete derived amino acid sequence of Clq. Biochem J. 274, 481^90. Sim, R.B., & Reid, K.B.M. (1991). CI: Molecular interactions with activating systems. Immunol. Today 12,307-311. Smith, C.A., Pangbum, M.K., Vogel, C.W., & Muller-Eberhard, H.J. (1984). Molecular architecture of human properdin, a positive regulator of the alternative pathway of human complement. J. Biol. Chem. 259,4582-4588. Takahashi, M., Takeda, J., Hirose, S. et al. (1993). Deficient biosynthesis of N-acetylglucosaminylphosphatidylinositol, the first intermediate of glycosyl phosphatidylinositol anchor biosynthesis, in cell lines established from patients with paroxysmal nocturnal hemoglobinuria. J. Exp. Med. 177,517-521. Taylor, M.E., Brickell, P.M., Craig, K.K., Summerfield, J.A. (1989). Structure and evolutionary origin of the gene encoding a human serum mannose-binding protein. Biochem. J. 262, 763—767. Taylor, P.W. (1983). Bactericidal and bacteriolytic activity of serum against gram negative bacteria. Microbiol. Rev. 47, 46-76. Taylor, P.W. (1993). Non-immunoglobulin activators of the complement system. In: Activators and Inhibitors of Complement (Sim, R.B., Ed.), pp. 37-68. Kluwer, Amsterdam. Till, G.O. (1986). Chemotactic factors. In: The Complement System (Rother, K., & Till, G.O., Eds.), pp. 354—367. Springer, Berlin. Turner, M.W. (1991). Deficiency of mannan binding protein—^a new complement deficiency syndrome. Clin. Exp. Immunol. 86(suppl.l), 53-56. Vik, D.P., Nunoz-Canoves, P., Chaplin, D.D., & Tack, B.F. (1990). Factor H. Curr. Topics Microbiol. Immunol. 153, 147-162.

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Weiler, J.M. (1987). Complement and the immune response. In: Complement in Health and Disease (Whaley, K., Ed.), pp. 289-316. MTP Press, Lancaster. Whaley, K. (1989). Measurement of complement activation in clinical practice. Complement Inflamm. 6,96-103. White, D.J.G. (1992). Transplantation of organs between species. Int. Arch. Allergy Appl. Immunol. 98, 1-5. Zalman, L.S. (1992). Homologous restriction factor. In: Current Topics in Microbiology and Immunology Vol. 178. "Membrane defenses against attack by complement and perforins" (Parker, C.J., Ed.), pp. 87-101. Springer-Verlag, Berlin. Ziccardi, R.J. (1983). The first component ofhuman complement (CI): Activation and control. Springer Semin. Immunopathol. 6, 213-231.

RECOMMENDED READINGS Morgan, B.P. (1990). Complement: Clinical aspects and relevance to disease. Academic Press, London. A monograph describing the complement system and focussing on the role of complement in disease. Law, S.K., & Reid, K.B.M. (1989). Complement in Focus. IRL Press, London. An excellent, concise summary of structural and functional aspects of the complement system (second edition may now be available). Rother, K., & Till, G.O. (1988). The Complement System. Springer-Verlag, Berlin. A multi-author text which provides an extremely detailed and complete review of all aspects of the complement system. Immunology Today (\99\). 12 (9), 291-342. A complete issue of this readable journal covering several important aspects of the complement system. Springer Seminars in Immunopathology (1994). 15 (4), 303—431. An issue dealing with various topical issues relating to the biology of the complement system.

Chapter 11

Phagocytes in Immunity and Inflammation PHILIP M. MURPHY

Introduction: General Role of Phagocytes in Homeostasis Historical Background Phagocytes and Infection Phagocytes and Immunity Phagocytes and Inflammation Phagocyte Development Phagocytes Differentiate from Totipotent Stem Cells in the Bone Marrow Mature Phagocytes Monocytes and Macrophages Specialized Functions of Phagocytes Cell Migration Phagocytosis/Opsonization Degranulation Production of Antimicrobial Oxidants

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Production of Pro-Inflammatory Mediators Immunoregulation Summary Recommended Readings

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INTRODUCTION: GENERAL ROLE OF PHAGOCYTES IN HOMEOSTASIS Historical Background The study of phagocytes in the 19^*^ century linked the phenomena of infection, inflammation and immunity, which had had quite separate histories. Prior to that time, epidemics of plague, pox, typhus, typhoid, and other scourges were usually attributed to pariahs, foreigners, or the wrath of God: contagion was visible, germs were not. Inflammation (from the Latin for "on fire") was defined by Celsus in the first century A. D. as a pathologic state characterized by redness, warmth, swelling and pain, a definition that has remained clinically useful to the present day. Inflammation was regarded correctly as a consequence of injury, but was not yet appreciated for its role in fighting infection. Immunity (from the Latin for "exemption") was the word given to describe resistance to contagion, although there was no understanding of its root causes. It was commonly recognized long before Jenner, for example, that milkmaids enjoyed unusually fair skin, because they were spared the scourges of the pox that afflicted nearly everyone else. We now know that exposure to cowpox, a viral disease of cattle that causes limited infections in humans, can protect an individual, as it did the milkmaids, from acquiring smallpox. It was only after the development of scientific methodology that such observations could be transformed through hypothesis, experiment and invention into theories of disease, enabling the intellectual histories of infection, inflammation and immunity to emerge from superstition, to become fully and mechanistically linked. The invention of the microscope by Leeuwenhoek in Holland, the development of aniline dyes in the nineteenth century by the new German chemical industry, and the invention of in vitro culture techniques by Koch and others, followed by their application to the study of pathologic specimens, led to the development of the germ theory of disease and the birth of the experimental science of microbiology, without any initial connection of infection to immunity or inflammation. The crown jewels of microbiology are Koch's postulates for determining the microbial etiology of specific diseases. Jenner, who knew nothing about germs, and later Pasteur, who shared in their discovery, subjected the phenomenon of immunity to the scrutiny of experimental test. Remarkably, the development of vaccination as a safe treatment modality, and the establishment of immunity as a scientific concept were both outcomes of the same series of experimental inoculations (Pasteur, 1880;

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Oppenheim and Potter, 1981). Jenner's simple experiment ultimately led to perhaps the greatest practical achievement of medical science: the eradication of smallpox as a threat to human health. Pasteur went further scientifically by showing that infection with microorganisms is causally linked to the development of immunity. However, the host factors responsible for the phenomenon remained elusive. "White corpuscles" were first discovered in the blood by William Hewson in the 18^*^ century (Robinson and Mangalik, 1975). In 1824, Dutrochet in France, and later Julius Cohnheim in Germany, accurately described the microscopic response of an animal to local experimental irritation (Dutrochet, 1824; Cohnheim, 1889). Adherence of leukocytes to blood vessels, and movement of both fluid and cells to the extravascular space led to local accumulation of leukocytes. Thus, leukocyteblood vessel interactions and leukocyte accumulation in the tissue spaces were proposed as the cellular bases of inflammation. The leukocytes were thought to respond to injury, but were not yet linked to host defense or immunity. In fact, leukocytes were thought by many to actually facilitate the dissemination of germs throughout the body. The next great intellectual contribution was that of Elie Metchnikoff, a Russian zoologist, who connected his experiments on the engulfment of rose thorns by the wandering phagocytes of transparent starfish larvae to Pasteur's observations on microbial pathogenesis in vertebrates. Metchnikoff s seminal proposal was that the relationship between phagocytes and microorganisms was not conspiratorial but was instead adversarial, and could explain immunity (Metchnikoff, 1905). Thus, Metchnikoff s study of phagocytes produced the theory of cell-mediated immunity that articulated the links among infection, inflammation and immunity. The work of Von Behring, Ehrlich and many others extended and corrected Metchnikoff s initial theory, showing that immunity can be the consequence of both cellular and humoral factors (Silverstein, 1979). Phagocytes are now known to lack the memory and target specificity characteristic of immunity, these being the province of lymphocytes. With this intellectual foundation firmly in place, the last 100 years have been spent in reductionist pursuit of the detailed molecular and cellular connections that exist among infection, inflammation and immunity. From the nineteenth century focus on microbes as the targets of the immune system, it is now known that non-self targets also include the cells and proteins of genetically distinct individuals (leading to serum sickness, tissue transplant rejection, and blood transfusion reactions), as well as inappropriate targets of the same individual (leading to autoimmune phenomena). Phagocytes may participate in the pathogenesis of all of these phenomena. See Table 1. Phagocytes and Infection

In order to accomplish its host defense mission, the phagocyte must quickly learn that a site of infection exists, then crawl to the site, bind to the offending cells, and finally engulf and digest them while ignoring nornial host structures. This process

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Table 1, Phagocytes and Human Disease Inflammation: diseases where the pathogenesis may be partly due to the presence of phagocytes and their products Rheumatoid arthritis Gout Neutrophilic dermatosis Myocardial infarction Inflammatory bowel disease Asthma Emphysema Vasculitis Glomerulonephritis Adult respiratory distress syndrome Dialysis-related leukoaggregation Malignant neoplasms at sites of chronic inflammation Impaired immunity: disorders of phagocytes associated with increased susceptibility to infection with pyogenic microorganisms I. Disorders of phagocyte number A. Decreased production -Acquired iatrogenic (chemotherapy, radiotherapy, drugs) nutritional infections myelophthisis drug-induced idiopathic -Congenital Cyclic neutropenia Kostmann's syndrome Reticular dysgenesis Chronic idiopathic neutropenia B. Increased destruction autoimmune neutropenia (e.g., Felty's syndrome, drug-induced) II. Disorders of phagocyte function -Acquired Association with numerous primary metabolic, neoplastic, and immunologic disorders (e.g., uremia, hypergammaglobulinemia and diabetes mellitus) Drug-induced Thermal injury -Congenital Leukocyte adhesion deficiency-1 Leukocyte adhesion deficiency-2 Neutrophil-specific granule deficiency Myeloperoxidase deficiency Chronic granulomatous disease Chediak-Higashi syndrome

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is not random but instead involves a highly regulated communication network and specific targeting events. Extracellular signaling molecules known as chemoattractants are generated at the target site by the microbe, as well as by the host in response to the microbe. As the chemoattractant diffuses out from the target site, phagocytes detect it by means of specific cell surface receptors. The spatial and temporal interaction of the receptors with the chemoattractant conveys information to the cell regarding the location of the target. In response to this information, the cell extensively remodels its cytoskeleton, and biases its otherwise random movements in the direction of higher concentrations of the chemoattractant, thereby moving towards the target. This is known as chemotaxis. Phagocytes leave the bloodstream by crawling between vascular endothelial cells, in a complex process known as diapedesis. The phagocyte recognizes and binds to the target most efficiently when it has first been decorated with molecular tags such as specific antibodies or generic complement fragments, for which it possesses additional sets of receptors. The tagging process is known as opsonization (from the Greek for "to prepare food"). The phagocyte then engulfs the bound target forming a cytoplasmic phagosome. Phagocytosis in turn triggers the delivery of antimicrobial proteins and digestive enzymes to the phagosome by fusion with cytoplasmic granules, and the production of an array of germ-killing oxidants by activation of a latent NADPH oxidase. Phagocytes and Immunity

Immunity can be operationally defined as the phenomenon whereby sublethal natural infection with a pathogenic microorganism, or exposure to its products, induces changes in the host that protect it from disease upon subsequent challenge with the same or a related organism. Vaccination is the intentional exploitation by man of this natural phenomenon. While the immune system can respond ferociously to non-microbial antigens, such as allergens or mismatched tissues, it is clear that these are evolutionary or iatrogenic sideshows to the main immunological event: defense of the host against microbial intruders. The hallmarks of the immune system—^fine target specificity, memory, and the ability to distinguish self from non-self—^are poorly-developed properties of phagocytes, but are highly-developed properties of lymphocytes. Nevertheless, the phagocytes, particularly macrophages, can distinguish normal cells from senescent cells, neoplastic cells and foreign cells. Specialized phagocytic receptors for the generic Fc portion of specific antibody molecules (FcRs), and for complement-derived opsonins, are critical determinants of target recognition by phagocytes (Ravetch and Kinet, 1991; Wright, 1992). Since they recognize many different targets indirectly by these generic adaptors, phagocytes can be regarded as performing a non-specific effector function on the efferent limb of the immune system. Nevertheless, phagocytes also serve as critical components on the afferent limb of the immune system by preparing complex microbial protein antigens for specific recognition by T lymphocytes, a process

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known as antigen presentation (Unanue and Allen, 1987). The engraftment in higher species of specific immune functions onto the more ancient scavenger functions of phagocytes over evolutionary time has produced a system of host defense and repair with greater flexibility and specificity, newly capable of identifying cryptic invaders such as viruses, and certain bacteria and protozoa that replicate inside of host cells, often inside of phagocytes themselves. Phagocytes and Inflammation Like livers and hearts, a healthy immune system usually functions imperceptibly: a silent war is waged and won against microbial challengers. When an encounter of the immune system with a microbe is clinically apparent, the condition is termed inflammation. From the perspective of antimicrobial host defense, inflammation can be regarded as an imperfection of the immune system, resulting from either an inefficient or overzealous reaction to the microbe. Inflammation occurs, in part, because many of the same oxidants, antimicrobial proteins, and degradative enzymes that damage microbes inside the protected environment of the phagosome, can also damage blood vessels, extracellular matrix and epithelial cells when they are released into the extracellular space by cellular degranulation or cytolysis. In fact, phagocytes and their products can fan the flames of an inflammatory reaction long after the microbial arson has itself been consumed. Poststreptococcal glomerulonephritis is a classic clinical example in which neutrophils contribute to the acute loss of kidney function when immune complexes become trapped in the subendothelial spaces of renal glomeruli, after the streptococcus has been eradicated from the pharynx or other local site. In addition to microbes and immunological insults, chemical and physical irritants can also trigger an inflammatory response. In these cases, inflammation can be regarded as a normal response of the immune system to acute tissue injury. Sunburn, blunt trauma, and cold-induced asthma are examples that differ dramatically in their clinical characteristics, and involve distinct effector components of the immune system. Natural inflammatory reactions may be acute and transient, or chronic and persistent; they may be systemic or localized, depending on the nature of the inciting irritant and individual host factors. Acute inflammation typically involves rapid changes in vascular smooth muscle tension and vascular permeability, as well as neutrophil accumulation and activation, all of which are induced by soluble inflammatory mediators. The complement system is a particularly important source of inflammatory mediators, since it can be activated by direct contact with immune complexes or target particles. A detailed description of the complement system can be found in Chapter 10 of this volume. Complement activation gives rise to partial cleavage products of specific complement proteins that acquire new functional properties, such as opsonization (C3b, C3bi), phagocyte chemoattraction (C5a), cytolysis (C5b-9, the membrane attack complex), and modulation of vascular permeability and vascular smooth muscle

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tension (C3a, C5a, the complement-derived anaphylatoxins). Acute inflammatory lesions can also occur without local accumulation of phagocytes, highlighting the importance of inflammatory mediators and vascular changes in inflammation (Ognibene et al., 1986). Chronic inflammation involves delayed and persistent tissue infiltration by macrophages and/or lymphocytes. Fibroblast and capillary proliferation and increased production of extracellular matrix proteins are also found. The granuloma is a classic histopathological lesion of chronic inflammation composed of macrophages and fibroblasts that often forms when macrophages cannot degrade an ingested particle. Intradermal antigenic challenges of sensitized subjects in vivo have been important in linking specific effector components of the immune system with specific types of inflammatory pathology and specific classes of antigens. By the turn of the century the Arthus reaction (IgG-immune complexes and neutrophils), immediate type hypersensitivity reactions (IgE and basophils), and delayed type hypersensitivity reactions (T lymphocytes) had been established as important experimental models of immunologically-mediated inflammatory reactions (Cohen etal., 1979).

PHAGOCYTE DEVELOPMENT Phagocytes Differentiate from Totipotent Stem Cells in the Bone Marrow

The development of phagocytes can be divided into mitotic and non-mitotic stages, lineage-committed and non-committed stages, and bone marrow, blood and tissue stages. Extensive overlap exists for these classifications (Figure 1). Thus, the mitotic stage occurs in two distinct operationally defined compartments of the bone marrow: the stem cell compartment, in which lineage decisions have not yet been made, and the progenitor cell compartment, in which the lineage of individual cells is defined. Furthermore, progenitor cells and possibly even stem cells can also be found in the peripheral blood. All of the formed elements of the blood, namely erythrocytes, leukocytes and platelets, are thought to arise from the proliferation and differentiation of single totipotent stem cells in the bone marrow. The first evidence for this idea came from experiments in which lethally irradiated mice were rescued by transfusion with very small numbers of syngeneic undifferentiated bone marrow-derived cells (Spangrude et al., 1988). Reconstitution has been achieved with as few as thirty donorderived cells. Given experimental inefficiencies, these experiments have been interpreted to mean that a single totipotent stem cell can fully reconstitute all cell lineages of the blood. Since it is impossible to completely ablate the recipient marrow without killing the animal by damaging other less radiosensitive organs, it has so far not been possible to rigorously define the very earliest totipotent stem cell. Nor has it been established whether the earliest stem cells are capable of

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self-renewal, or instead are selected for differentiation by stochastic processes from a cohort of identical totipotent cells that becomes depleted over time, much like gonadal oocytes. Bone marrow stromal cells contribute to a hematopoietic inductive microenvironment that may also play a role in lineage commitment. Cyclic neutropenia is an autosomal recessive condition in which the number of cells in all of the blood cell lineages decreases in a periodic manner owing to a transient maturation arrest (Wright et al., 1981). The period of each cycle is -21 days. Circulating neutrophil numbers are the most seriously affected because of their short half-life, and can be reduced to zero. Thus, the clinical features of the disease—periodic infections—^are determined by the degree of neutropenia. Transplantation studies in a natural animal model of cyclic neutropenia, the gray collie dog, have indicated that the defect is cellular and not humoral and that it probably lies in an early hematopoietic progenitor cell, possibly the totipotent stem cell. The rarity of the human disease and the lack of fortuitous cytogenetic abnormalities has impeded progress in defining the molecular genetics. While the stem cell compartment is defined by in vivo reconstitution experiments in lethally irradiated animals, lineage-specific progenitor cells are defined by panels of monoclonal antibodies for specific cell surface markers and by the ability to give rise to colonies of mature, morphologically identifiable blood cell lineages in vitro. The progenitor cells are designated colony forming unit-X (CFU-X), where X designates the lineage that arises. Three major cell lineage commitments occur early on: the erythroid lineage which leads to the production of red blood cells, the lymphoid lineage which leads to the production of T and B lymphocytes, and the myeloid lineage which leads to the production of granulocytes (neutrophils, eosinophils and basophils), monocytes and megakaryocytes. The molecular basis of lineage commitment is poorly understood, but appears to involve the expression of receptors for lineage-specific peptide growth factors that act at specific stages of differentiation (Golde and Gasson, 1988; Dexter et al., 1990). These include the colony-stimulating factors (CSF) for the myeloid series, Granulocyte-CSF (GCSF), Macrophage-CSF (MCSF), Granulocyte/Macrophage-CSF (GMCSF), and interleukin 3 (IL-3; granulocyte, macrophage, erythroid and mast cell colony-sfimulating factor). GMCSF and GCSF are multifunctional molecules that also potentiate certain functional properties of mature granulocytes. GMCSF and GCSF have important pharmacological properties and are clinically useful in restoring the neutrophil count to safe levels after cytoreductive chemotherapy (Groopman et al., 1989). Mature Phagocytes

The myeloid differentiation pathway is the most kinetically active, largely due to the rapid turnover of neutrophils, the most numerous cells of the myeloid lineage (Table 2). Two-thirds of bone marrow precursor cells can be shown to bear markers characterisfic of the myeloid lineage (Robinson and Mangalik, 1975).

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PHILIP M . M U R P H Y Table 2.

Differential Features of Phagocytes Phagocyte

Feature Specific growth factor Life-span Mitotic Nucleus ~% of blood leukocytes; Staining property Inflammation Microbial target Surface marker Granule marker Selective activator Phagocytosis Degranulation Antigen presentation Oxidant production ADCC Lipid mediator Cytokine production

Granulocyte

Monocyte/ Macrophage M-CSF^ months-years yes reniform 15% neutral chronic bacteria, fungi CD14 non-specific esterase MCP-1

++++

+ + + +

Neutrophil G-CSF days no multilobed 45% neutral acute bacteria, fungi

—

lactoferrin IL-8

+++ +

IL-5 months no

bilobed 5% acidic acute/chronic helminths FCgRII MBP, ECP, EDN, CLC, EPO IL-5

+ +-I-+

-

-

LTB4

LTB4

LTC4

++++

+

?

+ +

Basophil^

Eosinophil

+ +

IL-4 ?

no cleft 0.5% basic allergic ?

Fc,RI histamine allergen

-

++++

-

LTC4 ?

Notes: ^ Basophils are not phagocytic, but arise from a proximal progenitor cell, and have features in common with other granulocytes. Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; CSF, colony-stimulating factor; M, macrophage; G, granulocyte; IL, interleukin;CD, cluster designation; LT, leukotriene;MCP, monocyte chemoattractant protein; MBP, major basic protein; ECP, eosinophil cationic protein; EDN, eosinophil-derived neurotoxin; CLC, Charcot-Leyden crystal protein; EPO, eosinophil peroxidase.

Neutrophils, eosinophils and basophils are terminally differentiated granulocytes that can be distinguished by morphology and function. All have multi-lobed nuclei and conspicuous granules. Differences in granule content and in specialized surface receptors account in large part for the distinct functional properties of these cells. Neutrophils

The neutrophil has a high capacity for motility, phagocytosis and microbial killing. Sixty percent of nucleated cells in the bone marrow belong to the neutrophil series (Robinson and Mangalik, 1975). The mitotically active precursors of the neutrophil are the myeloblast (2%), promyelocyte (5%), and myelocyte (12%). The mitotic phase of neutrophil development lasts -7.5 days in the marrow. The post-mitotic phase lasts 6.5 days and includes the metamyelocyte and band forms (22%), and finally mature neutrophils (20%). Approximately 10'^ mature neutrophils are produced by the marrow each day. They do not return to the marrow. The

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marrow contains a large reserve of neutrophils that can be rapidly mobilized by pro-inflammatory stimuli. The blood pool is divided equally between a pool of circulating cells and a pool of cells that are adherent to endothelial cells in the microvasculature, commonly known as the marginated pool. Neutrophils in the marginated pool can be induced to demarginate by stress, exercise, glucocorticoids or epinephrine. In contrast, bacterial lipopolysaccharide, a potent pro-inflammatory component of the outer membrane of Gram negative bacteria that is also known as endotoxin, increases margination, transiently decreasing the total circulating neutrophil concentration. Later an increase in circulating neutrophils is observed in response to stimulation by endotoxin of neutrophil release from the marrow reserves. Glucocorticoids have a similar effect on the marrow reserves, but they further increase the circulating pool by decreasing both the marginated pool and diapedesis into the tissues. Neutrophil kinetics during challenges with bacterial endotoxin are similar to those that are observed during septic shock. After circulating for ~8 hours in the peripheral blood, neutrophils enter the tissue pool where they survive for 1 to 2 days. They are then cleared from the tissues in the spleen, gut and other sites. Abnormalities in the circulating concentration of neutrophils are common. Neutrophilia is a normal response to infection. Neutropenia is most commonly an acquired condition, especially as a consequence of cytoreductive chemotherapy or drug toxicity. Regardless of etiology, the risk of infection is particularly great when the circulating concentration of neutrophils is sustained below 500 cells/|Lil. In the few reported cases of inherited neutropenia, deficiencies of other blood cell types are also present. Kostmannn's syndrome is a particularly interesting autosomal recessive condition characterized by severe neutropenia. The bone marrow has normal cellularity, but has few or no myelocytes. Patients with Kostmann's syndrome typically die from overwhelming bacterial infections by 6 months of age (Kostmann, 1975). Eosinophils

Eosinophils have a characteristic bilobed nucleus and possess granules that stain red with the acid aniline dye eosin. Eosinophil and neutrophil precursors can be distinguished as early as the promyelocyte stage. Eosinophils represent only - 5 % of circulating leukocytes. Unlike neutrophils, they can live for weeks in the tissues. They are especially numerous at mucosal surfaces, such as the gut and respiratory tract. Eosinophil kinetics differ significantly from those of neutrophils in three respects: (1) unlike neutrophils, eosinophils can be found in the thoracic duct lymph and in lymph nodes, (2) unlike neutrophils, glucocorticoids, epinephrine and stress all decrease the circulating concentrations of eosinophils, and (3) unlike neutrophils, increased production of eosinophils is regulated in large part by the T cell cytokine IL-5. IL-3, IL-5, and GMCSF have important effects on both eosinophil development and functional activation. Of these three cytokines, only IL-5 is

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specific in its actions for eosinophils (Weller, 1991). The plasma membrane, the sensing surface of the cell, contains many of the same chemoattractant and phagocytic receptors in eosinophils and neutrophils, although the relative abundance of individual receptors may differ substantially. The eosinophil appears to be an important effector element in host defense against helminths (Butterworth, 1984; Weller, 1991). Natural helminth infections are associated with eosinophilia. Moreover, eosinophils are able to kill helminths in vitro, and rodents experimentally depleted of eosinophils are highly susceptible to helminth infections in vivo. High plasma levels of IgE are also characteristic of helminth infections. Although this finding is not fully understood, specific antiparasite IgE may serve as a physiologically important opsonin able to bind helminths to special IgE receptors on the surface of eosinophils (Capron et al., 1984). Blood eosinophilia and eosinophilic tissue infiltrates are also frequently found in allergic inflammation, and certain forms of malignancy. The Charcot-Leyden crystal is a hallmark of eosinophil-related disease. It is composed of a lysophospholipase from eosinophil granule and plasma membranes, and is most commonly found in sputum from patients with allergic asthma (Weller et al., 1980). Eosinophils and their products have been strongly linked to airway hyperreactivity, and increased mucus secretion in allergic asthma (Durham et al., 1989). Basophils Unlike other leukocytes of the myeloid lineage, basophils lack FCyRs and are unable to efficiently phagocytize particles. Basophils have a segmented nucleus and large cytoplasmic granules rich in histamine that stain purplish-red with blue aniline dyes, a property known as metachromasia. They have many structural and functional similarities with tissue mast cells (Metcalfe et al., 1992). Both cell types develop from hematopoietic stem cells, and both participate in immediate type hypersensitivity reactions. With the exception of the stomach and the central nervous system, blood basophils and tissue mast cells account for all of the histamine that is found in the blood and tissue. Basophils express high affinity receptors for IgE (FCgRI) that differ from the low affinity FCgR on eosinophils (FCgRII). FCgRI mediates basophil activation when it is cross-linked by IgE molecules bound to allergens (Ravetch and Kinet, 1991). Allergens are typically proteinaceous; polysaccharide antigens are poor allergens. After activation by allergen, basophils release preformed histamine from granules and newly synthesize a variety of inflammatory mediators such as leukotrienes, prostaglandins, and platelet-activating factor. Only a few FCgRs need to be cross-linked to obtain maximal amounts of histamine release. Basophils represent a small fraction (-0.5%) of circulating leukocytes yet play a paradoxically large role in human disease, owing to the potent vasoactive properties of histamine. Clinical examples of diseases in which basophils play an

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important role in pathogenesis include asthma, urticaria, anaphylaxis, and rhinitis. While their involvement in the pathogenesis of allergic inflammation is clearcut, the restorative and defensive roles of basophils have not yet been clearly identified. Studies with rodents have implicated basophils in host defense against ectoparasites such as ticks, although convincing data are lacking in humans (Brown et al., 1982). Allergy may be a disadvantageous remnant of the immune system that originally evolved to control parasites that may no longer confront the host because of extinction, or an altered host tropism. Thus, the basophil acts as an endogenous pathogen in allergic subjects. Monocytes and Macrophages

The promonocyte is the first morphologically identifiable cell of the mononuclear phagocyte system. Monocytes circulate in the blood, then enter and take up residence in the tissues where they differentiate into macrophages. Unlike granulocytes, mature cells of the mononuclear phagocyte system are capable of cell division, long life, and further differentiation in the tissues where they acquire generic, as well as tissue-specific functions (Adams and Hamilton, 1992). During differentiation, the cell changes dramatically: it more than doubles in size, increases its content of mitochondria, granules, and granule enzymes, and may acquire increased phagocytic and microbicidal capacity. Tissue macrophages (also known as histiocytes) may be adherent or free. Examples of adherent macrophages are Kupffer cells of the liver, osteoclasts, Langerhans cells of the skin, pulmonary alveolar macrophages, microglial cells of the brain, splenic macrophages, and multinucleated giant cells in granulomata. Examples of free macrophages are the fluid phase macrophages found in the peritoneal, pleural, and synovial spaces. There are structural and functional differences among macrophages from different sites. Together with the neutrophil, monocytes and macrophages are the major effector cells for killing bacteria and fungi. However, certain bacteria, such as the mycobacteria and Listeria monocytogenes, and certain protozoa, such as Leishmania spp. and Toxoplasma gondii, have evolved mechanisms for eluding the killing mechanisms of macrophages, and are in fact obligate intracellular pathogens of resting macrophages (Densen and Mandell, 1980; Cohn, 1986) (Figure 2). The killing capacity of the macrophage for these organisms can be increased dramatically by stimulation of the cell with interferon y, which markedly increases the ability of the cell to produce anti-microbial oxidants that can lethally damage the cell wall and other microbial structures (Nathan, 1987). Macrophages also kill tumor cells in vitro, although the physiologic importance of this function has yet to be convincingly demonstrated in vivo. Macrophages are also involved in the clearance of senescent cells and in detoxification of endogenous and exogenous wastes. They accumulate at sites of devitalized tissue (necrotaxis), and are involved in tissue remodeling and wound healing. In addition to their killing, scavenging and repara-

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tive actions, macrophages are the source of a wide range of soluble mediators that regulate the function of endothelial cells, lymphocytes and other phagocytes (Nathan, 1987). Macrophages or their products also regulate hematopoiesis, antigen presentation, coagulation, and lipid and iron metabolism (Adams and Hamilton, 1992). The diversity of macrophage functions is reflected in the complexity of its sensing surface, the plasma membrane, which deploys receptors for a phenomenal number of extracellular signalling molecules, including neurotransmitters, hormones, chemoattractants, immunoglobulins, adhesion molecules, carbohydrates, opiates, and cytokines.

SPECIALIZED FUNCTIONS OF PHAGOCYTES In the blood, phagocyte gene transcription, protein synthesis and oxygen consumption are all at very low levels: the cell circulates essentially as a quiescent sphere. Anti-microbial proteins and enzymes that were synthesized during maturation in the bone marrow are in a latent state in intracellular granules. The phagocyte's trigger is cocked; a chemoattractant or opsonized microbe pulls it. Nevertheless, unlike a gun that is fired, phagocyte activation is not a monotonous, uniform process, but instead is a complex web of metabolic changes that may occur in parallel or in series, that may vary in magnitude, and that can be dissociated from one another depending upon the identity and concentration of the stimulus. In general, however, phagocyte activation involves dramatic changes to cell membranes: granules fuse; pseudopods, phagosomes, and phagolysosomes are formed; and the plasma membrane becomes stickier. The cell becomes highly elongated in shape and highly polarized. Associated biochemical changes are equally dramatic with new production and release of oxidants and autacoids, mobilization of calcium, cytoskeletal remodeling, increased oxygen consumption, increased glucose metabolism, and increased lipid turnover. The cell biology of mature phagocytes has largely involved the study of their idiosyncratic functions, namely cell migration, phagocytosis, anti-microbial killing (degranulation and oxidant production), autocrine and paracrine regulation of the inflammatory response, and antigen presentation. Cell Migration

The study of cell motility in vitro has shown that the cell may undergo random displacements whose rate may be increased by extracellular stimuli, a process known as chemokinesis. If the stimulated displacement has a directional bias, it is then referred to as chemotaxis. When the extracellular stimulus is not soluble, but is instead a molecule bound to a surface, directed movement is referred to as haptotaxis. The relative contribution of random movement, chemokinesis, chemotaxis, and haptotaxis to the migration of cells in multicellular organisms in vivo is unknown. Directed movement of cells is the basis for physiologic processes as

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PHILIP M. MURPHY

diverse as nutrient acquisition by bacteria, stalk formation by slime molds, yeast mating, oocyte fertilization by spermatocytes, embryonal pattern formation, wound healing, tumor metastasis and pus formation. Prokaryotic cells and spermatocytes are propelled through an aqueous environment by extracellular appendages (cilia or flagella). In contrast, non-spermatocyte eukaryotic cells do not swim but instead crawl along surfaces by concerted movement of the plasma membrane and reorganization of the cytoskeleton (Stossel, 1993). The environment that is traversed, the type of molecular motor and the sensitivity of the cell to chemoattractants all greatly influence the nature and velocity of cell movement. The rapid, ameboid movements of phagocytes in response to chemoattractants enable them to serve as the first line of cell-mediated host defense against infection. The molecular basis of phagocyte activation has been the subject of intense study over the past 10 years. Greater than twenty different phagocyte chemoattractants Table 3.

Phagocyte C h e m o a t t r a c t a n t s

Chemoattractants and Secretagogues N-formyl peptides C5a Leukotriene B4 Platelet-activating factor Thromboxane B2 Histamine Pure Chemoattractants Substance P Transforming growth factor p^ lnterleukin-5 Chemokines (chemoattractant and cytokine activityf lnterleukin-8 (neutrophil-activating peptide 1) Neutrophil-activating peptide 2

GROa GROp GROy Epithelial-derived neutrophil activator-78 Y interferon-inducible protein-10 Macrophage inflammatory protein-1a Macrophage inflammatory protein-ip RANTES Monocyte chemoattractant protein-1 Monocyte chemoattractant protein-2 Monocyte chemoattractant protein-3 Note:

^ Chemokines are structurally and functionally related 70-78 amino acid peptides encoded by distinct genes on human chromosomes 4 and 17. A detailed description of the sources, cell targets, receptors and actions of chemoattractants can be found in O p p e n h e i m e t a l . , 1991 and Murphy, 1994.

213

Phagocytes in Immunity and Inflammation

have been purified (Table 3). Known phagocyte chemoattractants are mostly peptides or lipids. While different types of phagocytes respond to many of the same chemoattractants (e.g. C5a), cell-type specific chemoattractants are also known (e.g., histamine and IL-5 for eosinophils, and IL-8 for neutrophils) (Weller, 1991; Oppenheim et al., 1991). Many but not all of these molecules also activate other phagocyte functions such as degranulation (secretion) and oxidant production. With few exceptions (IL-5, transforming growth factor pi), phagocyte chemoattractants bind to structurally related receptors with seven predicted plasma membrane-spanning helices, that are functionally coupled to heterotrimeric GTP-binding proteins (G proteins) (Figure 3). Molecular cloning studies have shown that the G protein-coupled chemoattractant receptors are related to G protein-coupled neurotransmitter, hormone, and odorant receptors, and to the

Extracellular Milieu

Figure 3, Model for activation of phagocytes by chemoattractants. An extracellular ligand, IL-8 for example, binds to a G protein-coupled receptor with seven transmembrane helices, causing activation of the coupled G protein. Py subunits activate membrane phospholipases resulting in lipid remodeling, second messenger production, and mobilization of calcium stores from calciosomes. These events presumably activate other intracellular signalling molecules (black box) leading to the activation of specialized phagocyte functions. See text for details. C, C-terminus; N, N-terminus; 1-7, proposed transmembrane helices of the chemoattractant receptor; - , negative charge of the N-terminal segment of the IL-8 receptor; ++, IL-8 is a basic polypeptide; PIP2, phosphatidylinositol bisphosphate; IP3, inositol-1,4,5-trisphosphate; IP3R, IP3 receptor; DAG, diacylglycerol; PLC, phospholipase C; PLD, phospholipase D; PC, phosphatidylcholine; PA, phosphatidic acid; PPH, phosphatidate phosphohydrolase; PKC, protein kinase C.

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PHILIP M. MURPHY

opsins that mediate light reception. In some cases, a single receptor can specifically bind several different chemoattractants, and in other cases one chemoattractant can bind to several different receptors (Murphy, 1994). While specialized roles have been identified for certain chemoattractants, functional redundancy is a more prominent feature of the chemoattractants, perhaps serving to secure the whole system against inactivating mutations in any single component. Surprisingly, functional homologues of the IL-8 and the MIP-la/RANTES chemoattractant receptors have been found in Herpesvirus saimiri, a. transforming T lymphotropic primate virus, and human cytomegalovirus, respectively. Moreover, the Duffy red cell antigen that mediates invasion of red cells by the malaria parasite Plasmodium vivax, is also an IL-8 and RANTES binding protein. Thus, the chemoattractant receptors may have been turned to the host's disadvantage by these pathogens through molecular mimicry (reviewed in Murphy, 1994). The activated chemoattractant receptor catalyzes the exchange of GDP for GTP by the G protein, resulting in G protein activation. Many distinct G protein subtypes have been characterized. However, chemoattractant receptors probably are coupled to only a few of these. G^ appears to play a particularly prominent role. Upon activation, the Py subunit of the G protein dissociates from the a subunit. Py activates a phosphatidylinositol-specific phospholipase C, probably the P2 isoform, which catalyzes the hydrolysis of phosphatidylinositol bisphosphate, forming inositol-1, 4, 5-trisphosphate (IP3) and diacylglycerol (DAG). These molecules serve as intracellular second messengers: IP3 stimulates the release of stored calcium from structures known as calciosomes resulting in a rapid elevation of intracellular Ca^"^ concentration; DAG activates protein kinase C, which may regulate critical downstream signalling molecules by phosphorylation. A delayed but more abundant source of DAG is membrane phosphatidylcholine which is hydrolyzed by a phospholipase D. Early PLC- and late PLD-activation events have been associated with the migratory and cytotoxic responses of phagocytes, respectively (reviewed in Snyderman and Ewing, 1992). Phagocytes are incredibly sensitive to chemoattractant gradients: they are capable of sensing a gradient of less than 1% across the cell diameter (Zigmond, 1978). Consequently, the cell sends out a thin veil of membrane known as Sipseudopodium (or lamellipodium), which is highly ruffled. Cytoplasm then streams forward into this process bringing with it cytoplasmic granules which may fuse with the leading edge of the plasma membrane, thereby releasing proteolytic enzymes into the extracellular milieu. The formerly spherical cell thus becomes highly polarized; granules segregate towards the leading edge, whereas the nucleus is at the trailing edge or uropod (Snyderman and Goetzl, 1981; Stossel, 1993) (Figure 4). Paul Ehrlich first suggested that the release of granule contents during diapedesis could facilitate the movement of the phagocyte into the tissue (Ehrlich, 1900). It is now known that the released enzymes include elastase and coUagenase, which can

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degrade extracellular matrix proteins that form barriers to phagocyte movement (Janoff, 1985; Weiss, 1989; Henson et al, 1992). As first detailed by Julius Cohnheim in the nineteenth century, transendothelial migration of leukocytes (diapedesis) occurs in the post-capillary venules via specific leukocyte-endothelial cell contacts (Cohnheim, 1889) (Figure 4). Leukocytes in the marginated pool first make weak, reversible contacts that mediate rolling of the cell along inflamed endothelium. IL-1, bacterial endotoxin, and tumor necrosis factor are examples of stimuli that can promote the rolling phenomenon by activating endothelial cells. Dramatic advances in the molecular and cell biology of leukocyte adhesion have occurred in the last ten years, pioneered by the work of Springer, Weissman, Lasky, and their colleagues. Rolling is now known to be mediated by adhesive molecules known as selectins that are expressed on the plasma membrane of both leukocytes and activated endothelial cells, and that bind to specific sialylated, fucosylated carbohydrate counter-receptors expressed on the target cell surface (Lasky and Rosen, 1992). At least one of the natural ligands appears to be the sialyl Lewis^ blood group antigen. The selectins form a protein family with N-terminal lectin-like domains, complement domains and epidermal growth factor-like domains. E-selectin is expressed only on endothelial cells and requires de novo synthesis after activation. In contrast, P-selectin, which is also produced by platelets, is pre-synthesized and stored. L-selectin, is produced by leukocytes. Phaqocyte-Endothelium Interaction

Stage

Activator

Adhesins

Rolling

IL-1,TNF,lps

Selectins-CHO LAD-2 Cou nter-receptors

Firm Adhesion

Chemoattractant

lntegrins-ICAi\/ls

l-AD-1

Transendothelial Migration

Chemoattractant

PECAM

LAD-1, LAD-2

Degranulation

Chemoattractant

none

SGD

Disease

Intravascular Space

Extravascular Space

Figure 4. Model for the movement of phagocytes from the blood to the tissues. Ips, bacteria! lipopolysaccharlde; TNF, tumor necrosis factor; IL, interleukin; CHO, carbohydrate; ICAM, Intercellular adhesion molecule; PECAM, platelet-endothellal cell adhesion molecule; LAD, leukocyte adhesion deficiency; SGD, specific granule deficiency.

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PHILIP M. MURPHY

Selectin-mediated binding may be necessary for diapedesis, but it is not sufficient. A second stimulus directed at the leukocyte appears to be required to stimulate increased expression and functional activation of a second class of adhesion molecules known as the leukocyte integrins, a subset of the integrin superfamily of adhesion molecules (Springer, 1990). The leukocyte integrins are Mac-1, pi50,95 and LFA-1 which have distinct expression patterns on different leukocytes and mediate a broad array of adhesive functions, such as homotypic (leukocyte to leukocyte) and heterotypic (leukocyte to endothelium) aggregation, spreading on surfaces, and binding to opsonized particles. Mac-1 and LFA-1 bind tightly to endothelial cell counter-receptors known as intercellular adhesion molecules 1 and 2 (ICAM-1 and ICAM-2). The best candidates for the second stimulus for leukocyte-endothelial cell binding by integrins are chemoattractants, such as platelet-activating factor. After integrins become activated and bind to the endothelial counter-receptors, the spherical leukocytes stop rolling, rapidly flatten and begin to crawl between adjacent endothelial cells to arrive in the subendothelial space. The three leukocyte integrins are membrane heterodimers that share a common P subunit (CD 18) that is non-covalently associated with related but distinct a subunits (CDl la, b, and c) (Kishimoto and Anderson, 1992). Their endothelial cell counter-receptors, the ICAMs, are members of the immunoglobulin superfamily of proteins. Two experiments of nature, the human diseases designated leukocyte adhesion deficiency 1 and 2 (LAD-1 and LAD-2), have highlighted the importance of the integrins and the selectins, respectively, in the normal function of the immune system. Patients with LAD-1 have a mutation in the gene for the shared (3 subunit of Mac-1, pi50,95 and LFA-1 integrins that is transmitted as an autosomal recessive trait on chromosome 21. As a consequence, leukocytes from patients with LAD-1 fail to express functional heterodimeric leukocyte integrins (Springer et al., 1984; Anderson and Springer, 1987). Predictably, LAD-1 leukocytes fail to adhere to endothelial cells in vitro and in vivo, and therefore have a defect in transendothelial migration and fail to accumulate at sites of infection and inflammation. Moreover, phagocytes from patients with LAD-1 do not recognize C3bi-coated particles. The clinical expression of the disease is recurrent, life-threatening bacterial and fungal infections in children, often presenting in the neonatal period. Blood from uninfected patients with LAD-1 exhibits a sustained and marked leukocytosis, due to marked reduction in the size of the marginated pool. Leukocyte concentrations in the blood can elevate even ftarther during infectious episodes. Because of the inability of leukocytes to cross endothelial barriers, infected sites have delayed or insufficient inflammation (infection without pus), which often leads to life-threatening delays in diagnosis. There may be a history in patients with LAD-1 of impaired wound healing, often manifested as delayed separation of the umbilical cord after birth. In a reciprocal clinical condition, sera from patients with homozy-

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gous deficiency of C3 lack C3-dependent opsonic activity. As in LAD-1, C3 deficiency also results in recurrent infections with pyogenic micro-organisms. Only two patients have been described with LAD-2, both of whom were Arab children living in Israel who presented with severe bacterial infections. There was no clear history of parental consanguinity. Leukocytes from the patients expressed normal numbers of leukocyte integrins but failed to adhere properly to surfaces in vitro and in vivo. Both patients had deletions on chromosome 19, mental retardation and the rare Bombay blood group phenotype. The molecular defect appears to be in a fucosyl transferase responsible for the construction of the sialyl Lewis^ counter-receptor for the endothelial cell selectins. This enzyme is also required for formation of the Bombay blood group. Although it is theoretically possible to cure the adhesion defect by feeding the patients fucose, the new antigens thus formed may be recognized as foreign by the patients' immune system (Etzioni et al., 1993). The identification of the molecular basis of leukocyte adhesive phenomena has opened the door to the possibility of developing specific anti-adhesive treatments for acute and chronic inflammatory diseases, and for vascular occlusive phenomena, such as myocardial infarction, where phagocytes and their products may participate in pathogenesis. Phagocytosis/Opsonization

Phagocytosis involves the recognition, binding and internalization of particles (Stossel, 1974; Wright, 1992). While most cells can phagocytize particles to a limited extent, neutrophils and macrophages possess a high capacity for phagocytosis because of the expression of specialized receptors. Classical opsonic attachment occurs via receptors for antibody and the complement-derived opsonins C3b and C3bi (Ravetch and Kinet, 1991; Wright, 1992). Classical opsonization is particularly important for the phagocytosis of encapsulated bacteria such as Streptococcus pneumoniae. Two other types of attachment of phagocytes to microbes have been delineated: non-opsonic attachment of the integrins CR3, pi 50,95, and LFA-1 to bacterial lipopolysaccharide; and non-classical opsonic attachment via receptors for the plasma-derived opsonins mannose binding protein (MBP) and lipopolysaccharide-binding protein (LBP) (Wright, 1992) (Figure 5). Fewer binding sites are required for attachment of particles to phagocytes than for their ingestion. Complement receptors may be most responsible for binding, whereas FCyRs may be most responsible for internalization (Mantovani, 1975). The cell biology of phagocytosis can be conceptualized as localized cell motility, involving localized cytoskeletal remodeling. As the particle binds to and deforms the plasma membrane, oriented microfilaments surround the particle, and cytoplasm streams forward entraining granules that may fuse with the nascent phagosome. Formation of the phagosome requires energy, but can occur under anaerobic conditions. To form the phagosome, the plasma membrane fuses to itself, resulting in an inside-out membrane configuration.

218

PHILIP M. MURPHY

p150,95

Non-opsonic Attachment

CR3

Classical Opsonization

Non-classical Opsonization

\P\h^^®©^^(B Figure 5. Molecular apparatus for microbial attachment to phagocytes. Hair-like projections from the microbe represent bacterial llpopolysaccharlde. Ag, antigen; C3b, C3bi are complement-derived opsonins; LBP, lipopolysaccharide-binding protein; MBP, mannose binding protein; Man R, mannose receptor; CR, complement receptor; FcR, immunoglobulin Fc receptor (multiple subtypes for IgG and IgE); LFA, leukocyte function associated. Note that CR3 can bind directly to llpopolysaccharlde or to C3bi.

Three antigenically distinct Fcy receptors have been characterized on phagocytes. is a high affinity receptor (Kd~10-^M) for IgG v^hereas FCyRll and FCYRIII are low affinity receptors for IgG (K^-KT^M). Fc^Rs I and II are expressed on macrophages, whereas Fc^Rs II and III are expressed on neutrophils. Unexpected complexity has been identified in this system by molecular cloning of multiple distinct genes encoding additional Fc^R subtypes (Ravetch and Kinet, 1991). C3b binds to complement receptor type I (CRl), whereas C3bi binds to complement receptor type 3 (CR3). CR3 is identical to the Mac-1 integrin which as we have already seen is able to serve an alternative adhesive function, binding to ICAM-1 during transendothelial migration (Kishimoto and Anderson, 1992). Thus CR3 mediates both diapedesis and phagocytosis, illustrating at the molecular level the relationship between cell migration and phagocytosis. Adhesive and phagocytic ligands appear to bind to distinct sites on CR3. The phagocytic receptors explain the ability of phagocytes to target microorganisms, but less well their ability to distinguish normal from senescent, damaged and neoplastic host cells. Targetting may occur by additional adhesion systems that FCYRI

Phagocytes in Immunity and Inflammation

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involve the recognition of new carbohydrate structures or oxidized proteins on the surface of abnormal host cells by molecules such as the macrophage scavenger receptors or the mannose receptor (Stahl, 1990). Neutrophils are more phagocytic than monocytes, although the converse is true when the particles or cells are particularly large or numerous. Degranulation

One of the features that distinguishes phagocytes from lymphocytes is the abundance of granules packed into the cytoplasm. The granules contain the microbicidal weaponry of the cell that acts in the confines of the phagolysosome, but that also has the potential to damage macromolecules present on adjacent host cells (Gallin, 1984; Henson et al, 1992). In 1900, Paul Ehrlich proposed that neutrophil granules may contribute to inflammation by secreting their contents into the extracellular space. This hypothesis was examined by the work of Weissmann and his coworkers at New York University 70 years later, and has been extensively supported from the biochemical characterization of granule components (Table 4). To convey the destructive potential of their granules, Weissmann and others have referred to phagocytes as "secretory organs of inflammation" (Weissmann et al., 1973). Today the products of phagocyte granules have been implicated in the pathology of gout, rheumatoid arthritis, vasculitis, inflammatory bowel disease, a wide variety of dermatopathic disorders, periodontitis, myocardial infarction, asthma, emphysema, and the adult respiratory distress syndrome, among other human diseases (Malech and Gallin, 1987) (Table 1). In addition, granule proteins catalyze the production of anti-microbial oxidants that, in vitro, have been impliTable 4. Microbicidal and Degradative Proteins Contained in Neutrophil and Eosinophil Granules^ Microbicidal Granule

Oxidant

Production

Proteins Cationic

_ Proteins

Neutrophil Specific

flavocytochrome 6553 (O2) apolactoferrin (OH-) Azurophilic ; myeloperoxidase (HOC1)

defensins bactericidal/permeabilityincreasing protein (BPI)

Tertiary Eosinophil

Specific

eo-peroxidase (OH-) flavocytochrome bsss (O2)

Note:

eo-derived neurotoxin eo-cationic protein major basic protein

Degradative Enzymes

lysozyme collagenase acid hydrolases lysozyme elastase neutral proteases gelatinase acid hydrolases

^ Specific granules also contain chemoattractant receptors and integrins that are brought to the plasma membrane upon cell activation.

220

PHILIP M. MURPHY

cated in DNA damage and cellular transformation (Weitzman et al., 1985). This could explain the high incidence of carcinoma associated with chronic inflammation in vivo, as occurs in patients with chronic hepatitis B, ulcerative colitis, and scars from old tuberculous lung lesions. In addition to modifying the inflammatory site, degranulation adds new membrane and fresh supplies of chemoattractant receptors and phagocytic/adhesion receptors from storage pools in the granule to the leading or sensing edge of the cell, thereby recharging the sensing and effector pathways for phagocyte migration (Wright and Gallin, 1978; Henson et al., 1992). Exocytosis of granule contents may also be important for killing of helminths by eosinophils (Weller, 1991) (Figure 2). Finally, detonation of basophils during allergic reactions depends on rapid and massive fusion of granule membrane to plasma membrane (Metcalfe et al., 1992). Granule fusion appears to be regulated in part by calcium, calcium-binding proteins such as the synexins, GTP, low molecular weight GTP-binding proteins, and cytoskeletal remodeling by proteins such as gelsolin. Shortening of actin filaments by gelsolin may enhance the cytoplasmic motility of granules, permitting them to approach and fuse with other membrane-delimited compartments (Henson etal., 1992;Stossel, 1993). Subcellular fractionation techniques pioneered by deDuve, Palade and their collaborators at Rockefeller University in the 1950s and 1960s were applied to phagocytes, and led to rapid advances in understanding of the structure and biochemical composition of phagocytic cell granules. It is now known that neutrophils possess at least three types of granules (Baggiolini et al., 1969; Henson et al., 1992). The primary, or azurophil, granules appear during the promyelocyte stage of neutrophil maturation before the other granules are detectable. Azurophil granules are lysosomes that contain a variety of degradative enzymes such as neutral proteases, acid hydrolases and several highly cationic antimicrobial proteins such as the defensins and bactericidal/permeability-increasing protein. Myeloperoxidase is the most abundant primary granule enzyme, and is responsible for the green color of pus. Myeloperoxidase catalyzes the generation of hypochlorous acid, a potent antimicrobial oxidant, from hydrogen peroxide and chloride after activation of the respiratory burst oxidase (Klebanoff, 1992). A distinct and more abundant granule appears later in neutrophil development and contains a set of proteins that in most cases are unique to neutrophils. It has therefore been named the secondary or neutrophil-specific granule (Gallin, 1984). Specific granules contain apolactoferrin, collagenase, histaminase, chemotactic and adhesion receptors, and the flavocytochrome component of the NADPH oxidase (see below). Both primary and secondary granules contain lysozyme which breaks down bacterial cell wall proteoglycan. A third granule type, designated the tertiary granule, contains gelatinase. Membrane fusion events are under separate regulatory control for primary, secondary and tertiary granules. Thus, release of tertiary granule contents occurs

Phagocytes in Immunity and Inflammation

221

with minor perturbation of the cell membrane. In vitro and in vivo studies suggest that primary granules may preferentially fuse with phagosomes and play an intracellular, lysosomal, degradative role, whereas the contents of specific granules are more readily released into the extracellular milieu (Wright and Gallin, 1978; Henson et al., 1992). Specific granule products may amplify the inflammatory reaction by inducing the production of monocyte chemoattractants, thereby setting up a second, monocyte-dependent wave of inflammation (Gallin, 1984). In addition, specific granule proteases may degrade extracellular matrix proteins to facilitate movement of the cell through the tissues. While many details regarding the structure, ftmction and regulation of neutrophil granules remain unknown, their importance in homeostasis is most clearly established by the congenital disorder known as Neutrophil-Specific Granule Deficiency (SGD). SGD is a rare disorder of uncertain inheritance that is associated with recurrent bacterial infections. Leukocytes from affected patients are severely deficient in chemotaxis, adherence, and microbicidal functions when tested in vitro. Specific granule morphology is abnormal and biochemical markers of specific granules, such as lactoferrin, are absent or severely deficient. Defects in the production of azurophil and gelatinase-containing granule proteins, as well as in the production of eosinophil-specific granule proteins have also been reported in a patient with SGD (Rosenberg and Gallin, 1993). These findings plus the observation that lactoferrin is present in normal amounts in nasal secretions from patients with SGD, suggest that the underlying defect is in a phagocyte-specific regulatory factor controlling biosynthesis of the deficient proteins (Gallin et al., 1982; Gallin, 1985; Lomaxetal., 1989). The contents of eosinophil-specific granules are quite idiosyncratic (Weller, 1991). Lysosomal hydrolases and an NADPH oxidase are present in this granule, whereas in neutrophils they are separated into primary and secondary granules, respectively (Table 4). Like neutrophils, eosinophils also contain a peroxidase and highly basic anti-microbial proteins, although they are distinct from the neutrophil examples. The dense core of the eosinophil-specific granule is composed of crystals of major basic protein (MBP). The core is surrounded by a matrix composed of eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), eosinophil peroxidase (EPO), and several other lysosomal enzymes. The most potent antihelminthic products of eosinophils are MBP, ECP and EPO. MBP and ECP may amplify inflammatory responses by inducing histamine release from basophils and mast cells. Basophil granules are distinct from both eosinophils and neutrophils. They contain histamine and sulfated proteoglycans of uncertain function (Metcalfe et al., 1992). Production of Antimicrobial Oxidants

When phagocytes engulf particles, they dramatically increase their consumption of oxygen, a phenomenon known as the respiratory burst. This process leads to the

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PHILIP M. MURPHY

accumulation in the phagolysosome of a series of oxidants (e.g., superoxide anion, hydrogen peroxide, hypochlorous acid, hydroxyl radical) that chemically attack macromolecules of the ingested microbe (Babior, 1978) (Figure 6). Oxidant production also causes an increase in phagolysosomal pH to levels at which lysosomal enzymes are active, leading to degradation of the ingested microbe (Segal and Abo, 1993).

Specific Granule

NADP

NADPH Oxidase Assembly

^f^f^

NADPH

t

fHexose Monophos-'l I Dhate Shunt J

Azurophil Granule Figure 6. Oxidative killing mechanisms of phagocytes. Upon formation of the phagosome, a latent NADPH oxidase is activated by assembly of the indicated cytosolic and membrane components, and superoxide anion is produced, leading to the generation of a series of antimicrobial oxidants. The phagosome membrane is derived from the plasma membrane. Lactoferrin (LF) and myeloperoxidase (MPO) are delivered to the phagosome by fusion with specific and azurophil granules, respectively. The phagosomal membrane flavocytochrome component of the oxidase (gp91, p22) Is derived from both the plasma membrane and the specific granule membrane. See text for details and Table 4 for non-oxidatlve killing pathways. SOD, superoxide dismutase.

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The importance of oxygen and its toxic metabolites in the efficient killing of micro-organisms by phagocytes is most clearly revealed by Chronic Granulomatous Disease (CGD), a rare inherited human disorder of the phagocyte respiratory burst that is associated with recurrent life-threatening infections and high mortality rates in childhood. The phagocytes of patients with CGD ingest organisms normally but lack the ability to produce oxidants, and thus fail to efficiently kill certain bacteria and fiingi. Since micro-organisms themselves can produce hydrogen peroxide, phagocytes from patients with CGD can take advantage of this source to kill the organism by generating more toxic oxidants such as hypochlorous acid. Organisms that possess the enzyme catalase, however, metabolize H2O2 to O2 and H2O, thereby depriving the phagocyte of the H2O2 that they themselves produce. Thus, patients with CGD typically become infected with catalase positive organisms such as Staphylococcus aureus. The hallmark pathologic lesion of CGD, the granuloma, resembles granulomata formed during infection of macrophages by intracellular pathogens such as mycobacteria, which possess mechanisms for eluding the normal antimicrobial weaponry of the cell. Granulomatous inflammation persists even after the infectious process has resolved, suggesting the importance of oxidants in the resolution of the inflammatory response. The molecular details of oxidant production are now partly understood from combined genetic and biochemical studies of CGD (Rotrosen, 1992; Rotrosen, 1993). Upon cell activation by a chemoattractant or by phagocytosis, a multisubunit NADPH oxidase is assembled from three distinct cytosolic regulatory proteins, known as p47P'^°^, p67P^^^ and the low molecular weight GTP-binding protein rac-1, and a heterodimeric flavocytochrome located in the membrane of specific granules and plasma membranes. The flavocytochrome comprises the catalytic moiety of the oxidase enzyme. The fully assembled oxidase is an intramolecular electron transporter that transfers electrons from NADPH to flavin adenine dinucleotide (FAD), then to heme, and finally to molecular oxygen, thereby producing superoxide anion. NADPH is formed during the respiratory burst by Ci glucose metabolism via the hexose monophosphate shunt. Superoxide anion is rapidly converted by superoxide dismutase to hydrogen peroxide, a potent anti-microbial oxidant. Myeloperoxidase, which can be delivered to the phagosome by fusion with azurophil granules, then catalyzes the formation of HOCl from hydrogen peroxide and chloride. The microbicidal potency of HOCl (most commonly known as the active antiseptic ingredient in household bleach) is -^50 times greater than that of hydrogen peroxide. Lactoferrin, delivered to the phagosome from specific granules, may be the most important iron source for the Fenton reaction in which hydroxyl radical is formed from hydrogen peroxide in the presence of reducing agents such as superoxide (Ambruso and Johnston, 1981). The most common genetic mutations (--65%) found in patients with CGD occur in the gene for the large subunit of the flavocytochrome (gp9 P^^'^) which is located on the X chromosome. Thus inheritance of CGD in these families displays a classic

224

PHILIP M. MURPHY

X-linked Mendelian pattern, affecting -50% of males. The next most common mutations (--30%) are in the gene for p47P'^^^. These families display autosomal recessive inheritance. Mutations in the autosomal genes for p67P^^^ and the small subunit of the flavocytochrome are extremely rare accounting for only a few % of all cases of CGD (Clark et al., 1989). No mutations have thus far been reported for rac-1. All four disease-related genes have been cloned, paving the way for the development of gene therapy. While its importance in human phagocytes is not yet clear, the ability of murine macrophages to produce nitric oxide by the action of the enzyme nitric oxide synthase on L-arginine is a second oxidant-generating system of phagocytes that may be important for killing certain intracellular parasites, viruses, and tumor cells by macrophages (Liew and Cox, 1991). Although the phagocyte needs to produce oxidants to kill microbes, it also needs to control the level of oxidant production so as not to cause unnecessary damage to host structures. Oxidants can damage extracellular matrix proteins directly or by activating latent proteases such as collagenase, elastase and gelatinase (Weiss, 1989). The NADPH oxidase appears to be a highly regulated enzyme, lacking constitutive activity, and turned off with time after activation. Moreover, the phagocyte possesses several important mechanisms for reducing oxidant concentrations. These include catalase, superoxide dismutase, iron-free transferrin, and the glutathione redox system. NADPH, in addition to being the source of electrons for the production of toxic oxygen species, is also the reducing agent for production of reduced glutathione which in turn can detoxify hydrogen peroxide. Production of Pro-Inflammatory Mediators Phagocytes, especially macrophages, are important sources of cytokines, chemoattractants, and vasoactive mediators (Nathan, 1987). These substances have profound effects on humoral, cellular and vascular components of the inflammatory response. Local concentrations of mediators can be regulated at multiple levels, including gene transcription, latent enzyme activation and release of presynthesized products. IL-1, IL-6, and tumor necrosis factor are just three of the best characterized examples of multi-functional cytokines that participate in the pathogenesis of many aspects of inflammation from fever to septic shock. Activation of phagocytes also induces the synthesis and release of a variety of 20-carbon fatty acid derivatives of arachidonic acid, that upon cell activation is itself cleaved from membrane phospholipids by the action of phospholipase A2 (Samuelsson et al., 1987). Arachidonic acid is a substrate for the aspirin-inhibitable enzyme cyclo-oxygenase which catalyzes the production of cyclic endoperoxides. These can be further metabolized to produce either a series of prostaglandins or thromboxanes. Prostaglandin E2 is a particularly potent vasodilator that contributes to the edema and erythema present in inflammatory sites. Thromboxane B2, in contrast, is vasoinactive, but possesses chemotactic activity for phagocytes.

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Alternatively, arachidonic acid can be oxygenated by the enzyme 5-lipoxygenase to begin the metabolic pathway for the production of leukotrienes and lipoxins. Leukotriene B4 is the principal chemotactic leukotriene produced by activated neutrophils. Eosinophils produce primarily the sulfidopeptide leukotrienes C4, D4 and E4 from LTA4 and glutathione (Weller et al, 1983; Samuelsson et al., 1987; Weller, 1991). In contrast to LTB4, these substances are not leukotactic but instead are myotropic: they cause bronchoconstriction, increased mucus production and increased vascular permeability of post-capillary venules during late phase allergic reactions. For this reason they are also known collectively as the slow reacting substance of anaphylaxis. The lipid known as platelet-activating factor (PAF) is produced by activated phagocytes by replacing the 2-acyl group in 1-alkyl-2-acylglycerol phosphocholine, a membrane phospholipid, with acetate. PAF is a potent platelet secretagogue, a spasmogen for smooth muscle cells, and a chemoattractant and activating factor for phagocytes. Thus, activation of phagocytes leads to the production of peptide and lipid autocoids which amplify the signal that initiated the inflammatory reaction. Immunoregulation

Macrophages are unique among phagocytes in their ability to support a specific immune response by presenting complex protein antigens to T lymphocytes (Unanue and Allen, 1987). They do this by first engulfing antigens such as immune complexes or bacteria, and degrading them in the phagolysosome. Small antigenic fragments ~9 amino acids in length known as epitopes, then bind to a specific groove of class II major histocompatibility (MHC) molecules (Figure 2). The MHC-peptide complex is then transported to the cell surface where T lymphocytes bearing specific T cell receptors bind to the peptide and become activated. A detailed account of macrophages as antigen-presenting cells can be found in the earlier chapter of this volume.

SUMMARY Phagocytes represent the last vestige in higher species of the ancient cellular function for which they are named. Human genetic diseases indicate that phagocytosis and oxidant production are critical to the defense of the host against pathogenic bacteria and fungi, which undoubtedly served to select and maintain these functions over evolutionary time. Phagocytes may also be deleterious to the host, contributing to acute and chronic inflammation. Many chronic inflammatory conditions occur in adulthood after reproduction has occurred, and therefore do not impose selective pressure upon the germ line. Moreover, mechanisms for regulating phagocyte functions would by necessity evolve more slowly than the micro-organisms they are designed to control. Thus, inflammation may be an intrinsic property of a system

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PHILIP M. MURPHY

that only crudely responds over evolutionary time to hypermutable microbial targets, while awaiting establishment of a finely tuned, specific immune response.

ACKNOWLEDGMENTS I thank Sunil Ahuja, John Gallin, Helene Rosenberg, Fei Li, and Robert Sokolic for helpful comments about the manuscript.

REFERENCES Adams, D.O., & Hamilton, T.A. (1992). Macrophages as destructive cells in host defense. In: Inflammation. Basic Principles and Clinical Correlates, 2 edn. (Gallin, J.I., Goldstein, I.M., & Snyderman R., Eds.), pp. 637-662. Raven, New York. Ambruso, D.R., & Johnston, R.B. (1981). Lactoferrin enhances hydroxyl radical production by human neutrophils, neutrophil particulate fractions and an enzymatic generating system. J. Clin. Invest. 67, 352-360. Anderson, D.C., & Springer, T.A. (1987). Leukocyte adhesion deficiency: an inherited defect in the Mac-1, LFA-1, and pi50,95 glycoproteins. Ann. Rev. Med. 38, 175-194. Babior, B.M. (1978). Oxygen-dependent microbial killing by phagocytes. N. Engl. J. Med. 298, 659-668. Baggiolini, M., Hirsch, J.G., & DeDuve, C. (1969). Resolution of granules from rabbit heterophil leukocytes into distinct populations by zonal sedimentation. J. Cell Biol. 40, 529-541. Brown, S.J., Galli, S.J., Gleich, G.J., & Askenase, P.W. (1982). Ablation of immunity to Amblyomma americanum by anti-basophil serum: Cooperation between basophils and eosinophils in expression of immunity to ectoparasites (ticks) in guinea pigs. J. Immunol. 129, 790-796. Butterworth, A.E. (1984). Cell-mediated damage to helminths. Adv. Parasitol. 23, 143-235. Capron, M., Spiegelberg, H.L., Prin, L., Bennich H., Butterworth, A.E., Pierce, R.J., Ali Ouassi, M., & Capron, A. (1984). Role of IgE receptors in effector function of human eosinophils. J. Immunol. 132,462-468. Clark, R.A., Malech, H.L., Gallin, J.I., Nunoi, H., Volpp, B.D., Pearson, D.W., Nauseef, W.M., & Cumutte, J.T. (1989). Genetic variants of chronic granulomatous disease: Prevalence of deficiencies of two cytosolic components of the NADPH oxidase system. N. Engl. J. Med. 321, 647-652. Cohen, S., Ward, P.A., & McCluskey, R.T. (1979). Mechanisms of Immunopathology. Wiley and Sons, New York. Cohn, Z.A. (1986). Intracellular parasitism and the system of mononuclear phagocytes. In: Immunologic Diseases, 14th edn. (Samter, M., Frank, M.M., Austen, K.F., Talmadge, D., & Claman, H., Eds.). Raven, New York. Cohnheim, J. (1889). Lectures in General Pathology. Vol. 1. New Sydenham Soc, London. Densen, P., & Mandell, G.L. (1980). Phagocyte strategy vs. microbial tactics. Rev. Infect. Dis. 2, 817-838. Dexter, T.M., Garland, J.M., & Testa, N.G. (Eds.). (1990). Colony-stimulating factors. Marcel Dekker, New York. Durham, S.R., Loegering, D.A., Dunnette, S., Gleich, G.J., & Kay, A.B. (1989). Blood eosinophils and eosinophil-derived proteins in allergic asthma. J. Allergy Clin. Immunol. 84, 931-936. Dutrochet, M.H. (1824). Recherches, anatomiques et physiologiques sur la structure intime des animaux et des vegetaux et sur leur motilite. Bailliere, Paris. Ehrlich, P. (1900). La Leukocytose. In XIII Congr. Int. Med., Paris. Etzioni, A. (1993). Leukocyte adhesion deficiency type 2. N. Engl. J. Med. in press. Gallin, J.I. (1984). Neutrophil specific granules: A fuse that ignites the inflammatory response. Clin. Res. 320-328.

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Gallin, J.I., Fletcher, M.P., Seligmann, B.E., Hoffstein, S., Cehrs, K., & Mounessa, N. (1982). Human neutrophil-specific granule deficiency: A model to assess the role of neutrophil-specific granules in the evolution of the inflammatory response. Blood 59, 1317-1329. Gallin, J.I. (1985). Neutrophil specific granule deficiency. Ann. Rev. Med. 36, 263-274. Golde, D.W., & Gasson, J.C. (1988). Hormones that stimulate the growth of blood cells. Sci. Amer. 259, 62-70. Groopman, J.E., Molina, J-M., & Scadden, D.T. (1989). Hematopoietic growth factors. Biology and clinical applications. N. Engl. J. Med. 321, 1449-1456. Henson, P.M., Henson, J.E., Fittschen, C, Bratton, D.L., & Riches, D.W.H. (1992). Degranulation and secretion by phagocytic cells. In: Inflammation. Basic Principles and Clinical Correlates, Second Edition (Gallin, J.I., Goldstein, I.M., & Snyderman R., Eds.), pp. 511-540. Raven, New York. Janoff, A. (1985). Elastase in tissue injury. Ann. Rev. Med. 36, 207-216. Kishimoto, T.K., & Anderson, D.C. (1992). The role of integrins in inflammation. In: Inflammation. Basic Principles and Clinical Correlates, Second edn. (Gallin, J.I., Goldstein, I.M., & Snyderman R., Eds.), pp. 353-406. Raven, New York. Klebanoff, S.J. (1992). Oxygen metabolites from phagocytes. In: Inflammation. Basic Principles and Clinical Correlates, Second edn. (Gallin, J.I., Goldstein, I.M., & Snyderman R., Eds.), pp. 541-588. Raven, New York. Kostmann, R. (1975). Infantile genetic agranulocytosis. Acta Paediatr. Scand. 64, 362-368. Lasky, L.A., & Rosen, S.D. (1992). The selectins: Carbohydrate-binding adhesion molecules of the immune system. In: Inflammation. Basic Principles and Clinical Correlates, Second edn. (Gallin, J.I., Goldstein, I.M., & Snyderman R., Eds.), pp. 407-420. Raven, New York. Liew, F.Y., & Cox, F.E.G. (1991). Nonspecific defense mechanism: The role of nitric oxide. Immunol. Today 12,A17-A21. Lomax, K.J., Gallin, J.I., Rotrosen, D., Raphael, G.D., Kaliner, M.A., Benz, E.J., Boxer, L.A., & Malech, H.L. (1989). Selective defect in myeloid cell lactoferrin gene expression in neutrophil specific granule deficiency. J. Clin. Invest. 83, 514-519. Malech, H.L., & Gallin, J.I. (1987). Neutrophils in human diseases. N. Engl. J. Med. 317, 687-694. Mantovani, B. (1975). Different roles of IgG and complement receptors in phagocytosis by polymorphonuclear leukocytes. J. Immunol. 115, 15-17. Metcalfe, D.D., Costa, J.J., & Burd, P.R. (1992). Mast cells and basophils. In: Inflammation. Basic Principles and Clinical Correlates, Second edn. (Gallin, J.I., Goldstein, I.M., & Snyderman R., Eds.), pp. 70^726. Raven, New York. Metchnikoff, E. (1905). Immunity in Infectious Diseases. Cambridge University Press, Cambridge. Murphy, P.M. (1994). The molecular biology of leukocyte chemoattractant receptors. Ann. Rev. Immunol, in press. Nathan, C.F. (1987). The secretory products of macrophages. J. Clin. Invest. 79, 319-326. Ognibene, F.P., Martin, S.E., Parker, M.M., Schlesinger, T., Roach, P., Burch, C , Shelhamer, J.H., & Parrillo, J.E. (1986). Adult respiratory distress syndrome in patients with severe neutropenia. N. Engl.J. Med. 315, 547-551. Oppenheim, J.J., Zachariae, CO., Mukaida, N., & Matsushima, K. (1991). Properties of the novel proinflammatory supergene "intercrine" cytokine family. Ann. Rev. Immunol. 9, 617-648. Oppenheim, J.J., & Potter, M. (1981). Immunity and inflammation. In: Cellular Functions in Immunity and Inflammation. (Oppenheim, J.J., Rosenstreich, D.L., & Potter, M., Eds.), pp. 1-28. Elsevier/North-Holland, New York. Pasteur, L. (1880). Sur les maladies virulentes, et en particulier sur la maladie appelee vulgairement cholera des poules. C. R. Adac. Sci. Paris 40, 239-248. Ravetch, J.V., & Kinet, J-P. (1991). Fc receptors. Ann. Rev. Immunol. 9, 457-492. Robinson, W.A., & Mangalik, A. (1975). The kinetics and regulation of granulopoiesis. Semin. Hematol. 12,7-25.

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Rosenberg, H.F., & Gallin, J.I. (1993). Neutrophil-specific granule deficiency includes eosinophils. Blood 82, 268-273. Samuelsson, B., Dahlen, S.-E., Lindgren, J.A., Rouzer, C.A., & Serhan, C.N. (1987). Leukotrienes and lipoxins: structures, biosynthesis, and biological effects. Science 237, 1171-1176. Segal, A.W., & Abo, A. (1993). The biochemical basis of the NADPH oxidase of phagocytes. Trends Bioch. Sci. 18,43^7. Silverstein, A.M. (1979). Cellular versus humoral immunity: Determinants and consequences of an epic 19th century battle. Cell. Immunol. 48, 208-221. Snyderman, R., & Ewing, R.J. (1992). Chemoattractant stimulus-response coupling. In: Inflammation. Basic Principles and Clinical Correlates, Second edn. (Gallin, J.I., Goldstein, I.M., & Snyderman R., Eds.), pp. 421-440. Raven, New York. Snyderman, R., & Goetzl, E.J. (1981). Molecular and cellular mechanisms of leukocyte chemotaxis. Science 213, 830-837. Spangrude, G.J., Heimfeld, S., & Weissman, I.L. (1988). Purification and characterization of mouse hematopoietic stem cells. Science 241, 58-62. Springer, T.A. (1990). Adhesion receptors of the immune system. Nature 346, 425-434. Springer, T.A., Thompson, W.S., Miller, J., Schmalstieg, F.C., & Anderson, D.C. (1984). Inherited deficiency of the Mac-1, LF A-1, p 150,95 glycoprotein family and its molecular basis. J. Exp. Med. 160, 1901-1918. Stahl, P. (1990). The macrophage mannose receptor: current status. Am. J. Respir. Cell. Mol. Biol. 2, 317-318. Stossel, T.P. (1974). Phagocytosis. N. Engl. J. Med. 290, 717-723, 773-780, 833-839. Stossel, T.P. (1993). On the crawling of animal cells. Science in press. Unanue, E.R., & Allen, P.M. (1987). The basis for the immunoregulatory role of macrophages and other accessory cells. Science 236, 551-557. Weiss, S.J. (1989). Tissue destruction by neutrophils. N. Engl. J. Med. 320, 365-376. Weissmann, G., Zurier, R.B., & Hoffstein, S. (1973). Leukocytes as secretory organs of inflammation. Agents Actions 3, 270-279. Weitzman, S.A., Weitburg, A.B., Clark, E.P., & Stossel, T.P. (1985). Phagocytes as carcinogens: Malignant transformation produced by human neutrophils. Science 227, 1231-1233. Weller, P.F., Goetzl, E.J., & Austen, K.F. (1980). Identification of human eosinophil lysophospholipase as the constituent of Charcot-Leyden crystals. Proc. Natl. Acad. Sci. USA 77, 7440-7443. Weller, P.F. (1991). The immunobiology of eosinophils. N. Engl. J. Med. 324, 1110-1118. Weller, P.F., Lee, C.W., Foster, D.W., Corey, E.J., Austen, K.F., & Lewis, R.A. (1983). Generation and metabolism of 5-lipoxygenase pathway leukotrienes by human eosinophils: predominant production of leukotriene C4, Proc. Natl. Acad. Sci. USA 80, 7626-7630. Wright, D.G., & Gallin, J.I. (1979). Secretory responses of human neutrophils: Exocytosis of specific (secondary) granules by human neutrophils during adherence in vitro and during exudation in vivo. J. Immunol. 123, 285-294. Wright, D.G., Dale, D.D., Fauci, A.S., & Wolff, S.M. (1981). Human cyclic neutropenia: clinical review and long-term follow-up of patients. Medicine 60, 1-13. Wright, S. (1992). Receptors for complement and the biology of phagocytosis. In: Inflammation. Basic Principles and Clinical Correlates, Second edn. (Gallin, J.I., Goldstein, I.M., & Snyderman R., Eds.), pp. 477-496. Raven, New York. Zigmond, S.H. (1978). Chemotaxis by polymorphonuclear leukocytes. J. Cell Biol. 77, 269-287.

RECOMMENDED READINGS Klebanoff, S.J., & Clark, R.A. (1978). The Neutrophil: Function and Clinical Disorders. Amster-North Holland Biomedical, Amsterdam.

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Gallin, J.I., Goldstein, I.M., & SnydermanR. (Eds.). (1992). Inflammation. Basic Principles and Clinical Correlates, Second edn. Raven, New York. Mims, C.A. (1982). The pathogenesis of infectious diseases. Academic Press, London. Paul, W.E. (Ed.) (1988). Fundamental Immunology. Raven, New York. Williams, W.J., Beutler, E., Erslev, A.J., & Lichtman, M.A. (Eds.). (1990). Hematology, Fourth Edition. McGraw-Hill, New York.

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Chapter 12

Anaphylaxis CALMAN PRUSSIN and MICHAEL KALINER

Introduction Classification of Causes of Anapliylaxis IgE Mediated Anaphylaxis Complement Mediated Anaphylaxis Non-immunologically Mediated Mast Cell Reactions Clinical Findings Pathogenesis Differential Diagnosis Treatment Summary Recommended Readings

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INTRODUCTION Anaphylaxis represents the most impressive and potentially life threatening manifestation of immediate (IgE mediated) hypersensitivity. This syndrome can affect almost any organ system, although reactions involving the pulmonary, circulatory, cutaneous and gastrointestinal systems are most common. Reactions can vary from mild urticaria (hives) to anaphylactic shock and death. Portier and Richet used the term anaphylaxis to describe the fatal reaction induced by the introduction of minute amounts of antigen into dogs that had been previously sensitized to that antigen. Attempting to induce protective immunity to sea anenome toxin by prior injection, they unexpectedly observed that some of the dogs had an immediate fatal reaction when reimmunized. The dramatic and unexpected fatal response was the opposite (ana=Greek: backwards) of the expected protection (phylax=Greek: guard). Richet was subsequently awarded the Nobel prize in medicine and physiology in 1913 for his work in the field. Anaphylaxis is the clinical syndrome elicited in a hypersensitive individual on subsequent exposure to the sensitizing antigen. The necessary components of the anaphylactic response are: (1) a sensitizing antigen, usually administered parenterally, (2) an IgE class antibody response resulting in systemic sensitization of mast cells and basophils, (3) reintroduction of the sensitizing antigen, usually systemically, (4) mast cell degranulation with mediator release, generation, or both, and (5) production of several pathologic responses by the mast cell derived mediators manifested as anaphylaxis. Mast cell activation can be induced by a number of disparate stimuli. In classic IgE mediated anaphylaxis, activation is induced via cross linking of mast cell high affinity IgE receptors (FcSRI) by antigen. Upon activation, a number of both preformed and newly synthesized mediators with potent biological activity are released into the surrounding tissue. Mast cells are rich in granules containing a large number of preformed mediators, in particular histamine. Upon activation, the contents of these granules are exocytosed into the surrounding tissue with subsequent release of mediators. Activation causes the de novo synthesis and release of a number of active lipid mediators derived from arachidonic acid, including prostaglandin D2, Leukotriene C4 and platelet activating factor. These lipid mediators are all synthesized after activation. Recent reports demonstrate that mast cells are sources of both preformed and newly synthesized cytokines, such as tissue necrosis factor-a and interleukin-4. The significance of these mediators in anaphylaxis remains to be determined. Because the mediators that are released or generated by mast cells cause anaphylaxis, any event associated with mast cell activation may produce the same clinical picture. Anaphylaxis usually refers to IgE mediated, antigen stimulated mast cell activation. Whereas anaphylactoid reactions denote other non-IgE mediated responses where immunologic recognition of antigen does not occur; for example.

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those produced by chemical agents capable of directly causing mast cell degranulation (e.g., radiocontrast agents, opiates).

CLASSIFICATION OF CAUSES OF ANAPHYLAXIS IgE Mediated Anaphylaxis

IgE mediated anaphylaxis has been implicated in untoward reactions elicited by drugs, insect stings, chemicals, foods, preservatives, and environmental factors (Table 1). An agent capable of causing IgE antibody formation must be either a whole antigen or a hapten. For example, horse serum, formerly used as a source of antitoxin, contains many antigenic proteins. Subsequent reexposure to horse serum predictably caused anaphylaxis. Horse antisera are now rarely used therapeutically, an exception being antivenom therapy for snakebite. More common sources of antigens include allergenic extracts, insulin, Hymenoptera venom (e.g., bee sting), foods, latex rubber, seminal plasma, and chymopapain. Haptens are molecules that are too small to elicit an immune response by themselves; however, haptens may bind to endogenous serum proteins, such as albumin, and become antigenic. The most important haptens are penicillins and related antibiotics. Most instances of IgE mediated anaphylaxis are due to the administration of penicillin drugs. Complement Mediated Anaphylaxis

Anaphylactic reactions have been observed after the administration of whole blood or its products, including serum, plasma, fractionated serum and immunoTable h

Causes of Anaphylaxis/Anaphylactoid Reactions

IgE Mediated Reactions Antibiotics and other drugs Foreign Proteins (horse serum, chymopapain) Foods Allergen Immunotherapy Hymenoptera Stings Latex Rubber (gloves, medical supplies) Seminal Plasma Complement Mediated Reactions Blood, blood products Nonimmunologic Mast Cell Activators Radiocontrast Media Opiates Modulators of Arachidonic Acid Metabolism Nonsteroidal anti-inflammatory agents Exercise Induced Anaphylaxis Idiopathic Anaphylaxis Catamenial Anaphylaxis

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globulins. One of the mechanisms responsible for these reactions is the formation of immune complexes, resulting in the activation of the complement cascade. Many active species are generated by complement; the anaphylotoxins C3a, C4a, and C5a are capable of causing mast cell activation and consequent systemic reactions. In addition, anaphylotoxins may directly induce vascular permeability and contract smooth muscles. This complement mediated anaphylaxis is thus immunologically mediated, though not through IgE. Typical reactions are seen in blood transfusions in which the recipient has developed antibodies (IgG or IgM) to a component of the donor's blood, usually either cell surface antigens or serum proteins. An example of this is seen in ABO blood incompatibility reactions, in which the recipient has antibodies (typically IgG and IgM) to the donor ABO red cell surface antigens. Non-immunologically Mediated Mast Cell Reactions Direct mast cell activators can cause a syndrome clinically indistinguishable from anaphylaxis through release of mast cell granules. To differentiate these reactions from immunologically mediated (IgE, IgG/complement) reactions, they are commonly referred to as anaphylactoid reactions. Although the steps leading up to mast cell activation differ from classic anaphylaxis, release of mast cell mediators yields a syndrome that is clinically indistinguishable. Contrary to the pathogenesis of IgE mediated anaphylaxis, no priming is required, since the reaction is due to direct mast cell activation. The clinically most important agents include iodinated radiocontrast media, narcotics and neuromuscular blocking agents. Mild reactions are seen in approximately 5% of individuals receiving radiocontrast media. Severe systemic reactions occur in 1 in 1000 exposures. The pathogenesis of radiocontrast reactions is still uncertain. Contrast media is hypertonic and may cause direct mast cell degranulation via this mechanism; in addition, activation of the complement and coagulation systems with subsequent mediator release may play a role. Newer low osmolality non-ionic radiocontrast media have a significantly lower rate of reactions. Approximately 5-10% of asthmatic individuals react to nonsteroidal antiinflammatory drugs (NSAID), such as aspirin and indomethacin, with rhinorrhea, bronchospasm and rarely, vascular collapse. Typically these patients have the "aspirin triad"—^aspirin sensitivity, chronic sinusitis with nasal polyps and asthma. The exact mechanism underlying NSAID sensitivity is not yet known. The cause of this disorder appears at least in part to be due to a modulation of arachidonic acid metabolism by shifting from the cyclo-oxygenase to the lipoxygenase enzyme pathways. The consequences of this are: (1) a reduction in the production of prostaglandins and thromboxanes, and (2) enhanced production of leukotrienes and HETEs (hydroxy-eicosatetraenoic acids). These patients react to all NSAID's despite the diversities of their chemical structures. This is taken as ftirther evidence that the syndrome is not antibody

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mediated via immunologic recognition of the drug molecule, but rather through their common pharmacologic effects. Exercise induced anaphylaxis is a disorder in which individuals experience anaphylaxis with exercise. The pathogenesis of this unusual disorder is not clear. The syndrome is sometimes linked to ingestion of particular foods. A syndrome termed idiopathic anaphylaxis has been described in a group of patients who experience anaphylaxis due to no recognized cause. These patients present with classical anaphylaxis, yet despite extensive workup, no cause is found. The diagnosis is based on clinical signs and symptoms of anaphylaxis and the exclusion of known causes of anaphylaxis. The diagnosis of anaphylaxis can be confirmed in difficult diagnostic situations by performing a plasma tryptase determination (see differential diagnosis, below). A rare cause of repeated episodes of anaphylaxis without a readily identifiable etiology is catamenial anaphylaxis, a syndrome of hypersensitivity to endogenous progesterone secretion. Some, but not all, patients with catamenial anaphylaxis exhibit a cyclic pattern of attacks that intensifies during the luteal phase of the menstrual cycle. These patients have positive skin tests to medroxyprogestrone, experience systemic reactions to infusions of lutenizing hormone releasing hormone (LHRH), and respond favorably to ovarian suppression with LHRH agonists or oophorectomy. Clinical Findings

The primary organ systems involved in anaphylaxis are the cutaneous, gastrointestinal, respiratory and cardiovascular systems. Characteristically, patients describe an immediate sense of impending doom coincident with flushing, tachycardia and often pruritus. If left untreated, the initial signs and symptoms may rapidly evolve to urticaria, angioedema, rhinorrhea, bronchorrhea, asthma, laryngeal edema, abdominal cramps, cardiac arrhythmias, faintness, syncope, shock, and death. The organ systems involved in these responses contain the largest numbers of mast cells and thus are the most affected by mast cell activation. Urticaria is due to mast cell degranulation in the epidermis, with a consequent increase in vascular permeability and localized edema, causing the characteristic raised skin or wheal. Histamine is the mediator responsible for the associated pruritus. Angioedema has a similar pathogenesis; however, the location of mast cell degranulation is in the dermis or subcutaneous tissue. As such, the swelling is not as discrete and there is less pruritus, due to a relative lack of sensory nerve endings in this anatomic location. Pathogenesis

When mast cells degranulate, preformed and rapidly generated mediators are released into the connective tissue. Although many of these mediators induce

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236 Table 2,

Processes I n v o l v e d in t h e S y m p t o m s of A n a p h y l a x i s

Pathologic Process Vascular permeability Vasodilation Smooth muscle contraction

Sign or Symptom Urticaria, angioedema, laryngeal edema Flushing, headache

Tachycardia

Wheezing (asthma), gastrointestinal cramps diarrhea Palpitations

Reduced peripheral vascular resistance

Faintness, syncope, hypotension

Putative

Mediator

Histamine (Hi), leukotrienes, prostaglandins Histamine (Hi and H2), leukotrienes, prostaglandins Histamine (Hi), leukotrienes, prostaglandins Histamine (Hi) and leukotrienes Histamine (Hi and H2)

dramatic effects, few other than histamine are capable of entering the circulation in an active state. As summarized in Table 2, most of the changes occurring in anaphylaxis can be attributed to histamine (acting through Hi and H2 receptors), prostaglandins and leukotrienes. The consequences of mast cell mediator release include increased vascular permeability, contraction of smooth muscle and vasodilatation. Increased vascular permeability is due to the formation of intercellular gaps between endothelial cells in postcapillary venules. The increased vascular permeability causes tissue edema leading to urticaria (if the reaction is limited to the epidermis); angioedema (if the reaction is in the dermis or subcutaneous tissue); laryngeal edema; nasal congestion; and gastrointestinal swelling, with abdominal bloating and cramps. Contraction of smooth muscle leads to bronchospasm and asthma, as well as abdominal cramping and diarrhea. Vasodilation leads to flushing, reduced peripheral vascular resistance, hypotension and syncope. Cardiac histamine receptor stimulation leads to tachycardia and possible arrhythmias. Differential Diagnosis The diagnosis of anaphylaxis is usually based upon history and presenting signs compatible with a diagnosis of anaphylaxis. Patients typically note the onset within minutes following exposure to a provocative stimulus. Anaphylaxis is easily confused with a vasovagal reaction; distinction of the two is critically important as the prognosis and therapy of the two are quite different. Patients with vasovagal reactions typically present with pallor, diaphoresis and bradycardia; in contrast, flushing, urticaria, angioedema, pruritus, wheezing and tachycardia are typical of anaphylaxis. If laryngeal edema is the presenting problem, hereditary angioedema must be considered. Hereditary angioedema is not associated with flushing, asthma or urticaria. Systemic mastocytosis is characterized by a generalized overabundance

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of mast cells. These mast cells may degranulate, producing local effects or rarely causing systemic effects identical to anaphylaxis. Suspicion of the diagnosis should be raised with the recognition of the classic reddish-brown maculopapular lesions that urticate on trauma (Darier's sign), the history of flushing attacks, evidence of bone involvement, gastrointestinal pain and peptic ulcers. Increased urinary histamine and PGD2 metabolites are also seen. Bone marrow biopsy is usually diagnostic. In difficult diagnostic situations (e.g. critically ill or surgical patients), many of the signs of anaphylaxis may be obscured. In situations where the diagnosis of anaphylaxis itself is in doubt, measurement of plasma tryptase may confirm the diagnosis. Tryptase is an enzyme found in mast cell granules and is released into the circulation following systemic mast cell degranulation. Whereas the circulating half life of histamine is on the order of minutes, tryptase remains in the circulation long enough to be measured 3 to 4 hours later. Treatment

Anaphylaxis is an acute medical emergency requiring immediate attention. If possible, the source of antigen should be removed. Subcutaneous epinephrine is the mainstay of therapy. This drug maintains the blood pressure, antagonizes many of the mediators of anaphylaxis and reduces the subsequent release of mediators through its action on mast cells and basophils. Often both Hi and H2 antihistamines are given. Blood pressure should be maintained with fluid and pressors as needed. Corticosteroids have no immediate effect but should be administered to prevent prolonged or recurrent anaphylaxis. Treatment of anaphylaxis has been complicated by the increased use of betaadrenergic blocking agents in many patients. In the presence of beta-blockers, anaphylactic reactions may be more severe, prolonged and refractory to treatment.

SUMMARY Anaphylaxis is a major medical emergency, the pathogenesis of which is remarkably well understood. The signs and symptoms of anaphylaxis are due to mast cell activation with subsequent release of potent mediator substances. Activation is typically brought about by antigen binding to IgE on the surface of the mast cell. Anaphylactoid reactions differ in being caused by direct mast cell activators, which are not recognized by IgE. Histamine, prostaglandins and leukotrienes are the most significant mediators in the pathogenesis of anaphylaxis. Anaphylaxis is usually associated with an exposure to a causative substance such as medications, foreign proteins, foods, insect stings and blood products. Diagnosis is based largely on clinical findings, which include flushing, tachycardia, urticaria, angioedema, asthma, laryngeal edema, cardiac arrhythmias, shock and death. Treatment requires prompt diagnosis and institution of appropriate therapy.

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RECOMMENDED READINGS Atkinson, T.P., & Kaliner, M.A. (1992). Anaphylaxis. Med. Clin. North Amer. 76, 841-855. Bochner, B.S., & Lichtenstein, L.M. (1991). Anaphylaxis. New Engl. J. Med. 324, 1785-1790. Marquardt, D.L., & Wasserman, S.I. (1993). Anaphylaxis. In: Allergy. Principles and Practice. (Middleton, E., Ed.), pp. 1525-1536. Mosby, St Louis, MO.

Chapter 13

Autoimmunity and Autoimmune Disease SUDERSHAN K. BHATIA and NOEL R. ROSE

Introduction Autoimmunity Self and Non-Self Mechanisms of Positive Selection Negative Selection and Self-Tolerance Autoimmune Disease Autoimmune Thyroid Disease Incidence Histopathological Changes Autoantigens Animal Models Multifactorial Etiology Thyroglobulin Summary

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INTRODUCTION A cardinal property of the immune system is its ability to distinguish self from non-self The fundamental mechanisms involved in self-recognition are gradually coming to light. Clearly, more than one mechanism is responsible for this biologically important phenomenon. Yet, there are many instances where self/non-self discrimination breaks down and autoimmune disease results. In fact, it has been the study of these exceptions to the rule that has taught us most about the essential mechanisms required to maintain normal unresponsiveness to self In this chapter, we discuss the mechanisms known to underlie self/non-self discrimination. Then we describe in detail one of the best characterized examples of an autoimmune disease, autoimmune thyroid disease.

AUTOIMMUNITY A basic function of the immune system is to discriminate between self and non-self. Immunological unresponsiveness to self-antigens is termed self-tolerance. The importance of tolerance to self-antigens was first recognized at the advent of this century by Paul Ehrlich (see Himmelweit, 1901). "Horror autotoxicus" was the term that he used to imply that an immune response to self-antigens would be harmful. A major breakthrough in our current understanding of autoimmunity occurred in 1956, with the discovery of the induction of autoimmune thyroiditis in rabbits by immunization with rabbit thyroid extracts (Rose and Witebsky, 1956). At the same time, autoantibodies to thyroglobulin (Tg) were found in the serum of patients with Hashimoto's thyroiditis (Roitt et al., 1956; Witebsky et al., 1957). These events marked the beginning of our recognition of autoimmune disease as a major cause of human disease. In the latter (Witebsky et al., 1957), criteria, known as Witebsky's postulates, for the recognition of human autoimmune diseases were formulated. Based on these criteria, an autoimmune disease is characterized by: (i) identification of circulating or cell-bound antibodies in patients with the disease, (ii) demonstration of the specific antigen against which the immune response is directed, (iii) induction of antibody against this antigen in experimental animals, and (iv) development of pathological changes similar to the disease in question in the appropriate tissues of the experimental animal. As the recognition of self is an integral component of the immune response, the changing concepts of autoimmune disease over the years have enhanced our understanding of the immune response itself

SELF AND NON-SELF The mechanisms by which self/non-self distinction is achieved are now beginning to be understood. The cells of the immune system (the B cells and the T cells) are rendered tolerant to molecules of the host organism by three processes: one that

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results in elimination of the cells that would otherwise produce an anti-self response (clonal deletion) (Burnet, 1959); another that results in their inactivation (clonal anergy) (Nossal, 1983), and a regulatory mechanism of active suppression of autoreactive cells (clonal balance) (Rose et al., 1981). The major responsibility for distinguishing self from non-self lies with the T-cell receptor (TCR) (Sha et al, 1988). T cells recognize antigen exclusively in association with self-MHC^ (Bevan and Fink, 1978). This is not an inherent property of the developing T cells, but is "learned" during thymocyte maturation (Lo and Sprent, 1986). The "self refers to the MHC haplotype expressed on the thymic epithelium in which the thymocytes matured. When the thymic progenitor cells arrive from the bone marrow, they express no TCR. The TCR is a heterodimeric surface protein composed of an a and a P chain. Rearrangement of variable (V) diversity (D) and joining (J) gene segments at the P locus allows for cytoplasmic expression of the p chain. This event is followed by V-J rearrangement at the a locus, culminating in ap TCR expression on the cell surface of immature thymocytes (Fowlkes and Pardoll, 1989). Random rearrangement of TCR gene segments and combinatorial association of the two chains give rise to a large T-cell repertoire of at least 10^ different specificities. This randomly generated T-cell repertoire is shaped by two processes that occur in the thymus, positive and negative selection (Kruisbeek et al., 1992). Positive selection is responsible for generating a T-cell repertoire that has the ability to recognize antigenic peptides in association with self-MHC molecules. The other process is called negative selection, and assures that tolerance for self-antigens is achieved. During negative selection, potentially autoreactive T cells are actually deleted from the T-cell repertoire (Kappler et al., 1987) or are clonally inactivated (Ramsdell and Fowlkes, 1990).

MECHANISMS OF POSITIVE SELECTION The use of naturally occurring mouse mutants as well as genetically manipulated animals in the form of gene-deficient or transgenic mice has clarified many of the questions raised by earlier studies on positive selection in chimeric mice (von Boehmer, 1990). Immature T cells do not express the TCR or the auxiliary surface molecules, CD4 orCD8.'^ As described previously, lymphopoietic T-cell precursors first rearrange and express the TCR P gene and only later the TCR a gene (Raulet et al., 1985). TCR p can be expressed on the surface of immature, but not mature, T-cell lines in disulfide linkage to a developmentally regulated protein, named gp33 (Groettrup et al., 1993). The biological significance of this early P expression was confirmed by introduction of TCR p transgene into various rearrangement-deficient mice (von Boehmer, 1990). The results indicated that expression of TCR P favors the development of very immature CD4-, CD8-, TCR negative thymocytes into intermediate CD4+CD8+ thymocytes. The maturation due to this positive selection by TCR P chain involves signaling through a CD3 surface molecule and requires the cytoplasmic /c^-related tyrosine kinase (Levelt et al., 1993). Once the signal is

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delivered, TCR a rearrangement and expression are promoted, while the expression gp33 is terminated (Kishi et al, 1991; Goettrup et al., 1993). Therefore, further TCR P rearrangement is suppressed in the maturing cells, ensuring expression of only one TCR p allele. Further maturation and positive selection of the CD4+CD8+ thymocytes into functionally mature CD4+CD8- or CD4-CD8+ precursors of helper and cytotoxic T cells, respectively, are mediated by the ap TCR. The intrathymic ligands that must bind to the a p TCR to promote development are the polymorphic MHC molecules that are also involved in transport and presentation of antigenic peptides (von Boehmer, 1990). Experiments have shown that the specificity of the a p TCR for either class I or class II MHC molecules determines whether the rescued cells will be of the CD4-CD8+ cytotoxic or CD4+CD8- helper precursor phenotype (Scott et al., 1989). In accord with these conclusions, it was shown that class I MHC and class II MHC-deficient mice lack mature CD4-CD8+ and CD4+8- T cells, respectively (Cosgrove et al., 1991). Positively selected cells eventually leave the thymus and patrol the body as long-lived resting lymphocytes (von Boehmer and Hafen, 1993) that can only be activated by foreign peptides presented by self-MHC molecules. Similar to the role of TCR in positive selection of T cells in the thymus, the immunoglobulin (Ig) receptor has been shown to operate for positive selection of B cells in the germinal centers of spleen (von Boehmer, 1994). As a result of antigen binding and T-cell help, proliferating B cells undergo extensive somatic hypermutation of the variable regions of the Ig heavy and light chain genes (Jacob et al., 1991). Mutants that no longer bind the antigen presented on the follicular dendritic cells die (Weiss et al., 1992). A few cells, namely those with mutated Ig-receptor genes coding for a receptor that binds most efficiently to antigen, are rescued from programmed cell death (apoptosis) through the binding of their surface Ig receptor to antigen and binding of the CD40 molecule by the gp39 ligand (CD40 molecules are present on the surface of B cells, while their gp39 ligand is present on the surface of activated T cells) (Liu et al., 1989; Noelle et al, 1992). As a result, some of the rescued cells become long-lived memory cells (Schittek and Rajewsky, 1990) that, upon encounter with the same antigen, will produce high-affinity antibodies. The differences seen between the processes involved in T-cell and B-cell selection are that the starting point in B-cell selection is a mature rather than an immature cell and that the ligand that induces positive selection represents foreign rather than self protein (von Boehmer, 1994).

NEGATIVE SELECTION AND SELF-TOLERANCE A central tenet of the immune system is self-tolerance, or lack of immune responsiveness to self-components. A random germ line repertoire, positively selected to recognize antigen plus self-MHC, will certainly include autoreactive receptors. Negative selection describes the process whereby a lymphocyte/antigen interaction

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results in the death of autoreactive lymphocytes. Elimination of a self-reactive lymphocyte is termed clonal deletion. However, lymphocytes can be functionally silenced by antigen without being killed, a phenomenon termed clonal anergy (Nossal and Pike, 1980). Clonal deletion as a major mechanism of T-cell tolerance was first directly demonstrated by the absence of self-reactive clones in mice expressing the corresponding superantigen. For example, Vpi7a+ T cells, which frequently recognize MHC antigen I-E, were found to be virtually absent in the periphery of I-E expressing mouse strains (Kappler et al, 1987). Examination of TCR expression in the thymus of these animals showed that clonal deletion occurred during thymocyte development. Whereas there was significant expression of the autoreactive receptors in the immature thymocyte pool, expression was almost completely absent in the mature, medullary population of thymocytes. Clonal deletion was subsequently shown in other cases of Vp-dominated recognition of superantigens. For example, T cells that express Vp8.1, Vp6, and Vp9 are eliminated in mice expressing the superantigen/MHC combination with which they can react, Mis-la (the minor lymphocyte-stimulating antigen-1) plus the appropriate class II alleles (Kappler et al., 1988). Correspondingly, Vp3-expressing T cells are absent from Mls-2a- or Mls-3a-expressing mice (Pullen et al., 1988). In addition to Vpi7a-expressing cells, Vp5- and v p 11-expressing cells are deleted in most I-E-expressing strains of mice (Kappler et al., 1987). Clonal deletion in the thymus has also been induced experimentally in vivo. Injection from birth of Mis-la (MacDonald et al., 1988) or staphylococcal enterotoxin B (White et al., 1989) resulted in the efficient elimination of the mature thymocytes expressing Vps reactive with the antigens. Experiments demonstrated that the expression of different self-antigens causes deletion of thymocytes at different stages of development (Pircher et al., 1989). It is suggested that the timing of deletion is determined by the affinity of the TCR for antigen. Interaction of receptors with an antigen for which they have a high affinity causes earlier thymocyte deletion than interaction of receptors with a low-affinity antigen (Kappler et al, 1988). The affinity hypothesis proposes that whereas thymocytes with both high- and low-affinity receptors are positively selected on self-MHC, only the high-affinity clones are subsequently deleted (Sprent and Webb, 1987). In other words, the affinity theory suggests that very weak engagement is positively selective, whereas stronger engagement is lethal. The question arises as to how tolerance to self-antigens that are not constitutively expressed in the thymus is accomplished. According to one view, circulating self-antigens may be captured by the thymic epithelial cells and effectively presented, in complex with their cell surface MHC, to the developing T cells in the thymus, subsequently resulting in negative selection (Blackman et al., 1990). Another view is that nondeletional mechanisms of peripheral tolerance, such as clonal anergy or suppression, may operate (Kruisbeek et al., 1992).

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Immunological self-tolerance comprises a summation of all of the regulatory influences that prevent autoimmunity. It is not fully clear why some self-antigens induce tolerance by one mechanism or the other. Nevertheless, some potentially autoimmune T cells slip through the traps of selection to escape and give rise, at a later date, to autoimmune disease. The importance of the thymus in inducing tolerance to self-antigens is strengthened by the evidence that neonatally thymectomized mice develop a variety of tissue-specific autoimmune diseases later in life (Kojima and Prehn, 1981). Neonatal thymectomy (NTx) probably interferes with clonal deletion and permits self-reactive T cells to escape. It may also eliminate a population of regulatory or suppressor T cells. Although factors controlling these autoimmune diseases are poorly understood, defects in clonal selection and clonal deletion have been assigned to be the major causes of (NTx) autoimmune diseases (Smith et al., 1989). Tolerance induction in B cells was documented long ago (Nossal and Pike, 1975; Metcalf and Klinman, 1977). Immature B cells in the bone marrow were considered especially sensitive to tolerance induction by one nondeletional mechanism— clonal anergy (Nossal and Pike, 1980). Mature B cells could also be rendered tolerant if they encounter antigen in the absence of T-cell help or other co-stimulatory influences, but only at much higher antigen concentrations (Pike et al., 1981). In recent years, with the help of transgenic mouse technology, it has been revealed that all three mechanisms of tolerance induction (deletion, anergy and clonal balance) may be involved in B-cell tolerance (reviewed in von Boehmer, 1994). During development, some lymphocytes with self-reactivity ignore a self-antigen because of low affinity, poor accessibility, lack of suitable presentation, or absence of appropriate help. Such lymphocytes may induce strong autoimmune responses following changed operational circumstances (Nossal, 1994).

AUTOIMMUNE DISEASE Autoimmune disease results from an attack on the host's own tissues by elements of the immune system. Examples of diseases with an autoimmune component range from multi-system disorders, such as systemic lupus erythematosus or rheumatoid arthritis, to localized diseases, such as multiple sclerosis, Type I diabetes, and thyroiditis (Basten, 1989). Many different pathways can lead to the development of autoimmune disease. The central event is the proliferation of high-affinity self-reacfive T cells by a variety of inifiators. Molecular mimicry of self-antigens with bacterial, viral or other external antigens may be one such triggering event (Penhale and Young, 1988; Oldstone, 1989). Alterafion of self-antigens by chemicals, heavy metals or other toxic environmental substances may also potentially contribute to induction of an autoimmune response (Weigle and High, 1967). Aberrant expression of class II MHC has been proposed to play a role in the development of autoimmune diseases (Bottazzo et al., 1983).

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Once the T cell has been stimulated, it can induce autoimmune disease through a variety of effector mechanisms. It may produce lymphokines that directly injure surrounding cells (Sinha et al., 1990); cytotoxic cells directly produce tissue destruction (Sarvetnick et al., 1988); T-cell and B-cell interaction may activate self-reactive B cells to generate autoantibodies (Mamula et al., 1992), and the autoantibodies may directly affect target organs (Stanley et al., 1984); or antibody may cooperate with complement or "killer" cells to produce tissue injury (Mamula etal., 1994). The immunological recognition of an antigen by T-helper cells is directed to a limited number of sites on the antigen molecule. All antigenic proteins have stretches of amino acids which, in peptide form, are able to fit a common peptidebinding site on the class II molecule recognized by the T-cell receptor (Buus et al., 1986). The identification of these antigenic determinants of autoantigens could enable the development of effective therapies to block the antigen-specific generation of the autoimmune response.

AUTOIMMUNE THYROID DISEASE Autoimmune thyroid disease (AITD) is a prototype of an autoimmune disease, where the thyroid gland is targeted by the immune system. AITD clinically presents in different forms; the two most commonly encountered are Hashimoto's thyroiditis (HT), usually causing hypothyroidism, and Graves' disease (GD) in the form of hyperthyroidism. The recognition of HT as a classical example of an autoimmune disorder is credited to the independent discoveries of experimental thyroiditis and anti-Tg autoantibodies by Rose and Witebsky (1956) and Roitt et al. (1956). The concept that GD can also be included in the group of thyroid autoimmune disorders stems from the discovery by Adams and Purves (1956) of the long-acting thyroid stimulator (L ATS) and the subsequent demonstration that L ATS is a thyroid-stimulating autoantibody responsible for the hyperthyroidism of GD. Incidence

HT occurs more frequently in females than in males, with a ratio of about 4:1. The peak incidence is between 30 to 50 years of age (Bigazzi and Rose, 1985). A juvenile variant occurs in children and adolescents. The prevalence of thyroiditis in the general population varies between 0.5% to 1.2%. Epidemiological studies have demonstrated that the incidence of HT is increasing, especially in younger age groups (Safran et al., 1987). Histopathological Changes

The thyroid glands of patients with AITD are usually enlarged. In HT, the thyroid gland shows diffuse mononuclear cell infiltration, consisting mainly of lymphocytes. These cells may aggregate to form lymphoid follicles with germinal centers.

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Plasma cells and macrophages are also present. The glands may show parenchymal damage, hyperplasia of thyroid epithelial cells and variable degrees of fibrosis. In GD, the thyroid is diffusely enlarged owing to parenchymal hypertrophy and hyperplasia. Lymphocytic and plasma cell infiltration often occur. The lymphocytic infiltrates are composed of approximately equal proportions of T cells and B cells (Totterman, 1978). The percentages of CD4+ and CD8+ cells, as reported by various studies, vary considerably (Del Prete et al., 1986; Bagnasco et al, 1987; Margolick et al., 1988; Cohen and Weetman, 1988), but CD8+ lymphocytes seem to predominate in HT (Mariotti and Pinchera, 1990). Cytotoxic T cells have been cultured from lymphocytic infiltrates, and their phenotypes have been mainly CD4-CD8+. Aberrant expression of class IIMHC by thyroid cells is an observation by some investigators (Davies et al., 1988). MHC class II antigens are not detectable on the surface of normal thyroid epithelial cells; however, HLA-DR and -DQ molecules have been found on thyroid epithelial cells from patients with AITD (Most et al., 1986). This led to the hypothesis that such epithelial cells could become APCs, capable of stimulating autoreactive T cells by presentation of endogenous autoantigens (Bottazzo et al., 1983; Weetman et al., 1986). However, the idea that this phenomenon could be the initiating event in AITD seems unlikely, as the phenomenon is dependent on local T-cell infiltration (Cohen et al., 1988). Nevertheless, class Il-expressing thyrocytes may play a role in perpetuating the autoimmune response (Weetman, 1992). Autoantigens Three different autoantigens, namely, thyroglobulin (Tg), thyroid peroxidase (TPO) or microsomal antigen and thyroid-stimulating hormone receptor (TSHR) have been recognized as playing a role in the development of AITD. Autoantibodies against these antigens have been detected in patients suffering from HT and GD. Until recently, Tg was the only antigen known to induce experimental autoimmune thyroid disease in animal models. TPO was recently reported to have induced experimental murine thyroiditis (Kotani et al., 1990). Among other antigens to which autoantibodies have been detected in thyroiditis patients are the second colloid antigen (Bigazzi, 1990); autoimmune thyroid disease related autoantigen (ATRA-1), recognized by about 27% of Hashimoto's sera (Hirayu et al., 1987); and a 70 kD antigen (Chan et al., 1989). The significance of these antigens is unknown. Antibodies against T4 and T3 have also been detected, although infrequently, in patients. They may represent a subset of anti-Tg antibodies, specifically recognizing a short Tg sequence containing iodothyronine (Pearce et al., 1981). There may be other, as yet unknown, cell surface antigens involved in AITD.

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Animal Models

Several animal models of experimentally induced thyroiditis (EAT) or spontaneously induced thyroiditis (SAT) have been described (Kuppers et al., 1988). EAT may be induced by injection of homologous or autologous Tg in many animal species, including rabbit, guinea pig, mouse, rat, dog, monkey, chicken, and cat. T cells play a central role in the mechanisms responsible for EAT, as the disease can be induced by adoptive transfer of T cells (Felix-Davies and Waksman, 1961; Twarog and Rose, 1970; Romball and Weigle, 1987). SAT has been described in chickens, rats, and dogs. The OS chicken is the best studied model of human HT, being characterized by spontaneous appearance of high titers of antibodies to Tg. The lymphocytic infiltration of the thyroid gland and the subsequent destruction of thyroid follicles presents similarities to the human disease. Another model of spontaneous thyroiditis was introduced by Penhale et al. (1973); it involves neonatal thymectomy and irradiation of Wistar rats. Recently, thyroiditis has been reported to develop in NOD mice (Bernard et al., 1992). It might prove to be a good model to study human AITD as a large collection of murine immunological reagents is available. So far, no counterpart of GD in experimental animals has been established. Multifactorial Etiology

Evolving knowledge of the human immune system and the results of studies on thyroid autoimmunity suggest that the cause of autoimmune disease is multifactorial. The development of autoimmune disease involves genetic and environmental influences that amplify self-reactivity. The complex interplay between these multiple factors may shift the balance and contribute to the pathogenesis of AITD. The OS chicken is one of the best established animal models for a spontaneously occurring autoimmune disease. Much of our knowledge of a multifactorial etiology of AITD comes from this model of SAT. The various factors that seem to be involved in the autoimmune process in the OS chicken include: (i) immune dysregulation, including severe mononuclear cell infiltration, induction of anti-Tg autoantibodies, hyper-responsiveness to ConA stimulation, decreased suppression, (ii) genetically determined susceptibility of the target organ (thyroid gland) such that there is a lower threshold of thyroid epithelial cells for MHC expression via gamma IFN, which may perhaps take over the role of antigen presentation from the initially involved macrophages and endothelial cells, (iii) hormonal influences— presence of high amounts of corticosteroid which, in turn, leads to a heightened immune responsiveness in the OS chicken, and (iv) environmental factors, including endogenous virus (ev 22) and dietary iodine (Wick et al, 1986). Similar mechanisms may be operative in HT.

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Immunological Response

Humoral and eel I-mediated immunity. Antibodies to Tg are present in 8090% of patients with HT, and 50-60% of patients with GD (Amino et al., 1976). B cells secreting anti-Tg antibodies have been demonstrated in the peripheral blood and among the intra-thyroidal lymphocytes of patients with AITD (McLachlan, 1983; litaka et al., 1988). Since there are relatively few circulating Tg-specific antibody-secreting B cells, it has been suggested that antibody formation occurs mainly in the thyroid, where antibody-secreting cells are more easily demonstrated (McLachlan et al., 1979; Weetman et al., 1982). Marked dissociation has been observed between the extent of histological thyroiditis and the levels of autoantibodies. The natural experiment of transplacental antibody passage from mother to the fetus suggests that antibody alone cannot destroy the thyroid gland (Parker et al., 1961). Autoantibodies to Tg in combination with mononuclear cells can produce antibody-dependent cell-mediated cytotoxic activity (Calder et al., 1974) or may cause injury by forming immune complexes and activating the complement system (Kalderon et al., 1977). In animal model studies of OS chickens, autoantibodies have been shown to increase the severity of mild thyroiditis but, by themselves, do not have the ability to induce disease (Jaroszewski et al., 1978). Isotype restriction of Tg autoantibodies has also been reported. One study states that anti-Tg antibodies in HT patients are primarily IgG4 and IgGl (Thompson et al., 1983). Another study found that, although IgG4 is over-represented, it accounts for around only 30% of the Tg antibody contained in total IgG (Weetman et al., 1988). Recently, Kuppers et al. (1993) have reported that all four IgG subclasses were used in the response to human Tg, although the pattern of usage varied between AITDs. Patients with HT showed a higher proportion of IgG2 antibody than did GD patients. The predominance of a particular subclass of autoantibodies may have diagnostic significance as a predictor of disease. There is speculation that Tg autoantibodies of different IgG subclasses interact with different epitopes on Tg (Fukuma et al., 1989). Animal experiments have demonstrated that autoimmune thyroiditis can be induced by transfer of Tg-specific T cells into naive syngeneic animals (FelixDavies and Waksman, 1963; Twarog and Rose, 1970; Romball and Weigle, 1987), thus providing evidence for a T-cell-mediated immune mechanism as a cause for the development of autoimmune thyroid disease. A specific T-cell reaction to Tg has generally been found in AITD patients (Weetman and McGregor, 1984), although optimal antigen concentration appears to be variable. Peripheral and intra-thyroidal T cells proliferate in vitro when cultured with autologous DR+ thyroid cells (Del Prete et al., 1987). The precise characterization of antigenic determinants recognized by the T cells needs to be investigated. Many studies have examined T-cell subsets in thyroid tissues of patients with active autoimmune thyroid disease. Increased CD8 (cytotoxic or suppressor) T cells and increased CD4 helper T cells have been reported in GD patients (Margolick et al., 1984). Increased

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proportions of activated cytotoxic/suppressor T cells, expressing HLA-DR antigen, have been found in HT patients (Canonica et al, 1984). Role of cytokines. Cytokines produced by T cells, macrophages and other cell lines regulate the function and activity of T cells. There is evidence of the production of gamma interferon (IFN-y) and alpha tumor necrosis factor (TNF-a) in intrathyroidal lymphocyte infiltrates (Del Prete et al., 1989). In situ production of IFN-y in the thyroid gland of AITD patients has ak D been demonstrated (Margolick et al., 1986). The increased production of IFN-y has been shown to induce HLA-DR antigen expression on the surface of human thyroid cells. The aberrant expression of class II MHC antigens on thyroid epithelial cells has been hypothesized to be an early event of thyroid autoimmunity (Hanafusa et al., 1983). Production of IL-1 and IL-6 by the thyroid cells has also been demonstrated (Zheng et al., 1991). This phenomenon could be particularly relevant, as a DR+ thyroid cell producing IL-1 and IL-6 could be considered a fully functioning antigen-presenting cell, able and sufficient to trigger autoimmune reactions. IL-6 is one of the most important cytokines affecting B-cell development. The production of IL-6 is enhanced by TNF and IL-1 (Weetman et al., 1990). As thyroid cells are exposed to these cytokines in thyroiditis, it is likely that the enhanced IL-6 production within the diseased gland, in turn, stimulates plasma cell formation, leading to localized autoantibody formation. In addition, both IL-1 and IL-6 activate T cells; their localized production by thyrocytes may further promote the development of thyroiditis by stimulating cell-mediated effector mechanisms. Recent reports demonstrate that patients receiving cytokines as therapeutic agents seem unusually prone to develop thyroiditis. The immunological effects produced by IL-2 and GM-CSF therapy sometimes result in the common development of autoimmune thyroiditis (Burman et al., 1986; Von Leissum et al., 1989; Hoekman et al., 1991). Suppressor cell defect A partial antigen-specific defect in suppressor T lymphocytes as the basis for AITD has been suggested by Volpe (1991). Self-tolerance to Tg is hypothesized to be maintained by an immunoregulatory balance between helper and suppressor T cells (Volpe, 1988). Volpe suggests that an HLA-related gene abnormality in antigen presentation to T lymphocytes may result in inadequate activation of antigen-specific suppressor T cells. Environmental factors adversely affecting overall suppressor T-cell function may serve to precipitate AITD. The consequent activation of specific helper T cells leads to cytokine and thyroid antibody production, and the combined effects of cytokines, thyroid antibodies and cellular components on thyrocytes complete the pathological picture. Recently, Volpe's group has demonstrated that peripheral blood mononuclear cells (PBMC) from HT and GD patients, when stimulated in culture with Tg, have markedly higher proliferative responses when they are CD8 depleted and CD4 enriched (Akasu et al., 1993). This finding suggests that suppressor CD8 cells play a role in

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preventing CD4 cells from proliferating due to Tg-stimulation. Other investigators do not agree on the role of antigen-specific T-suppressor cells (Weetman, 1989). Some have even questioned the very existence of suppressor cells (MoUer, 1988). Genetic Predisposition HT and GD frequently occur in the same family and several cases of twins with either of the two conditions have been reported (Hall et al., 1972; Volpe et al., 1973; Doniach, 1975). Thyroid antibodies are found in approximately 50% of first-degree relatives of patients with AITD (Farid et al., 1977). Using more sensitive assays, segregation analysis of families with thyroiditis suggested that the inheritance of thyroid autoantibodies is a Mendelian dominant trait in women, with reduced penetrance in men (Philips et al., 1990). HL A genes are supposed to control immune responsiveness to a variety of foreign and self-antigens. In many autoimmune conditions, associations have been reported with certain MHC alleles. An association of HLA-DR5 with HT was reported (Farid et al., 1981). In one small study from Newfoundland, HT was associated with DR4 (Thompson and Farid, 1985). Another study in patients from Toronto and Denmark reported an association with DR5 (Vargas et al., 1988), while from Hungary DR3-association was reported (Stenzsky et al., 1987). The DNA-restriction fragment length polymorphism (RFLP) method of DR typing, which is less prone to error, has shown that DR3 was more frequent in English patients with HT (Tandon et al., 1991). An RFLP study from the USA found no HLA-DR associations with HT (Mangllabruks et al., 1991). Transracial comparisons of HLA association with HT are inconsistent. HLA-B17 and HLA-Bw46 have been reported in blacks and orientals, respectively (Hawkins et al., 1987). In Japanese, B16 is increased and DR2 is decreased (Nakao et al., 1978; Sakurami et al., 1982). In a more recent report from Japan, an association with HLA-DRw53 was noted (Honda et al., 1989). In contrast, a significantly increased prevalence of HLA-B8 and -DR3 has been reported in Caucasians with GD (Farid, 1981). Thus, HLA associations are either weak or inconsistent between populations. As described previously, the T-cell receptor (TCR) is a heterodimer, composed of two chains, a and p. Another heterodimer, 76, also exists but its ftinctional significance in autoimmune disease is not clear. Restricted heterogeneity of the gene for the variable regions of the a-chain of the human TCR has also been reported (Davies et al, 1991). Restriction of the T-cell repertoire at the site of autoimmune attack would imply a role for a selected subset of T cells in the pathology of the disease. Such restriction would have therapeutic implications: therapy with monoclonal antibodies to specific TCR, peptide fragments of the TCR or with the T cells themselves might be possible. However, recent reports by other investigators have failed to show evidence of limited variability of antigen receptors on intrathyroidal T cells (Mcintosh et al., 1993).

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Animal studies also lend support to the role of genetic control of susceptibility. Variation in the susceptibility of various strains of mice to induction of experimental autoimmune thyroiditis (EAT) indicates the influence of genetic factors. The response was found to be predictable on the basis of their MHC (H-2) genotype (Vladutiu and Rose, 1971). A number of other genes, such as the IgG heavy chain allotypes, may also be involved (Kuppers, 1988). Recently, Gleason et al. (1990) investigated the binding specificities and nucleotide sequences of murine monoclonal anti-Tg antibodies. They report no evidence for preferential use of any V-region family or gene segment, suggesting that the antigenic epitopes on mouse thyroglobulin (mTg) elicit a very diverse autoantibody response that is derived from a large number of V-region gene segments. A recent study in humans (Chazenbalk et al., 1993), using another thyroid antigen—^TPO, demonstrated that the putative germ line genes used by the TPO human autoantibodies involved only five different heavy and light (H and L) chain combinations. In addition, the study found that the same H and L combinations were used by mukiple patients. Further analysis revealed that the F(ab) proteins expressed by these genes define two major, closely associated domains in an immunodominant region on TPO. These domains contain the binding sites of 80% of IgG-class TPO autoantibodies in the sera of patients with autoimmune thyroid disease, thus indicating that there is a restriction in H and L chain usage in relation to the interaction with specific antigenic domains in human, organ-specific autoimmune disease. Genetic defects, such as haplotypes associated with hyper-responsiveness of the immune system (evidenced by high Tg autoantibody titers and severe SAT) observed in OS chickens, also contribute to our understanding of the role of genetic loci for the altered reactivity of the immune system and the susceptibility of the target organ for autoimmune effector mechanisms (Wick et al., 1986). Endocrine Abnormality

A hypothesis presented by Wilkin (1989) argues that the primary lesion of autoimmune disease is in the target organ. According to this theory, a heightened immune response is a physiological reaction to the excessive production and turn-over of the antigens from the primary lesion. Hence, autoimmune thyroiditis is a result of the excessive production of Tg by the thyroid gland; on the other hand, there are some reports that raising the serum Tg levels can downregulate autoimmune thyroiditis in mice (Lewis et al., 1987). Structural abnormality of Tg has been suggested to be a possible factor in the induction of autoimmune disease, but no link so far of any heterogeneity at the molecular level has been observed in AITD. A mutation within exon 10 of the Tg gene has been demonstrated in patients with non-toxic goiter, leading to less efficient production of thyroid hormone (Corral et al., 1993). Hormones also appear to play an important role in the regulation of the autoimmune process. A strong female:male predominance for autoimmune thyroid disease

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suggested an influence by sex hormones. The sex hormones have a variety of effects on the immune system. Investigations showed that estrogens exacerbate experimental autoimmune thyroiditis (EAT), whereas testosterone ameliorates it (Okayasu et al., 1981; Weetman, 1991). Prolactin may have a role because asymptomatic autoimmune thyroiditis is more common in hyperprolactinemic disorders (Ferrara et al., 1983). Altered levels of these hormones and other changes which accompany pregnancy presumably account for the remission seen in the last trimester, and the subsequent exacerbation of subclinical thyroiditis and rise in autoantibody levels which constitute the entity of post-partum thyroiditis (Lazarus and Othman, 1991). Similarly, after onset of puberty, the prevalence of thyroid autoimmunity rose among females (Burek et al., 1982). Environmental Factors

Infectious agents. Immune reaction to an environmental antigen could lead to antibodies that cross-react with thyroglobulin or other thyroid antigens. Molecular mimicry between bacterial and viral antigenic determinants and thyroid autoantigens could lead to the induction of an immune response against the target organ and to the development of autoimmune disease. A high frequency of antibodies directed to human thyroid cell cytoplasm has been observed in sera of patients with Yersinia enterocolitica infection (Lidman et al., 1976). Conversely, a high frequency of antibodies to Y. enterocolitica has been reported in patients with various thyroid diseases (Safron, 1987). TSH-specific binding sites sharing several properties with the human TSH receptor have been found in Y. enterocolitica and to a lesser extent in other bacteria (Weiss et al., 1983). A similar phenomenon has also been observed in patients with infections due to leishmania and mycoplasma (Safron, 1987). Virus infections of thyroid cells have been frequently suggested as potential triggers of AITD through induction of structural alterations of autoantigens or enhancement of antigen presentation to the immune system (Joasoo et al., 1975), but evidence in favor of this hypothesis has been difficult to obtain. Epstein-Barr virus infection has been implicated in the development of autoimmune thyroiditis in three patients (Coyle et al., 1989). A recent report of nucleotide and amino acid homology between human thyrotropin receptor and the HIV-1 nef protein suggests an avenue through which a shared response against an HIV-1 related retrovirus could play a role in the pathogenesis of GD (Burch et al., 1991). Role of iodine. Various reports on the role of increased dietary iodine consumption in humans suggest that a significant relationship exists between high iodine intake and increased prevalence of autoimmune thyroid disease (Gaitan et al, 1985; Tajiri et al., 1986; Roti et al, 1987; Koutras et al., 1987). The exact mechanism of this relationship is not well understood. Weetman et al. (1983) have demonstrated that iodine enhances IgG synthesis by human peripheral blood

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lymphocytes in vitro. Studies of animal models provide ample evidence for the pathogenic role of iodine in AITD. A significant increase in lymphocytic thyroiditis in young BBAV rats was observed with increased iodine ingestion (Braverman, 1989). Similar observations have been made in the spontaneous thyroiditis model in OS chickens (Bagchi et al., 1985). Experimental evidence suggests that incorporation of dietary iodine into thyroglobulin increases its immunogenicity (Sundick etal., 1987). Current information suggests that iodine is a major contributing environmental factor in the pathogenesis of the multifactorial etiology of AITD. More detailed investigations are needed to establish a correlation and to understand the immunological effects of iodine. Other Factors

Stress. The role of stress in triggering and modulating autoimmune mechanisms responsible for GD has been envisaged (Safran et al., 1987). Physical and psychological stressful events have been associated with the onset of GD (McKenzie and Zakarja, 1989). It is hypothesized that stress could aggravate the putative defect of both nonspecific and thyroid-specific T-suppressor lymphocytes (Volpe, 1986). It may also induce changes in levels of cytokines (Abraham, 1991). Drugs. Serum anti-thyroid antibodies and overt clinical AITD have been reported to be increased in patients receiving lithium therapy (Safran et al., 1987). The in vitro activity of lithium on some immune parameters is consistent with augmentation of helper T-cell activity and macrophage phagocytosis (Safran et al., 1987). Animal experiments demonstrate that lithium may increase anti-Tg antibodies in rat EAT but does not induce thyroid antibodies in normal nonimmunized animals (Hassman et al., 1985). Treatment with amiodarone (an iodine-containing drug) has been reported to affect thyroid function, inducing either hyper- or hypo-thyroidism (Martino et al., 1984). Thyroid autoantibodies developed in several patients treated with amiodarone, but disappeared after withdrawal of the drug (Monteiro et al., 1986). Amiodarone has also been shown to affect T-cell function, increasing the number of both helper and suppressor/cytotoxic T cells (Rabinowe et al., 1986). The cause of this abnormality, whether due to amiodarone itself or to its iodine content, remains to be elucidated. Toxins. Several environmental toxins have been proposed as potential inducers of AITD. They include polybromated biphenyls, phenols, thiocyanate, hydroxypyridines, substituted dihydroxybenzenes, and resorcinol derivatives (Safran et al., 1987). Carcinogens, such as methylcholanthrene and dimethylbenzanthracene, have also been shown to induce EAT in certain genetically susceptible

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strains of rats (Reuber, 1970). Similar factors in humans genetically predisposed to the development of AITD could be the initiating factors of the disease.

THYROGLOBULIN Human Tg is a huge molecule of 660,000 molecular weight. It is a homodimer, with each chain containing 2748 amino acids. The nucleotide structure of the mRNA is composed of 8448 base pairs. The gene sequence of Tg is comprised of over 300 kb, with 37 exons (Malthiery et al., 1989). The only known function of this protein is serving as a prohormone for thyroxine, the hormone which regulates the vital metabolic functions of the body. Of 110 tyrosine residues in each chain, only four have been shown so far to be involved in the formation of the hormone. The rest of the structure of this huge molecule is not known to serve any other function, except for conserving iodine for efficient production of the hormone. Some structural homology of Tg has been seen with the acetylcholinesterase molecule (Schumacher et al., 1986), and the alternatively expressed domain of the invariant chain of the la molecule associated with the MHC system (Koch et al., 1987). Recently, McLachlan and Rapoport (1989) showed evidence of structural homology of a T-cell epitope between human TPO and human Tg, and possibly certain bacterial proteins. They suggest that this common T-cell epitope may play a role in the pathogenesis of AITD. From a pathological aspect, Tg is the major antigen involved in autoimmune thyroid disease. In order to understand the process of antigen recognition for production of autoantibodies and to determine the immunodominant regions of Tg involved in T-cell activation, it is essential to characterize the B-cell and T-cell epitopes of Tg. The results of such an investigation can aid in the understanding of the immunological process of the autoimmune disease. The knowledge so obtained can further be extended for designing preventive measures and immunotherapeutic approaches for autoimmune thyroid disease. Overall, it can form a basis for the study of other, more crippling, autoimmune diseases. Investigations reported to date on the characterization of B-cell epitopes of Tg initially suggested 40 antigenic sites (Heidelberger, 1938) on the basis of recognition by xenogeneic antibodies. Analysis performed by competitive binding between autoantibodies and monoclonal anti-Tg antibodies suggests 4 to 6 major antigenic determinants of Tg (Nye et al., 1980). Results also suggest different determinants for pathological and natural autoantibodies (Piechaczyk et al., 1987; Bresler et al., 1990). Species cross-reactivity has also been utilized to demonstrate multiple epitopes of Tg (Chan et al, 1987). Some experiments suggest that autoantibodies recognize only the conformational epitopes (Kondo and Kondo, 1984). Recent reports, using recombinant peptides of Tg, have shown that Tg autoantibodies are highly heterogeneous (Henry et al., 1990). There is some evidence of certain epitopes being more frequently involved in autoimmune reactions.

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Recently, the identification of three different T-cell epitopes of Tg have been reported by three different investigators. All of these epitopes are in different regions of Tg. A 40 amino acid synthetic peptide in the 1672—1711 residue region of human Tg was shown to be recognized by a human Tg-specific cytotoxic hybridoma (Texier et al., 1992). Another group of investigators (Hutchings et al., 1992) report a 12 mer synthetic peptide containing a thyroxine at position 2553 in Tg to be recognized by two clonotypically distinct murine Tg autoreactive T-cell hybridomas. This peptide could not directly induce thyroiditis in susceptible mice, but adoptive transfer of in vivo primed lymph node cells boosted in vitro with peptide could transfer thyroiditis in naive recipients. A third group of investigators (Chronopoulou and Carayanniotis, 1992) has reported a 17 mer peptide corresponding to amino acids 2495 to 2511 of the human Tg, with one residue difference at position 2510. This peptide has been shown to induce mononuclear cell infiltration of the thyroid gland in susceptible mice.

SUMMARY The mechanisms for achieving self/non-self distinction include clonal deletion, clonal anergy, and clonal balance. Clonal deletion results in the elimination of self-reactive T cells during their maturation in the thymus. T cells reactive with self-antigens not represented in the thymus, and probably some low-affmity T cells, escape clonal deletion and populate peripheral lymphoid tissues and blood. These self-reactive T cells are normally held in check by clonal anergy or clonal balance. Anergy occurs when a T cell receives its antigen-specific signal in the absence of required co-stimulatory signals. Clonal balance depends upon the ratio of stimulatory and inhibitory signals delivered to the T cells. Self-tolerance of B cells probably depends upon similar mechanisms, but is less effective. Autoantibodies, therefore, are more common than self-reactive T cells. Autoimmune disease results from an attack on the host's own tissues by self-reactive T cells or antibodies. Autoimmune thyroid disease is a well-characterized paradigm of disease caused by autoimmunity. Graves' disease results from an over-active thyroid gland produced by autoantibodies directed to the receptor for the thyroid-stimulating hormone. Hashimoto's thyroiditis is associated with thyroid damage produced by self-reactive T cells directed to thyroglobulin or, possibly, thyroperoxidase. The disease can be reproduced in experimental animals by immunization with autologous thyroglobulin, providing firm evidence that the thyroiditis is caused by autoimmunization. The etiology of autoimmune disease is multifactorial and includes both genetic and environmental factors. Several genes are involved, affecting both immune regulation and vulnerability of the target organ. Among the environmental factors are infectious agents, drugs, diet, and toxins. Hormones and even emotional status also play a role. Both T cells and antibodies are involved in the pathogenesis and the demonstration of autoantibodies is useful in diagnosis. Future treatment and

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prevention of autoimmune disease depends upon precise delineation of the pathogenic determinants of the antigens.

NOTES 1. MHC = major histocompatibility complex. See Chapter 7. 2. CD = cluster determinants. See Chapter 3.

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Chapter 14

Cell Death and the Immune System R.M. KLUCK and J.W. HALLIDAY

Introduction Cell Death Definitions Morphology of Apoptosis Biochemistry of Apoptosis Fate of the Apoptotic Cell Cell Death of Immune Cells Death During Development and Maturation of Immune Cells Death of Mature Immune Cells Cell Death Induced by the Immune System—^Target Cell Death Apoptosis in Clinical Immunology Inflammation Infection Cancer Host-Graft Interactions Autoimmune Disease Summary Recommended Readings

Principles of Medical Biology, Volume 6 Immunobiology, pages 265-280. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-811-0

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INTRODUCTION Cell death plays a critical role at several levels of immune function. It is integral to the development of immune cells of lymphoid and myeloid origins, as well as to the effects of mature immune cells and lymphokines on their targets. The form of cell death shown to predominate in all of these processes is known as apoptosis, an "environmentally friendly" form of cell death, as opposed to necrosis. Significant recent findings in the relationship of apoptosis to the immune system have provided a novel and fruitful approach to the understanding of immune function as well as new avenues for the clinical treatment of immune-related diseases.

CELL DEATH Definitions Until quite recently, all cell death was considered to take the form of necrosis, where gross damage results in lysis of the cell membrane and dispersal of the cell contents, initiating further damage and inflammation. Another form of cell death was described by Kerr, Wyllie and Currie (1972). This was termed apoptosis (pronounced apo-TOE-sis), a Greek word meaning "falling off as of the leaves from trees, descriptive of its central role in the homeostasis of tissues. In vivo, apoptosis is often inconspicuous as it affects isolated cells, it proceeds rapidly, and has limited sequelae to indicate its passage. It is the physiological form of cell death, and has been referred to as "altruistic suicide" to distinguish it from the accidental or "destructive" death of necrosis. Necrosis often follows severe environmental trauma, which either directly damages the plasma membrane or interferes with the generation of energy by blocking the synthesis of ATP. Apoptosis in the immune system is often referred to as "programmed cell death" in reference to either its regulation by gene expression (Schwartz and Osborne, 1993), or to a perceived role in the survival of the organism as a whole (Cohen, 1993). Morphology of Apoptosis Apoptosis has characteristic morphological features (Figures 1 and 2), which include compaction of chromatin leading to uniformly dense, well circumscribed masses abutting the nuclear membrane, nucleolar disintegration, cell shrinkage with preservation of organelles, detachment from surrounding cells, and nuclear and cytoplasmic budding to form membrane-bound fragments, known as apoptotic bodies (Kerr et al., 1995). These are rapidly phagocytosed by adjacent parenchymal cells or by macrophages. Necrosis, in contrast, exhibits non-uniform chromatin density, swelling of organelles, lysis of membranes, and dispersion of the cellular

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System

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Figure 1, The morphological characteristics of apoptosis and necrosis as seen by electron microscopy. Cytotoxic T cell killing results in apoptosis (A) of a mastocytoma target cell (X6000). Note the electron dense chromatin lying in crescent shapes inside the nuclear membrane. Two normal cytotoxic T cells (T) adjacent to the apoptotic cell are also seen. In contrast, transformed B cells in late necrosis (N) exhibit marked disruption of chromatin, membranes and organelles (X8000). (Courtesy of Professor j.F.R. Kerr, Department of Pathology, University of Queensland.)

Table 1. Characteristics of Apoptosis and Necrosis Apoptosis Chromatin DNA Nucleus Cell size Cytoplasm Organelles

Cell membrane Cell fate

- dense sharply delineated masses adjoin the nuclear envelope - cleavage between nucleosomes => ladder on electrophoresis - commonly buds - decreases - condensation due to extrusion of water - well preserved

- convolutes and buds - loss of microvilli - rounded apoptotic bodies

engulfed by monocytic macrophages without inflammation

Necrosis -scattered mild clumping - random degradation => smear on electrophoresis - never buds - increases - swelling as membrane pumps fail - swelling of endoplasmic reticulum and mitochondria - ribosomes disappear -cell lysis - cell debris inititiates acute inflammatory response

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contents which initiate further cell damage and inflammation, with eventual clearance by macrophages (see Table 1). Biochemistry of Apoptosis The biochemistry of apoptosis needs to be considered from two perspectives, (1) the molecular changes that either predispose a cell to or protect it from apoptosis and (2) the molecular changes that occur in the cell when it undergoes apoptosis. Distinguishing between these two areas is difficult because the biochemical stage at which an individual cell is irreversibly committed to undergo apoptosis is unknown. Arends and Wyllie (1991) conceptualize cells "primed" for apoptosis as those that have a molecular profile which makes them susceptible to the irreversible "triggering" of the apoptotic pathway. In 1980, Wyllie observed that DNA extracted from apoptotic thymocytes was cleaved at linker regions between nucleosomes, possibly by a Ca^'*"-dependent endonuclease. This cleavage results in a characteristic ladder pattern on gel electrophoresis (Figure 2) and contrasts with the random DNA degradation seen following necrosis (Figure 2). More recently, it has been shown that DNA cleavage to large fragments of 50 and 300 Kb in size is an earlier and more universal feature of apoptosis. This cleavage of DNA in the dying cell provides a means of deleting potentially dangerous genetic material. Inhibitors of protein synthesis inhibit, or delay, apoptosis in some cells encouraging the concept of apoptosis as an active, and programmed, process. However, an essential role for any specific newly synthesised protein has not been verified (Schwartz and Osborne, 1993). The susceptibility (priming) of cells to apoptosis has been shown recently to be influenced by the activity of several proteins in the cell. Three of these proteins are products of cellular oncogenes c-myc, bcl-2, andp55 found in all cell types and are involved in intracellular biochemical pathways. Activity of these proteins is determined by both the rate of gene expression and the rate of degradation, with genetic mutations commonly affecting activity. Susceptibility to apoptosis has been associated with high c-Myc, high p53 and low Bcl-2 protein levels; current investigation of the biochemical properties of these and other proteins suggests great potential for manipulating the apoptotic process. With respect to immune cell development, survival of immature B and T cells seems to require increased bcl-2 expression. The expression of the tumor suppressor gene p5 3 increases in response to DNA damage with a resulting increase in cell death by apoptosis. In mice in which both copies of the p53 gene have been deleted, apoptosis following DNA damaging agents does not occur. Somatic mutations leading to increased c-Myc, increased Bcl-2, and to mutant p53, have been implicated in the etiology of cancer, possibly consequent upon apoptosis failure. Thus a complex network of gene expression is likely to be involved in priming cells for apoptosis. In addition to the expression of intracellular proteins, the expression of receptor proteins on the cell surface of immune cells and their target cells plays a crucial

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role in apoptosis during immune cell development and function. For example, failure to express the CD95 (Fas/APO-1) receptor protein results in decreased thymocyte apoptosis, and a lymphoproliferative disorder in mice akin to that in autoimmune diseases. Following the application of various apoptotic stimuli there is no known invariable sequence of biochemical events, but such events may encompass changes in intracellular ionized Ca^^, H"*", ceramide, oxidative stress, tyrosine kinase activation, as well as new protein synthesis. As these same messengers can also mediate cell activation and proliferation in the same cells, it is likely that the ultimate triggering of apoptosis is determined by a complex interplay of positive and negative influences. The trigger point at which a cell becomes committed to undergo apoptosis is currently not known. However, it is likely to be prior to, or even at, the initiation of a series of proteolytic events involving the recently discovered ICE/ced-3/CPP32 family of cysteine proteases (Femandes-Alnemri et al., 1994). One or more of these proteases exist in the cytoplasm of all cells in an inactive pro-form. Apoptotic signals lead to their conversion to active proteases which subsequently cleave a range of substrate proteins in the nucleus (lamins, poly (ADP-ribose) polymerase, U1 snRNP) and cytoplasm (actin, fodrin). It is yet to be ascertained how the cleavage of these structural and catalytic proteins may relate to the structural and functional collapse of the cell to form apoptotic bodies. Fate of the Apoptotic Cell The removal of apoptotic cells is accomplished via sloughing from the tissue surfaces or via phagocytosis by neighboring normal tissues and macrophages with subsequent degradation by lysosomes. Changes in the apoptotic cell membrane which are recognized by macrophages may include removal of sialic acid from glycoproteins on apoptotic thymocytes, involvement of particular non-protein anionic groups on the apoptotic neutrophil surface, or flipping of inner membrane surface phosphatidylserine to the outer surface of thymocytes (Savill et al., 1993). Because no cell contents are released and the membranes remain intact prior to phagocytosis, there is no inflammation or stimulation of cytokine release. This lack of inflammation is a most important part of the physiological nature of cell death by the apoptotic mechanism.

CELL DEATH OF IMMUNE CELLS Death During Development and Maturation of Immune Cells Cell death of great biological significance occurs during the development of immune cells. In theory, gene rearrangement of B and T cell antigen receptors provides a repertoire of cells with the potential to recognize every possible protein,

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Cell Death and the Immune System

including those peculiar to itself. The latter cells must obviously be removed and this is achieved by their deletion by apoptosis. Selection in immune cell development is analogous to selection in nature, in that both have an inherent requirement for death. The finely tuned selection of mature B and T cells, if imbalanced, results in disease of either immuno-deficient or autoimmune nature. T Cell Selection in the Thymus

Approximately 5x10^ immature CD4~8~ thymocytes are generated from cortical germ cells every day. As a result of differentiation and then selection which is generally believed to occur by massive apoptosis, only 1—5 per cent of these are exported from the thymus (Surh and Sprent, 1994). Immature thymocytes begin expressing CD4, CDS molecules and rearranged T cell receptor (TCR) gene products on their surface. Binding of these cell surface receptors to a range of molecules expressed on thymic epithelial cells determines the fate of thymocytes (Figure 3). CD4"'8"' members of the new TCR repertoire with TCR that do not bind self-MHC (major histocompatability complex) (85-95%) and other epithelial cell surface molecules, will not receive a positive signal, and it is thought that they undergo apoptosis or "death by neglect" over the next 3 ^ days. Those that recognize self MHC molecules (5—15%) will be positively selected as a result of receptor binding, and differentiate into either CD4'^ or CD8"^ mature T cells.

CD4-8- thymocytes arrive from the blood and proliferate in the thymic sub-capsular region, expressing CD4 and CDS as well as a diverse array of TCR. Within the cortical epithelium, ceils expressing TCR that Is either aberrant or fails to recognize self-MHC antigens present on local thymic cells, do not receive positive signals and undergo apoptosis - 'byneglecP.

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Cells with TCR that do recognise self-MHC antigens receive positive signals to multiply and differentiate, Into either CD4+ or CD8+ cells - 'posftfve selection',

Positively selected single positive T cells migrate towards the medulla, where those cells whose TCR recognise self-antigens presented by interdigitating cells are stimulated to undergo apoptosis • 'negative selection'.

Figure 3. T cell development in the thymus.

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In the thymus a second round of apoptosis called negative selection or clonal deletion occurs when either €04"^ or CD8"^ cells bind via their specific TCR to self-MHC with self-antigens displayed in the thymic medulla. This thymic clonal deletion is thought to be the major mechanism of self-tolerance, i.e., failure to produce circulating self-reactive T cells. It does appear, however, that some self-reactive T cells succeed in reaching the peripheral circulation in the absence of autoimmune disease, implying the existence of peripheral tolerance. It is unclear whether these cells are inactive due to activation induced cell death (AICD)(KabeIitz et al., 1993), to down-regulation of TCR and CDS, or to clonal anergy. Thus, in mature T cells which recognize antigens in association with self-MHC molecules, signaling via the CD3/TCR complex mediates a variety of T cell outcomes i.e., proliferation, differentiation, positive selection or death by apoptosis. These altemate outcomes are influenced by extracellular signals from associated receptors on the cells presenting antigen, from cytokines such as IL-2, as well as by differences in intracellular signaling pathways. A role for circulating glucocorticoid, and even toxic agents, influencing thymocyte deletion has also been suggested. Stromal thymic cells can supposedly trigger positive or negative selection, depending on the layer of primitive fetal tissue from which they derived i.e., endoderm, mesoderm, or ectoderm. One of the possible key intracellular regulators is the Bcl-2 protein, as high levels are found in T cell developmental stages that are not susceptible to apoptosis (Nunez et al., 1994). B Cell Selection An analogous process of selection and deletion takes place during B cell clonal development, first in the bone marrow, then in germinal centers of peripheral lymphoid organs (McCarthy et al., 1992). Seventy-five percent of pre-B cells maturing and undergoing Ig gene rearrangement in the bone marrow do not reach the circulation, because they undergo apoptosis and are phagocytosed by bone marrow macrophages. If pre-B cells fail to associate with stromal cells in the bone marrow, because they have no productive immunoglobulin (Ig) gene rearrangement, they die by "neglecf (Figure 4). Alternatively those pre-B cells which rearrange their Ig genes in such a way as to produce surface Ig (sig) that associates with stromal cells, are protected from apoptosis by positive clonal selection. If the sIg then reacts strongly with self antigens either in the bone marrow, or in other lymphoid organs, the cells undergo negative selection by clonal deletion and die. Circulating B cells which become activated by antigen (and T helper cells) undergo a further selection process in germinal centers. Here B cells undergo active hypermutation of their Ig variable region genes, producing antibody of greater or lesser affinity for the antigen. Cells which either fail to modify or actually decrease the affinity of antibody undergo apoptosis, a form of clonal deletion. This is seen in the large numbers of apoptotic cells which appear within tingible body macro-

Cell Death and the Immune System

Pro-B cells

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In the bone marrow pro-B cells undergo immunoglobulin gene rearrangement and express cell surface antibodies. Cells with nonsense Ig mutations undergo apoptosis due to neglect Cells that bind strongly to self-antigens expressed on follicular denrltic ceils undergo apotposis.

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phages during peak germinal center activity (i.e., day 5 to 10 post-antigenic challenge). B cells which have Ig v^ith increased affinity for the antigen survive, but for continued survival they need to further interact with helper T cells and cytokines to differentiate to plasma cells and perhaps memory cells. As in T cell development, the rescue of B cells from apoptosis by crosslinking receptors has been associated with increased bcl-2 expression. The small number of self-reactive B cells that elude negative selection appear to be eliminated by AICD in the periphery. Other Cell Types

Immune cells other than B and T cells do not express a diverse repertoire of antigen-specific receptors, and their development does not involve massive cell deletion. Death of Mature Immune Cells

Population numbers of peripheral (mature) immune cells fluctuate according to need. Cell numbers are determined by a complex interplay of all components of the immune system, i.e., antigen, antibody, and cytokine levels and, in particular, T helper cell activity. Normal hemopoietic cells require cytokine viability factors

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including colony stimulating factors and interleukins, and undergo apoptosis when they are withdrawn. There is evidence that circulating T cells can survive for up to 10 years, possibly contributing to T cell memory, although most have much shorter lifespans. Stimulation with bacterial superantigens, separate ligation of CD4 and TCR molecules, or activation with cytokines such as IL-2 have all been shown to trigger apoptosis in mature T cells under defined conditions. Once activated, CDS"^ cells can initiate cytotoxicity in a number of target cells without becoming effete, although it seems a fraction are killed (by AICD) upon a second encounter with the specific antigen. The need to terminate a T cell response is thought to be accomplished largely by apoptosis of the IL-2 dependent activated CD8"^ cells which results from decreasing IL-2 levels. This is reflected in the apoptosis of lymphocytes from virus-infected patients on in vitro isolation and concomitant depletion of IL-2. There is evidence that the majority of peripheral B cells have very long lifespans; that only about 2% of the cells are rapidly turning over; and that the lifespan in vivo correlates with bcl-2 gene expression (Haury et al., 1993). The daily adult human production of approximately 5 x 10'^ neutrophils and 1.2 X 10'^ monocytes, implies a continuous deletion of cells. The average life expectancy of polymorphonuclear neutrophils is about one day. Unless sfimulated by factors such as bacterial products, they become senescent and undergo apoptosis. Activated monocytes are deleted by apoptosis, unless rescued by bacterial products or proinflammatory cytokines (IL-1 and TNFa). Senescent megakaryocytes also undergo apoptosis. Thus, cytokines and other immune factors regulate the immune system by stimulation of proliferation and differentation, as well as by modulation of apoptosis.

CELL DEATH INDUCED BY THE IMMUNE SYSTEM—TARGET CELL DEATH The desired end result of an immune response is the elimination of targets expressing "foreign" antigens. Such targets include infectious cellular organisms, damaged or virally infected autologous cells, cancer cells, and transplanted tissue. Unfortunately, autologous cells which apparently express only self antigens are also targets in the case of autoimmune disease. Of the immune elements which operate to eliminate foreign elements, the innate immune system can destroy many pathogens on first encounter. For example, complement can bind to proteins on invading cells and induce direct membrane destruction with ensuing necrosis and inflammation. In addition the direct recognition of polysaccharides and other molecules on the surface of some bacteria by macrophages and neutrophils can lead to their engulfment and neutralization. Tumor necrosis factor from macrophages can induce apoptosis in virus-infected

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Figure 5. A variety of receptor combinations provide recognition of a target cell by cytotoxic T cells and phagocytes. Cytotoxic T cells recognize antigen presented with M H C molecules. Natural Killer cells recognize tumour specific antigens. Killer cells, and any cytotoxic cell with Fc receptors can target antibody-coated cells. Lectins bind protein receptors non-specifically.

cells. The non-cellular immune components i.e., lymphokines and antibodies, also have a major indirect contribution to the immune response via the modulation of cell-mediated cytotoxicity. Cell-mediated cytotoxicity requires the cell-cell interactions of recognition and binding of an immune cell to a target cell, v^hich trigger a unidirectional signal for

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its death. While the target cell dies by apoptosis in most cases, the immune cell remains undamaged and available for further cell-killing. The immune system has evolved an array of cell types and receptor recognition mechanisms that take part in cell-mediated cytotoxicity (Figure 5). It is now known that individual cell types are not restricted to one type of cell killing e.g., K cell killing (antibody dependent cell cytotoxicity [ADCC]) can be initiated by cytotoxic T cells, natural killer cells, monocytes, macrophages and polymorphs, all of which have Fc receptors for immunoglobulin. While the specificity of bonding between the cytotoxic cell and the target is provided by the TCR or antibody, associated receptor—ligand interactions help to stabilize the bond, and can even help to trigger the killing event. For example, the addition of anti-CD3, anti-CD2 or anti-CD 16 antibodies to CD8+ cells can trigger them to kill the target cells to which they are bound. In vivo such physiological ligands are likely to be another of the many modulators of cell mediated cytotoxicity (Roitt, 1993). Macrophages utilize tumor necrosis factor, nitric oxide and other factors to induce apoptosis in their targets, and may themselves succumb to nitric oxide-induced apoptosis. Cytotoxic T lymphocyte (CTL) and natural killer (NK) cells can kill their targets by at least two mechanisms (Liu et al., 1995; Smyth and Trapani, 1995). One mechanism involves granule exocytosis of soluble mediators such as perforin and granzymes from the cytotoxic cell into the contact space next to the target cell. While perforin alone can form pores in the target cell membrane and lead to necrosis, perforin in combination with granzyme B leads to apoptosis of the target cell. This may occur via the demonstrated ability of granzyme B to directly activate members of the ICE/ced-3/CPP32 family of cysteine proteases as well as other proteins. A second mechanism by which lymphocytes can mediate apoptosis in target cells is via the binding of CD95 ligand protein on the killer cell to the CD95 antigen on the target cell. This interaction of the CD95 antigen with its ligand then signals apoptosis in the cell expressing the CD95 antigen. It is possible that other mechanisms also participate in CTL-killing. CTL-killing has Ca^'^-dependent and Ca^'^-independent components and some CTL targets show mixed features of apoptosis and necrosis. Important features of CTL-killing are that it does not require protein synthesis, it is not inhibited by bcl-2 expression, and it cleaves the target cell DNA.

APOPTOSIS IN CLINICAL IMMUNOLOGY The immune system impacts on a host of clinical diseases. What is considered to be normal immune activity is seen in inflammation and graft rejection; inadequate immune function contributes to chronic infections and some cancers, while overactivity generates hypersensitivity and autoimmune disease. Apoptosis occurring in, and induced by, the immune system is pivotal in these disorders.

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Inflammation

Resolution of inflammation exhibits the elegant application of apoptosis to limit the activities of neutrophils. At the end of an inflammatory response there is a decrease in pro-inflammatory lymphokines and bacterial products, on which activated neutrophils are dependent. As a result, they undergo apoptosis with simultaneous membrane changes, which identify them to macrophages for phagocytosis before the lytic release of the toxic neutrophil contents. If lytic release does occur, further tissue damage may evolve to chronic inflammation and possibly autoimmune disease. Macrophages present at sites of inflammation also undergo apoptosis and are removed. One of the early descriptions of apoptosis was in the "piecemeal necrosis" which is characteristic of the active inflammation in chronic hepatitis of the autoimmune type (Kerr et al, 1979). In this condition lymphocytes may be seen surrounding hepatocytes, and apoptotic bodies are clearly visible in the areas of "piecemeal necrosis" seen in stained biopsies. Infection A major function of the cellular immune system, in particular T cells, is the control of viral infections. Cells infected with virus often present portions of viral proteins on their surface in association with MHC molecules which results in CTL recognition of the cell as foreign. Apoptosis and removal of the infected cell follows. Several viral genes have also been shown to directly regulate apoptosis of their host cell. Mechanisms include Bcl-2-type activity of the Epstein Barr virus gene BHRFl, and inhibition of cysteine proteases by the baculovirus gene p35 and the cowpox virus gene crmA. An additional family of viral proteins (which have mammalian homologues) called apoptosis inhibitory proteins (lAPs) also inhibit apoptosis perhaps via inhibiting receptor signaling (Uren et al., 1996). Thus it appears that viruses can regulate apoptosis to optimize viral replication. HIV infection attacks the immune system, primarily by the depletion of 004"*" helper cells. Several mechanisms relating to apoptosis have been advanced. There is some hope that manipulation of apoptosis may inhibit the immune deficiency, if not eliminate the HIV infection. Cancer

Tumor growth results from an excess of cell proliferation versus cell death. Many immune system malignancies are associated with changes in gene expression making lymphoid and myeloid cells less susceptible to apoptosis (Kerr et al., 1994). For example, virtually all follicular lymphomas contain a translocation of the bcl-2 gene so that its new location next to an antibody gene means that it is turned on indefinitely in B cells, leading to protection from apoptosis. Transgenic mice carrying an immunoglobulin enhancer-activated c-myc developed B and pre-B

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lymphoma at a frequency of 90-95% while bcl-2/c-myc doubly transgenic mice developed tumors more rapidly than c-myc transgenic mice. Increased apoptosis evident in tumors outside the immune system may result from immune recognition of tumor antigens, as well as from susceptibility to apoptosis following altered gene expression (e.g., c-myc, K-ras, c-abl, bcl-2, p53). In theory, if the balance of apoptosis and mitosis in tumors could be tipped towards apoptosis by drug or gene therapy, tumor progression could be halted (Area et al., 1996). Host-Graft Interactions Apoptosis is observed in vivo in graft-vs-host disease and in immune rejection of allografts. Hope for minimizing this pathology comes from work where transplanted tissues engineered to express CD95 ligand avoid rejection by binding CD95 antigen on cytotoxic cells and inducing their apoptosis (Vaux, 1995). Autoimmune Disease All autoimmune diseases have associations with MHC alleles, although it is not understood how these alleles contribute to "over-recognition." Under normal conditions, immature lymphocytes that bind self antigens present on self-MHC, undergo negative selection by apoptosis in the thymus. A defect in this deletion is seen in mutant Ipr/lpr mice. The absence of the normal Ipr protein CD95 results in large numbers of circulating CD4~8~ immature T cells, and the animal develops an autoimmune disease similar to lupus erythematosus (Mountz and Talal, 1993). The relatively recent recognition of apoptosis has inspired investigations which hint at a multitude of apoptotic stimuli and likely interactive apoptotic mechanisms. We are now approaching some basic understanding of the control of essential immunological phenomena. Manipulation of the opposing forces of mitosis and apoptosis offers one of the most promising areas for future therapy of immune disorders and of malignancy. With the use of the powerful tools of somatic gene therapy such manipulation points to exciting new therapeutic horizons.

SUMMARY Organism survival implies a balance of life and death on a cellular level. With respect to the immune system, cell death occurs during the development of mature B and T cell repertoires, and is an important aspect of their reactivity against foreign targets. This cell death primarily takes the form of apoptosis. Apoptosis, also referred to as programmed cell death, differs from the random destructive nature of necrosis, in that the dying apoptotic cell undergoes chromatin condensation, maintains intact membranes and organelles, and buds to form apoptotic bodies which are then phagocytosed without inflammation. Biochemically, apoptosis is regulated by several types of proteins including the Bcl-2 and lAP protein families,

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and involves the activation of pre-existing proteases which then cleave several nuclear and cytoplasmic proteins. Developing B and T cells that express immunoglobulins or T cell receptors that recognise self antigens, undergo apoptosis via negative selection, their elimination resulting in immunological tolerance. Only a small percentage of developing cells are rescued from apoptosis by positive selection to become mature circulating immune cells. The primary function of the immune system is the elimination of foreign invaders such as infected cells, tumor cells and transplanted cells. This is accomplished by killing these foreign targets, often via apoptosis which results in a controlled "environmentally friendly" target cell death. Cytotoxic T cells induce apoptosis in their targets via an as yet undefined mechanism, which may or may not involve membrane-lytic proteins such as perforin. Other cytotoxic agents employed by the immune system such as natural killer cells, lymphokine activated killer cells and cytokines such as tumor necrosis factor induce apoptosis in their targets, while complement induces necrosis. The production each day of large numbers of monocytes and neutrophils, as well as B and T cell proliferation are balanced by their elimination via apoptosis. Elegant mechanisms for the removal by apoptosis of activated T cells and neutrophils have been shown to be essential for the resolution of immune responses. The many clinical conditions in which the immune system plays a major role, for example, viral infections, cancer, autoimmune disease and graft versus host disease, all involve the induction of apoptosis. The role of apoptosis in immune function, and the authority of immune ftinction over disease, is currently inspiring the design of therapeutic interventions centered both on apoptosis regulation by the immune system and on immune regulation by apoptosis.

ACKNOWLEDGMENTS Supported by funding from the National Health and Medical Research Council of Australia, and the Queensland Cancer Fund.

REFERENCES Area, M.J., Mule, J.J., & Change, A.E. (1996). Genetic approaches to adoptive cellular therapy of malignancy. Semin. Oncol. 23, 108-117. Arends, M.J., & Wyllie, A.H. (1991). Apoptosis: mechanisms and roles in pathology. Int. Rev. Exp. Pathol. 32, 223-254. Cohen, J.J. (1993). Apoptosis. Immunol. Today 14, 126-130. Femandes-Alnemri, T., Litwack, G., & Alnemri, E.S. (1994). CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukinip-converting enzyme. J. Biol. Chem. 269, 30761-30764. Haury, M., Freitas, A., Hermitte, V., Coutinho, A., & Hibner, U. (1993). The physiology of be I-2 expression in murine B lymphocytes. Oncogene 8, 1257-1262.

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Kabelitz, D., Pohl, T., & Pechhold, K. (1993). Activation-induced cell death (apoptosis) of mature peripheral T lymphocytes. Immunol. Today 14, 338-339. Kerr, J.F.R., Wyllie, A.H., & Currie, A.R. (1972). Apoptosis: a basic biological phenomena with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239-257. Kerr, J.F.R., Cooksley, W.G.E., Searle, J., Halliday, J.W., Halliday, W.J., Holder, L., Roberts, I., Burnett, W., & Powell, L.W. (1979). The nature of piecemeal necrosis in chronic active hepatitis. Lancet Oct. 20, 827-828. Kerr, J.F.R., Gobe, G.C., Winterford, CM., & Harmon, B.V. (1995). Anatomical methods in cell death. Methods Cell. Biol. 46, 1-27. Kerr, J.F.R., Winterford, CM., & Harmon, B.V. (1994b). Apoptosis: its significance in cancer and cancer therapy. Cancer 73, 2013-2026. Liu, C-C, Walsh, CM., & Young, J.D-E. (1995). Perforin: structure and function. Immunol. Today 16, 194-201. McCarthy, N.J., Smith, C.A., & Williams, G.T. (1992). Apoptosis in the development of the immune system: Growth factors, clonal selection and bcl-2. Cancer Metastasis Rev. 11, 157—178. Mountz, J.D., & Tatal, N. (1993). Retroviruses, apoptosis and autogenes. Immunol. Today 14,532-536. Nunez, G., Merino, R., Grillot, D., & Gonzalez-Garcia, M. (1994). Bcl-2 and Bcl-x: regulatory switches for lymphoid death and survival. Immunol. Today 15, 582-588. Roitt, I.M., Brostoff, J., & Male, D.K. (1993). Immunology. 3rdedn. Churchill Livingstone, New York. Savill, J., Fadok, V., Henson, P., & Haslett, C (1993). Phagocyte recognition of cells undergoing apoptosis. Immunol. Today 14, 131—136. Schwartz, L.M., & Osborne, B.A. (1993). Programmed cell death, apoptosis and killer genes. Immunol. Today 14,582-590. Smyth, M.J., & Trapani, J.A. (1995). Granzymes: exogenous proteinases that induce target cell apoptosis. Immunol. Today 16, 202-206. Surh, CD., & Sprent, J. (1994). T-cell apoptosis detected in situ during positive and negative selection in the thymus. Nature 372, 100-103. Uren, A.G., Pakusch, M., Hawkins, C.J., Puis, K.L., & Vaux, D.L. (1996). Cloning and expression of apoptosis inhibitory protein homologs that function to inhibit apoptosis and/or bind tumor necrosis factor receptor-associated factors. Proc. Natl. Acad. Sci. USA 93, 4974-4978. Vaux, D.L. (1995). Ways around rejection. Nature 377, 576-577. Wyllie, A.H. (1980). Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555—556.

RECOMMENDED READINGS Carson, D.A., & Ribeiro, J.M. (1993). Apoptosis and disease. Lancet 342, 1251-1254. Osborne, B.A, (1995). Induction of genes during apoptosis: examples from the immune system. Semin. CancerBiol. 6, 27-33. Wyllie, A.H. (1992). Apoptosis (The 1992 Frank Rose Memorial Lecture). Br. J. Cancer 67, 205-208.

Chapter 15

Designer Antibodies ANDY MINN and JOSE QUINTANS

Introduction General Antibody Structure and Biosynthesis Introduction Antibody Fragments Constant Domains and Effector Functions A Brief Review of Antibody Biosynthesis Monoclonal Antibodies in Therapeutics Uses in Cancer and Allograft Rejection Other Uses of Monoclonal Antibodies Concerns and Limitations of Monoclonal Antibody Therapy Chimeric and Humanized Antibodies Expression of Antibody Fragments in Escherichia Coli Advantages Technical Overview Immunoglobulin Fragment Combinatorial Libraries Making an Immunoglobulin Fragment Expression Library Mimicking Antigen-Driven Selection Mimicking Antibody Secretion vs. Cell Surface Expression

Principles of Medical Biology, Volume 6 Immunobiology, pages 281-302. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-811-0 281

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Bypassing Immunization Mimicking Affinity Maturation Murine Expression Systems Using SCID Mice and Transgenic Mice Designer Antibody-Targeted Effector Functions Immunotoxins Other Antibody-Effector Molecule Conjugates Bispecific Antibodies Intracellular Antibodies Conclusion Recommended Readings

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INTRODUCTION The term antibody was introduced in the second half of the 19th century as part of a circular definition involving antigens and antibodies. At the time, antibodies were substances produced by the experimental animals injected with antigens, usually microbial products. Antibodies appeared to be highly specific and in spite of the lack of knowledge about their biochemical properties they were soon used therapeutically, often with great success. In the first decades of this century, the clinical applications of heterologous antisera led to the early appreciation of their antigenicity and the serious consequences of serum sickness. The first clue about the biochemistry of antibodies was provided in the 1930s when antibody activity was associated with the gamma globulin fraction of serum proteins; a decade later it was learned that antibodies were made by plasma cells. In spite of concerns about its relevance to normal structure, much work on antibodies secreted by plasma cell malignancies, called myelomas, was carried out in the following two decades. That plasma cells represented the differentiated progeny of B lymphocytes was not known until the 1960s, a period that saw the elucidation of the biochemistry of antibodies as immunoglobulins, glycoproteins made up of a basic unit consisting of 4 polypeptide chains arranged in two symmetrical halves, each containing a heavy and light chain. The modular construction of Ig chains became apparent in the 1970s when the concepts of Ig domain and Ig superfamily were introduced. In the middle of the decade the revoluntionarily simple hybridoma technology was developed and soon widely applied to make monoclonal antibodies of desirable specificities for experimental and diagnostic uses (Kohler, 1975). For technical reason, hybridomas of human origin could not be made easily and as a result, the therapeutic applications of monoclonal antibodies were hampered by the use of rodent antibodies. Unlike the situation at the beginning of the century, immunologists now possessed detailed knowledge of the modular nature of immunoglobulins and their genes and saw great promise in the application of the genetic engineering technology available in the 1980s. As a consequence of this convergence of

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immunological and molecular interests, the era of designer antibodies dawned. This chapter summarizes the most recent developments in the area. Due to space limitations, a detailed discussion of antibody structure and genetics cannot be given here. The reader is referred to recent publications on these topics (Mayforth, 1993: Paul, 1993).

GENERAL ANTIBODY STRUCTURE AND BIOSYNTHESIS Introduction

Antibodies, or immunoglobulins (Ig), are glycoproteins that bind specific fluidphase antigen. Ig molecules are composed of four polypeptide chains, two identical heavy chains and two identical light chains (Figure 1, top left). Each heavy chain is bonded to a light chain via hydrophobic interactions and a covalent disulfide bond. The heavy chain/light chain complexes are similarly joined by hydrophobic interactions and disulfide bonds. The heavy chain and light chain are composed of variable (V) domains and constant (C) domains. The heavy chain has one V domain (VH) and four C domains (CH) while the light chain has one v domain (VL) and one C domain (CL). Both V and C domains have the characteristic structure of immunoglobulin domains. Ig domains are 90-110 amino acids long and form seven or nine (3 strands folded into two tightly packed antiparallel beta sheets, usually stabilized by an intrachain disulfide bridge. The strands are 5-10 amino acids long and arranged in such a way that the interior of the fold is hydrophobic and the exterior is hydrophilic. Although most of the amino acids in the immunoglobulin domain are well conserved, there are three regions in the V domain that show extensive variability between different antibody molecules. These regions are known as hypervariable regions (Figure 1, top right). Structurally, the hypervariable regions are loops that sit on top of the P-pleated sheets and connect adjacent or distant P strands together. Functionally, the hypervariable regions make-up the antigen binding site. (Hypervariable regions are also known as complementarity determining regions, or CDR for short). Thus, the complete antigen binding site is assembled from six loops, three from VH and three from VL, which rests on a scaffold made by the P-pleated sheets. Since antibody molecules are made up of two identical heavy chains and two identical light chains, this antigen binding site is represented twice. The antigen binding site made by the loops has a high stereochemical complementarity with the antigenic determinant—depressions in one are fitted with protuberances in the other. Those residues not involved in loop main chain conformation are free to vary and modulate the surface topography and charge distribution, leading to different antigen binding properties. Residues with different bulk, charge, or hydrogen bond properties can alter the binding affinity of the antigen binding site.

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Like many types of proteins, the domain organization of antibody molecules makes them amenable to engineering because domains are modular. These modular properties include: (1) Both heavy and light chains can be altered by replacing domains within each chain since the heavy and light chains are built up from several immunoglobulin domains. (2) The relative spatial position and surface topology of VH and VL is well conserved. This allows different VH to combine fairly freely and interchangeably with different VL domains. (3) The double P-pleated sheet framework made by VH and VL form a nearly constant scaffold on which the antigen binding site sits. This allows grafting of CDRs from one antibody molecule to another without losing antigen binding specificity. Antibody Fragments

Proteolytic cleavage of antibody molecules results in discrete fragments (Figure 1, middle). Papain cleaves an antibody molecule on the N-terminal side of the interchain disulfide bond to give two Fab fragments and one Fc fragment. The Fab fragment consists of the light chain and the Fd fragment, which is the VH and CHI region of the heavy chain, along with part of the hinge region. Since the Fab fragment still contains the antigen binding site, it binds antigen in a similar fashion to the parent antibody molecule. Pepsin cleaves just the C-terminal of the interchain disulfide bond to give a divalent fragment that contains both antigen binding sites, called F(ab')2. The F(ab')2 fragment can be further cleaved to generate Fab', which is similar to Fab except for an additional stretch of C-terminal amino acids in the heavy chain part. More extensive digestion of Fab' with pepsin generates Fv fragments, which consist of only the VH region noncovalently linked to the V^. Constant Domains and Effector Functions

The light chain has one constant domain (C) while the heavy chain is made up of three or four. Different C regions in the heavy and light chains determine which isotope class an antibody belongs to: IgM, IgD, IgG, IgA; or IgE. Isotypes are species specific. Minor differences in antibody isotypes result in subclasses of immunoglobulins, for example, IgG 1, IgG2, IgG3 and IgG4 in humans. In addition, allelic differences within isotope classes and subclasses constitute allotypes. Dif-

Figure 1, Antibody Structures. Top. Left. General antibody structure, a disulfide linked dimer of a heterodimeric light chain and heavy chain. (HV) hypervarlable region, (V) variable region, (C) constant region. Right. A ribbon diagram of the variable domain. Notice the hypervarlable regions are loops that rest on top of a scaffold created by two layers of beta pleated sheets. Middle. Structure of antibody fragments. F(ab')2 Is produced by pepsin cleavage. Further cleavage can produce Fab' and Fv. Papain cleavage generates Fab and Fc. (Continued.)

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Figure 1. (Continued.) An Fd fragment (not shown) can be generated by reducing the disulfide bond of the Fab. scFv is a V from the heavy chain (VH) linked to V from the light chain (VL) by a short peptide linker (11-18 amino acids). Bottom. Structure of selected designer antibodies. For the chimeric and humanized antibodies, the shaded regions are from rodent and the open regions are from human. For the bispecific antibody and the diabody, different shades represent regions from different antibodies.

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ferent isotope classes and subclasses of antibodies confer distinct properties to the antibody molecule. Some of these properties include the following: (1) Serum half life. IgGs, the dominant class during a secondary immune response, have the longest serum half lives and comprise the majority of the immunoglobulin pool; (2) Ability to fix complement. The complement cascade involves a system of functionally linked proteins that interact with one another in a highly regulated manner to provide many of the effector functions of humoral immunity and inflammation, including opsonization, cytolysis, chemotaxis, etc. IgM and IgGs are good at activating complement; (3) Reactivity with Fc receptors on leukocytes. The binding of antibodies to Fc receptors can lead to phagocytosis, release of cell toxins, or release of inflammatory mediators. For example, the binding of IgE to FceRI on mast cells leads to release of histamine and other mediators of hypersensitivity. A Brief Review of Antibody Biosynthesis The transcriptional unit encoding the VH domain is actually made up of three gene segments called the V, D, and J gene segments. VL domain is made up of V and J gene segments only. The V gene encodes for CDRl, CDR2, and the intervening framework region, while the D gene and/or J gene encodes for CDR3. To form the VH domain, one of more than 100 V genes is brought next to a D gene segment and/or J gene segment during the process of B cell lymphopoiesis. The various combinations of different V(D)J joinings along with other mechanisms give rise to a large and diverse antibody repertoire, with each B cell expressing antibodies of only one specificity. During an immune response, those B cells that bind to antigen with the highest avidity proliferate optimally. Some of the selected B cells give rise to effector cells, while others give rise to long term memory cells. A process called affinity maturation allows responding B cells to develop a greater affinity to the antigen so that a second encounter with the same antigen will be more effectively handled. Affinity maturation is driven by somatic hypermutation, a unique process that introduces point mutations into the gene segments encoding for the hypervariable regions of antibodies. B cells with a mutated receptor of higher affinity will be selectively expanded.

MONOCLONAL ANTIBODIES IN THERAPEUTICS As mentioned in the introduction, because of the high degree of specificity in antibody mediated recognition, the clinical usefulness of antibodies has long been recognized. Applications of engineered antibodies include cancer, allograft rejection, autoimmunity, tissue damage, diagnostic imaging, etc. Advances in molecular biology make it possible to modify antibody genes almost at will so that the resulting product is optimally designed for a particular clinical application.

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Uses in Cancer and Allograft Rejection In the case of cancer, antibodies can be used to target specific antigenic determinants found on tumors and mark their destruction. Although these tumor specific antigens are relatively rare, recent research is uncovering more and more tumor rejection antigens. Antibody therapy of cancer relies on enhancing various natural effector mechanisms of the host and/or interfering with cell function. For example, antibodies can mediate antibody dependent cytotoxicity through their Fc receptors, or they can kill the target by recruiting the complement cascade. Antibodies that recognize particular cell surface signaling molecules can neutralize target cells even without recruiting host effector functions. For example, antibodies against the Fas antigen have been found to induce apoptotic cell death, and certain antibodies directed against the Her-2/neu cell surface receptor can decrease the growth rate of the tumor cells by signaling them to differentiate. Antibodies against Ig idiotypes on B cell lymphomas have been found to induce an otherwise aggressive lymphoma into a state of dormancy. As an alternative to relying on natural effector mechanisms, antibodies can also be conjugated to various effector molecules. Toxins from plants and bacteria can be linked to antibodies, creating an immunotoxin, which can deliver the toxin to the target cell. Radioisotopes have been successfully used in patients with nonHodgkin's lymphomas and appear to be better than immunotoxins or unmodified antibodies in treating this particular malignancy. In cases of allograft rejection and autoimmune disease, it is feasible to suppress the immune system to the point where it does not reject the targeted tissue. Most cell surface antigens targeted for this purpose are molecules involved in cell signaling. Lymphocyte activation requires T cell receptor engagement and a secondary signal known as costimulation. 0KT3, which is the only murine antibody approved for clinical use, binds a T cell signaling molecule called CD3 and acts as an immunosuppressant by blocking T cell receptor signaling. Antibodies that block important costimulatory signals (e.g.; CD28 and B7) lead to immunologic tolerance. Qin et al. recently provided evidence that antibodies used to block certain T cell signaling interactions may actually be able to guide the immune system into a feedback loop that can maintain tolerance to the antigen even after the antibody is removed. Other Uses of Monoclonal Antibodies Monoclonal antibodies have many other applications besides cancer and transplantation and will be briefly mentioned here. Drug toxicity and gram negative sepsis can be treated by using antibodies that can bind and neutralize the toxin. An antibody against the lipid A domain of endotoxin can reduce mortality in patients with Gram-negative bacteremia. Infiltrating leukocytes secreting cytokines and inflammatory mediators often cause tissue damage. Antibodies specific for leuko-

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cyte adhesion molecules such as LFA-1 or ICAM-1, can reduce tissue damage by preventing the accumulation of leukocytes. Finally, diagnostic imaging can be greatly improved if radiolabeled or heavy metal-labeled antibodies could target the tissue of interest. Antibodies can be used to image tumors as small as 0.5 cm, a size that is sometimes missed by other radiological methods. Concerns and Limitations of Monoclonal Antibody Therapy One obstacle limiting the use of antibodies in therapeutics is obtaining enough quantities of antibody with the right specificity. Hybridoma technology almost resolved this problem by providing an unlimited source of monoclonal antibodies for human therapy. Although the technique works well when murine B cells are fused to mouse myeloma cells, the fusion of mouse myeloma cells with human cells often leads to the preferential loss of human chromosomes and instability of the hybrids. For ethical reasons, humans cannot be immunized with tumor antigens. Thus, obtaining a large quantity of a monoclonal antibody with the right specificity has not been an easy task. The most convenient method has been to immunize mice and make mouse hybridomas. However, there are several concerns when using murine antibodies in humans. Since murine antibody molecules are considered foreign by the human immune system, a human anti-mouse antibody response (HAM A response) results in neutralization of the therapeutic effects of the antibody and quick clearance from the body. The patient can also get serum sickness, which in rare cases can lead to anaphylactic shock. Yet another drawback of developing a HAM A response is that it can prevent future usage of mouse antibodies because the immune system has been primed against it. As with any drug, unwanted side effects are always a concern. Unmodified antibodies are able to recruit effector functions, but these are not always desirable (When effector functions are desirable, it must be kept in mind that murine and rodent antibodies are poor at eliciting human effector functions). If a toxin is conjugated to an antibody, non-specific toxicity must be reduced. These non-specific effects can be caused by lack of specificity in the recognized antigen. As mentioned already, tumor specific antigens have been relatively elusive. Immunotoxins have caused neurologic damage in patients due to a lack of exactitude in antibody targeting. The relative lack of success of monoclonal antibody therapy in cancer over the last decade highlights another obstacle in antibody therapy. Many of the reported instances of complete tumor remission have occurred with tumors that are accessible to the circulation (e.g.; lymphomas and leukemias), making tumor cell accessibility an important determinant of success. Cancers that are highly vascularized probably respond better than cancer cells growing in solid tumor parenchyma. Monoclonal antibodies are relatively large molecules that have difficulty reaching certain tumors due to elevated interstitial fluid pressure, basement membrane

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barriers, intercellular tight junctions, and the long distance through interstitia that antibodies must travel after extravasation. The focus of much of the recent research in designer antibody technology has been to overcome many of these limitations of monoclonal antibodies for use in therapy. The rest of this review will focus on these efforts and the promises that they hold.

CHIMERIC AND HUMANIZED ANTIBODIES The 1980s saw the successful introduction of molecular biological techniques to overcome the problems imposed by using rodent and murine antibodies for human therapy. The two main advances were chimeric and humanized antibodies. These technologies are reviewed extensively elsewhere (Mayforth and Quintans, 1991) and will only be summarized here. In 1984, Morrison and colleagues used genetic engineering to combine the genes for rodent VH and VL domains with the genes for human CH and CL domains (Morrison et al., 1984). The heavy and light chain constructs were then transfected into a nonsecretor myeloma cell. The result was a secreted antibody that was only about 25% rodent in origin (Figure 1, bottom). In addition to the reduction in antigenicity, these chimeric antibodies could be further engineered to tailor the effector functions of the antibody to give the most appropriate response. For example, a complement fixing Fc domain could be selected for a tumor seeking antibody, while a nonfunctional Fc domain could be used in tumor imaging. Although the first clinical trials with chimeric antibodies showed some promise, the results were not always successful. It became evident that a HAMA response could still develop, despite the fact that most of the chimeric antibody was human in origin. The murine V regions could still be immunogenic. Only two years after Morrison developed the first chimeric antibody. Winter's group reduced the immunogenic potential even more by grafting only the gene segments of murine CDRs onto a human antibody framework (Jones et al., 1986). This decreased the mouse portion of the antibody molecule to 10% (Figure 1, bottom). In 1988, Reichmann et al. created a humanized Campath-1 antibody, which was a rat monoclonal antibody that recognized an antigen on human lymphocytes. Humanized Campath-1 was used in a clinical trial to treat nonHodgkin's lymphoma. In two patients tested, no HAMA response was observed and both patients showed tumor regression. Although there is no doubt that a humanized antibody is better than a chimeric antibody at reducing immunogenicity, a HAMA response can still occur if the allotype of the humanized antibody is not matched to that of the recipient. In addition, unique antigenic features of the V domains, called idiotypes, can also cause a HAMA response. Anti-idiotypic response can effectively eliminate the therapeutic effect of the antibody by blocking the antigen binding sites.

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The efficacy of humanized antibodies relies on certain principles that have been mentioned above. It requires that different framework regions be structurally conserved in both the orientation of the 2 P-pleated sheets and in the packing of the VH and VL domains, such that the CDRs are supported in the same way. Also, the transfer of CDRs to another framework requires that the framework regions not contribute in a significant way to the antigen binding properties. Although these requirements are generally true, it has been found that residues in the framework region can affect the antigen binding site and that the conformation of the loop is not entirely dependent on its intrinsic amino acid sequence. Tertiary interactions with residues in the framework region can be important. Thus, humanized antibodies can have a decreased affinity as compared to the murine version.

EXPRESSION OF ANTIBODY FRAGMENTS IN ESCHERICHIA COU Advantages Initially, genes for chimeric and humanized antibodies were genetically engineered and then transfected into a eukaryotic cell called a transfectoma, a myeloma cell that can produce 1-20 micrograms per milliliter of the recombinant antibody into the culture supernatant. However, the production of antibody molecules in E. coli bacterium is technically simpler, more economical, and can yield more antibody. In addition, there are many advantages in using the same host organism for both genetic manipulation and protein production. The main advantage is that antibody genes can be much more readily manipulated and engineered. The main problem in using a bacterial expression system is that whole antibody molecules cannot be produced. So far, only antibody fragments have been successfully manufactured in E. Coli. However, depending on the application, antibody fragments can often be just as good, if not better, than the whole molecule. Antibody fragments are potentially useful in therapeutics because they retain the antigen binding specificity of an antibody molecule but have pharmacokinetic properties that can be better suited for tumor imaging and targeting. Compared to whole antibody molecules, antibody fragments have a shorter serum half-life and are able to penetrate deeper into tissue. Also, since the fragments lack Fc regions, they can neither bind to cells with Fc receptors nor mediate effector frinctions. These properties, along with the amenability of a bacterial expression system has brought excitement and promise to the field. Technical Overview To produce antibody fragments in E. coli, both heavy and light chain genes are co-expressed and their protein products co-secreted by the same bacterium. A bacterial signal peptide DNA sequence is glued to the DNA encoding the N-termi-

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nal end of the mature antibody proteins to direct the proteins into the periplasm of the bacteria. Unhke the cytoplasm, the periplasm is an oxidation redox environment where disulfide bonds can form during protein folding and association. The antibody fragment can be recovered by liberating the protein from the periplasm as a functional and soluble protein, or the protein can be recovered from inclusion bodies in an insoluble form and renatured in vitro. Fv fragments are the smallest Ig fragments with an antigen binding site. They consist of a noncovalently associated VH and VL domains and generally have the same affinity as the whole Ig. Fv fragments can be produced in high yields in a bacteria expression system; however, the association of the two V domains can vary in stability. Single-chain Fv fragments (scFv) are covalently linked VH and VL domains (Figure 1, middle). The covalent linkage is provided by a flexible peptide segment fused to the N-terminus of the VH domain and the C-terminus of the V^ domain (or vice versa). The covalent linkage resolves the stability problem and is easy to produce. However, the yield of scFv fragments is generally lower than Fv fragments, and the linker peptide often leads to lower affinities due to sterics or conformational change of the antigen binding site. Fab fragments consist of the entire light chain and VH and CHI regions of the heavy chain. These fragments have identical antigen binding properties as whole Ig and are more stable and rigid than smaller antibody fragments. Unfortunately, there is a lower yield of correctly folded protein compared to the smaller fragments when using a bacterial expression system. A whole antibody molecule is divalent. It is often desirable to maintain this characteristic because it contributes to the avidity of the molecule, and also because crosslinking of cell surface receptors is often necessary for physiologic effects. One way to accomplish this is to express a Fab' fragment (same as Fab fragment except that it contains the protein segment necessary for interchain disulfide bond formation) and dimerize it in vitro with a chemical coupling reaction. For some reason dimerization does not proceed in the periplasm. Recently, dimerization of scFv fragments was done in vivo in E. coli by including an amphipathic helix fused to the C-terminus of the scFv fragments. The helix seems to give the disulfide bond time to form in the periplasm. With a divalent scFv fragment, it may be possible to generate a hetero-biftmctional antibody fragment. For example, one scFv fragment can bind to a tumor antigen while the other scFv fragment can recruit a cytotoxic T cell.

IMMUNOGLOBULIN FRAGMENT COMBINATORIAL LIBRARIES If anytime along the B cell maturation process, a B cell expresses a self-reactive receptor, it needs to be eliminated or anergized in order to prevent autoimmunity. Although this negative selection process is good to avoid autoimmune disease, many of the therapeutic applications of monoclonal antibodies rely on the antibody

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being specific for a cell surface antigen or other self antigen. Therefore, the production of antibodies with specificity to self antigens presents a problem because even if ethical considerations were not an issue, one could not produce many of the antibodies that are specific for cell surface antigens by immunizing a human. Of course, assuming the human form of the antigen is immunogenic in mice, murine monoclonal antibodies can be made, but as discussed already, murine antibodies present other serious problems. Recently, molecular immunologists have succeeded in cloning immunoglobulin genes into filamentous bacteriophage and making an expression library. The principal idea behind an immunoglobulin expression library is very similar to B cell maturation. The first step is to express the antibody on the surface of the phage. Then, one screens for those phages with antibodies of the desired specificity and selectively expands this population by infecting bacteria. Finally, the antibody can be purified from the bacterial supernatant by chromatography. However, for reasons described already, the phage expression library is limited to producing antibody fragments like Fab and a covalently linked single-chain Fv (scFv). Despite this limitation, the in vitro system has the ability to mimic key features of the immune system such as antigen driven selection of antibodies, affinity maturation, cell surface display vs. secretion of antibodies, but is not constrained by mechanisms which eliminate self-reactive antibodies. Furthermore, the in vitro system has the potential to generate even more diversity than the in vivo immune system. Making an Immunoglobulin Fragment Expression Library In order to clone Ig genes into bacteriophage, mRNA from B lymphoc)^es from a mouse or human is isolated and reverse transcribed to make cDNA (Figure 2A). Heavy chain cDNA is cloned out by using universal primers that recognize conserved sequences in the 5' V domain. The choice of 3' primer depends on whether one wants to make a Fab or Fv expression library. Light chain cDNA is cloned in a similar fashion. In this way, the entire rearranged Ig gene repertoire can be isolated. The use of filamentous phage to display the antibody fragments encoded by the cloned cDNA has become a popular method because of the advantages offered in screening for antibody specificity. Filamentous (Ff) phage are viruses that infect bacteria, replicate themselves, and bud from the bacteria (without lysing it) to move on to infect other cells. Ff phage use the host replication machinery to replicate their DNA and make various proteins needed for assembly and packaging of virus. pIII and pVIII are two coat proteins that are targeted to the periplasmic space ofE. coli and anchored to the inner membrane by a signal peptide. During phage assembly, these coat proteins are then expressed on the surface of the budding phage. Both pIII and pVIII can be fused with proteins to display the proteins on the surface of phage (Figure 2B). By taking the heavy chain cDNA and linking it to the gene for one of the coat proteins, the antibody chain is anchored in the

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periplasmic space. The light chain is secreted into the periplasm, where it associates with the anchored heavy chain. The dimer is then displayed on the surface of the budding phage. Likewise, it is also feasible to fiise scFv genes to coat protein genes, circumventing the requirement for association between heavy chains and light chains (shown in Figure 2). Mimicking Antigen-Driven Selection

To screen for those phage which display the desired antigen specificity, several methods have been employed. Antigen can be bound to dishes or a column matrix. Phages that bind with low affinity are washed off (Figure 2C). Those that bind with high affinity can be eluted by acid or alkali and used to reinfect E. coli. This cycle can be repeated many times to select phages that display antibodies with increasingly higher affinities. An enrichment factor of up to 10^ can be obtained by a single round of selection and phage with as little as 2-4 fold differences in affinity can be selected for. Mimicking Antibody Secretion vs. Cell Surface Expression

Once the desired antigen specificity is selected and enriched, the antibody fragments need to be purified from the phage surface. There is a way to mimic antibody secretion in the phage expression system, allowing for easier purification. This is accomplished by cloning into a vector that contains an amber stop codon (the name for stop codon UAG) between the antibody gene and the gene encoding the pIII coat protein (Figure 2B). When surface expression of the antibody fragment is desired, the phage is grown in a supE suppressor strain ofE. coli. This strain has a mutation in an anticodon loop of a tRNA and reads the amber stop codon as glutamine instead of a translational stop, allowing the generation of the fusion protein. When secretion into the culture media is desired, the phage can be grown in a non-suppressor strain where the amber stop codon is read as a translational stop, preventing linkage of the antibody fragment with the pIII coat protein. By taking the bacterial supernatant which contains the antibody fragment and using affinity chromatography, purified antibody fragments can be isolated. Bypassing immunization

Burton and colleagues demonstrated the promise of combinatorial libraries by making Fab fragments against a plethora of viral pathogens (Williamson et al., 1993). Using the bone marrow of two HIV seropositive donors which had antibody titers to a variety of viral antigens, including HIV (human immunodeficiency virus), CMV (cytomegalovirus), HSV (herpes simplex virus), and others, a Fab combinatorial library was prepared. To see if Fab fragments specific for the seropositive viruses were recovered, ELIS A wells were coated with either purified viral antigen or viral lysates. After various rounds of panning and enrichment, Fab fragments

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Clone into Vector Produce Random scFV ^

VH

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Isolate B Cell mRNA and Make cDNA

Figure 2. Diagram of the basic strategy used in making and screening an immunoglobulin fragment combinatorial library. Read from bottom up. A. B cell mRNA is isolated and reverse transcribed to make cDNA. "Universal" primers to the V H and VL gene regions are used to amplify these respective genes. V H and V L genes are randomly ligated together to make a random scFv library. B. The random scFv library is cloned into an expression vector, upstream of a gene v^^hich encodes for a phage coat protein. Between the scFv gene and the gene for the coat protein is an amber mutation, which is suppressed when grown in a supE bacterial strain, allowing for production of the an scFv fused to the coat protein. (Continued.)

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specific for all of the viral antigens were obtained. In addition, some of the Fab fragments were tested and found capable of neutralizing virus infectivity. Work such as this provides a way to obtain human monoclonal antibody preparations for disease therapy and prophylaxis. In the above example, the combinatorial library was generated from rearranged Ig genes of human donors that were seropositive for antibodies against viral antigens. The donor immune system was biased because it contained memory B cells specific for viral antigens. Thus, the combinatorial library generated from these donors was not naive and would be expected to produce heavy and light chain combinations specific for viral antigens. The isolation of high affinity antibody fragments still depended on either a donor that was seropositive for desired antigen-specific antibodies, or making a combinatorial library from an animal immunized with the antigen of interest. In order to bypass immunization altogether, a combinatorial library should be made from a naive repertoire. Winter and his colleagues did just this by making a library which contains, VH, V^, and V^ genes cloned from the mRNA encoding IgM of 10^ peripheral blood lymphocytes from two healthy human volunteers (Marks et al., 1991). From this library, soluble antibody fragments were isolated which bound to 15 different antigens. These antigens included, 2-phenyloxazol-5one, turkey egg-white lysozyme, bovine serum albumin, and bovine thyroglobulin. More importantly, human antibody fragments to self-antigens were also isolated, which included TNF-a, two tumor markers, and T cell antigen CD4. Human antibodies to these self-antigens would be extremely difficult, if not impossible, to obtain by conventional procedures. The affinities of these antibodies was similar to that of antibodies produced from a primary immune response. Even combinatorial libraries made from an unimmunized animal or donor are not truly naive because the animals have received many antigenic challenges. In addition, recent evidence seems to indicate that the use of antibody chains is not as random as previously thought. One solution to these biases in the immune system is to make a synthetic V gene repertoire whereby some or all of the CDR's are

Figure 2. (Continued.) This fusion protein is targeted to the membrane and eventually displayed on the surface of the phage. Expression in a nonsuppressor strain causes the amber mutation to be read as a stop codon, truncating the fusion gene prior to the coat protein region. As a result, the scFv is secreted, allowing for easier purification. C. Selection for the phage that display antibodies with the right specificity can be done by panning or by using a column bound with the antigen of interest. Non-specifically bound phage are washed off, while specifically bound phage are eluted and used to reinfect, enriching for phage that express scFv of the correct specificity. In order to mimic affinity maturation, the scFv gene from phage of interest can also be isolated and subjected to mutagenesis or chain shuffling. Phage which display a scFv with a greater affinity are selected.

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encoded by synthetic nucleic acids randomized at most positions. In a sense, this method is mimicking the germline rearrangement of Ig genes. Such a Ubrary has a potential diversity greater than that of animals. Winter's group imitated the rearrangement of 49 cloned human germline VH gene segments in vitro by adding random synthetic CDR3's of five or eight residues and a J region (Hoogenboom and Winter, 1992). These "rearranged" heavy chain gene segments were cloned with human V^s light chain genes to generate scFv fragments. Antibody fragments specific for two haptens and human TNF-a were isolated. Mimicking Affinity Maturation Since the antibody fragments produced from a combinatorial library made from a naive repertoire have affinities similar to those of antibodies produced during a primary immune response, a method to mimic affinity maturation is necessary to produce antibodies with higher affinities. The process of somatic hypermutation can be mimicked by using error-prone Polymerase Chain Reaction (PCR) to introduce random mutations in the V genes. Alternatively, synthetic oligonucleotides can be used to specifically target mutations to the CDRs. A third process called chain shuffling can also increase the affinity of antibody fragments (Figure 2C). In chain shuffling, the heavy chain or light chain of an antigen-reactive antibody is shuffled with a library of light chains or heavy chains (Marks et al., 1992). Recently, this was done with a human antibody against the hapten phenyloxazolone isolated from a phage library, resulting in a 300 fold improvement in affinity. These methods of mimicking affinity maturation often lead to antibody fragments with affinities comparable to those from hybridomas made during a tertiary immune response, while bypassing immunization. Finally, although not directly related to affinity maturation, it is worthwhile to note that the filamentous phage library allows selection from a much greater repertoire of antibody fragments than is possible in vivo. The immune system is limited by the number of B cells of a given specificity available at a given point in time, usually > 10^. However, a phage library can be much larger than this, potentially yielding antibodies with higher affinities simply because the affinities of antibodies isolated from a library is thought to be proportional to the size of the library. Murine Expression Systems Although the bacterial expression system is an excellent tool to express engineered genes, it cannot surpass natural immunization for the ease and efficiency of obtaining monoclonal antibodies because of the laborious screening and manipulation process. One could avoid this problem if it were possible to endow a mouse with a human immune repertoire, thereby obtaining human monoclonal antibodies directly. For this approach to work, the mouse needs to tolerate the human proteins

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in order to prevent rejection. Mice that are immunocompromised or mice carrying the human antigens during development are capable of tolerating an artificially endowed human immune repertoire. Using SCID Mice and Transgenic Mice Severe combined immune deficiency (SCID) mice have a defect in immunoglobulin gene rearrangement, resulting in mice without T or B cells. These mice have recently been populated with human peripheral blood leukocytes (PBL) from donors that have not been boosted with tetanus toxoid (TT) antigen for 17 years (Duchosal et al., 1992). Immunization of the human PBL-populated SCID mice (hu-PBL-SCID mice) with a tetanus toxoid booster shot resulted in a significant rise in serum IgG anti-TT levels. B cell RNA was extracted from these mice and used to make a combinatorial library. Screening of the Fab library demonstrated Fab fragments with affinities equal to those antibodies derived directly from boosted individuals. This method of generating high affinity antibody fragments for hu-PBL-SCID mice could be useful in the production of human monoclonal antibodies for therapy. For example, expanded repertoires of anti-HIV antibodies against specific epitopes could be generated by peptide stimulation of SCID mice populated with PBL from seropositive donors, or human autoantibodies could be obtained using appropriate autoimmune donors. Transgenic mice are another valuable tool to molecular biology that could help in the production of human monoclonal antibodies. Unrearranged human antibody genes can be injected into fertilized mice ova. The resulting transgenic mice can then be immunized to produce human antibodies. The spleen cells can be fused to mouse myeloma cells to generate hybridomas that secrete human antibodies. One potential drawback of this approach is that the size of the human Ig gene repertoire in the transgenic mice may be limited by the amount of new DNA that can be carried by the transgenic animal.

DESIGNER ANTIBODY-TARGETED EFFECTOR FUNCTIONS Another rapidly developing field in designer antibodies is the use of antibodies to target an effector molecule to a specific cellular antigen. Our immune system does this naturally in the form of antibody-dependent cell-mediated cytotoxicity and complement-mediated lysis. With the creation of fiision proteins between antibodies and effector molecules such as toxins, radioisotopes, cytokines, enzymes, and other antibodies, novel effector functions can be created. Recent advances have even allowed for targeting antibody molecules to specific intracellular locations. Immunotoxins An immunotoxin is an antibody chemically or genetically conjugated to a toxin for the purpose of delivering a "lethal hif to a specific cell type (Figure 1, bottom).

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Generally these toxins are of plant or bacterial origin. Ricin is derived from the beans of the Ricinus communis plant, and is capable of inhibiting protein synthesis by decreasing the affinity of 28S ribosomal RNA for elongation factor-2, rendering the ribosome nonfunctional. Pseudomonas exotoxin and diptheria toxin are two bacterial toxins that also prevent protein synthesis by inactivating elongation factor-2 through ADP-ribosylation. When conjugated with an antibody, all of these toxins can be specifically targeted to cells through antibody binding and endocytosis. Often the toxin must be modified to reduce non-specific targeting. For example, sugar moieties on the toxins can bind to receptors on hepatocytes and cells of the reticuloendothelial system; therefore, these sugar residues must be removed. Another problem that is not solved so easily is that the toxin or the antibody used can be immunogenic. However, humanization of immunotoxins has been described (Rybak et al., 1992). A chimeric antibody to the human transferrin receptor was fused to angiogenin, a human homologue of pancreatic RNase, and was effective at inhibiting protein synthesis in cells specifically expressing the transferrin receptor (This receptor is found in high numbers on rapidly proliferating cells). Since this immunotoxin is a chimeric antibody fused to a human protein, it may bear importantly in designing human therapeutic strategies for reducing potential immunogenicity in traditional immunotoxins that incorporate plant or bacterial toxins. Complete tumor regression by immunotoxins has been observed in some animal studies; however, overall, immunotoxin therapy seems to work best with tumors such as leukemias, that are readily accessible to the circulation. Immunotoxins have been rather ineffective in killing solid tumor masses. Additionally, the side effects of immunotoxins remain a significant impediment to their use in human therapy. Indeed, unconjugated antibodies are sometimes better suited than immunotoxins as therapeutic agents. Other Antibody-Effector Molecule Conjugates Radioisotopes can also be effector molecules. In a recent phase I study, a radioiodinated B cell specific antibody was successfully used in 10 patients with non-Hodgkin's lymphomas unresponsive to primary chemotherapy (Kaminski et al., 1993). Four of the treated patients had complete remissions, with no evidence of myelosuppresion. This type of treatment holds promise for improving standard chemotherapy by decreasing toxic side affects and increasing efficacy. As an attempt to reduce the risk of non-specific toxicity of immunotoxins, antibodies have been conjugated to enzymes that convert a prodrug into its active form. In this way, the antibody is first injected and given enough time to find its target and for the excess antibody to clear. The prodrug is then injected and converted to its active form only at the sites where the antibody is bound. Such antibody targeted prodrugs have been shown to be more effective in eliminating tumors than the chemotherapeutic drugs alone.

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Although some cancers may have tumor-specific antigens, the immune system might not be effective in rejecting the tumor because the antigens do not elicit strong immune responses. Recently, Tao and Levy (1993) demonstrated a promising way to improve the potency of the immune system against weak tumor immunogens. They fused the gene for GM-CSF, a cytokine thought to augment antigen presentation in a variety of cells, to the gene for an antibody specific for the variable regions of the immunoglobulin molecules expressed on malignant B cells in order to increase the immunogenicity of the tumor. The fusion protein elicited a strong antibody response and protected mice from subsequent challenge with tumor cells bearing the tumor antigen. These results could have important implications in the design of cancer vaccines. Bispecific Antibodies

A bispecific antibody has two specificities, resulting from the fusion of two hybridomas, or chemical conjugation of two antibodies with different specificities (Figure 1, bottom). Bispecific antibodies can have useful therapeutic applications. For example, having one antigen binding site that is specific for a toxin, drug, enzyme, or radioisotope, can provide an alternative to chemical conjugation of effector molecules. A less obvious use is recruiting effector cells to cellular targets. One antigen binding site can be used to locate a tumor cell, while the other antigen binding site can be used to recruit a cytotoxic T cell or NK cell by recognizing a cell surface protein on the cytotoxic effector cell. Bispecific antibodies may enable the killing of target cells by cytotoxic T cells in an MHC independent manner if they engage a common cell surface antigen such as CD3 (a molecular complex involved in T cell signal transduction). This approach could prove useful in killing tumor cells that have lost MHC expression and as a result are capable of escaping CTL surveillance. The advantages of antibody gene fragments ranging from the ability to use a bacterial expression system to the elimination of Fc reactivity, are also available for use with bispecific antibodies. Winter's group has produced bivalent and bispecific antibody fragments called "diabodies" (Holliger et al., 1993). Diabodies are made by linking a VH and VL domain from different antibodies by a short polypeptide chain (similar to scFv fragments) that is too short to allow pairing of the two V domains (Figure 1, bottom). The two domains are then forced to associate with a complementary Vn-short linker-VL chain that is coexpressed in a bacterial expression system. Winter's group made a double construct: they combined a VH domain gene segment from a monoclonal antibody specific for phenyloxazolone with the gene for the VL domain from a monoclonal antibody specific for hen egg lysozyme, and reciprocally, combined the VL domain gene of the phenyloxazolone antibody and linked it to the VH domain gene of the hen egg lysozyme antibody. The two scFv associated and were shown to bind specifically to phenyloxazolone and/or hen egg lysozyme.

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Normally, antibodies are secreted and bind to antigens in the extracellular milieu. The idea of using an antibody to bind an intracellular target has been known for more than a decade; however, it was not until recently that advances in antibody engineering made the use of intracellular antigens much more feasible and promising. Marasco et al. (1993) have illustrated the use of intracellular antibodies by designing an antibody that acts intracellularly to prevent the processing of the HIV envelope protein gpl20, a crucial protein the HIV virus uses to attach and infect target cells. Marasco accomplished this by designing a single chain Fv fragment from an antibody known to bind gpl20 and linking it to a leader sequence so as to target it to the endoplasmic reticulum, the site where gpl20 is processed from its precursor, gpl60. Once expressed in the endoplasmic reticulum of a cell expressing high levels of the envelope protein, the single-chain antibody bound gpl60, prevented its cleavage to gpl20, and inhibited gpl20 mediated syncytial formation by 80-90% (some researchers think syncytial formation is at least partly responsible for the loss of immune cells in AIDS). When the intracellular antibody was expressed in cells that were infected with the entire HIV genome, the virus particles released from the cells were more than 1000 times less infectious than ordinary HIV. Also encouraging was the fact that the intracellular antibody appeared to have no toxic effects on cells. Work on designer antibodies such as this illustrates the prospect of using antibodies in gene therapy. Besides the possible approach to AIDS therapy, intracellular antibodies may be beneficial in cancer therapy as many oncoproteins traverse through the ER. Intracellular antibodies that target other intracellular locations besides the ER are also available and may be exploited. One day, antibodies may prove to be important in both humoral and cellular immunity.

CONCLUSION Paul Ehrlich, who was one of the first to appreciate the specificity of antibodies, became particularly intrigued with using antibodies as weapons against cancer and dubbed them "magic bullets." Although few would dismiss Ehrlich's early visions as unrealistic, attaining the vision has not been without formidable obstacles. Fortunately, recent research has brought a plethora of new tools and ideas that are making the effective therapeutic use of monoclonal antibodies much more of a reality. Antibody molecules are well suited for genetic engineering. Their multichain, domain structure, as well as the fact that the antigen binding sites are loops that rest on top of a scaffold of beta pleated sheets, give them modularity. This modularity was first exploited in the forms of chimeric and humanized antibodies, with the goal of ameliorating such side effects as a HAMA response. The expression of engineered antibody genes in prokaryotes is a development that has the potential to bypass animal immunization altogether and allow us to mimic the generation of

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diversity, selection, and affinity maturation. With these tools, antibodies can be manipulated to change their antigenicity, effector functions, pharmacokinetics, localization, and expression. Basic science is providing knowledge of biological mechanisms giving us insight into how to design antibodies to enhance or interfere with these mechanisms to produce desired results. It would seem that Paul Ehrlich's "magic bullets" are closing in on the bull's eye.

ACKNOWLEDGMENTS We would like to thank Phillip Funk for a critical reading of this manuscript.

REFERENCES Duchosal, M.A., Eming, S.A., Fischer, P., Leturcq, D., Barbas, C.F., McConahey, P.J., Caothien, R.H., Thorton, G.B., Dixon, F.J., & Burton, D.R. (1992). Immunization of hu-PBL-SCID mice and the rescue of human monoclonal Fab fragments through combinatorial libraries. Nature (Lond.) 355, 258-262. Holliger, P., Prospero, T., & Winter, G. (1993). "Diabodies": Small bivalent and bispecfic antibody fragments. Proc. Natl. Acad. Sci. USA 90, 6444-6448. Hoogenboom, H.R., & Winter, G. (1992). Bypassing immunisation: Human antibodies from synthetic repertoires of germline V^ gene segments rearranged in vitro. J. Mol. Biol. 227, 381-388. Jones, P.T., Dear, P.H., Foote, J., Neuberger, M.S., & Winter, G. (1986). Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature (Lond.) 321, 522-525. Kaminksi, M.S., Zasadny, K.R., Francis, I.R., Milik, A.W., Ross, C.W., Moon, S.D., Crawford, S.M., Burgess, J.M., Petry, N.A., Butchiko, G.M., Glenn, S.D., & Wahl, R.L. (1993). Radioimmunotherapy of B-cell lymphoma with ['^^I]anti-Bl (anti-CD20) antibody. The New England J. of Medicine 329, 459-465. Kohler, G., & Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256,495-497. Marasco, W.A., Haseltine, W.A., & Chen, S.Y. (1993). Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gpl20 single-chain antibody. Proc. Natl. Acad. Sci. USA 90, 7889-7893. Marks, J.D., Griffiths, A.D., Malmqvist, M., Clackson, T., Bye, J.M., & Winter, G. (1992). By-passing immunization: Building high affinity human antibodies by chain shuffling. Biotechnology 10, 779-783. Marks, J.D., Hoogenboom, H.R., Bonnert, T.P., McCafferty, J., Griffiths, A.D., & Winter, G. (1991). By-passing immunization: Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222, 581-597. Morrison, S.L., Johnson, M.J., Herzenberg, L.A., & Oi, V.T. (1984). Chimeric human antibody molecules: Mouse antigen-binding domains with human constant region domains. Proc. Natl. Acad. Sci. USA 81, 6851-6855. Rybak, S.M., Hoogenboom, H.R., Meade, H.M., Raus, J.C.M., Schwartz, D., & Youle, R.J. (1992). Humanization of immunotoxins. Proc. Natl. Acad. Sci. USA 89, 3165-3169. Tao, M., & Levy, R. (1993). Idiotype/granulocyte-macrophage colony-stimulating factor fusion protein as a vaccine for B-cell lymphoma. Nature (Lond.) 362, 755-757. Williamson, R.A., Burioni, R., Sanna, P.P., Partidge, L.J., Barbas, C.F., & Burton, DR. (1993). Human monoclonal antibodies against a plethora of viral pathogens from single combinatorial libraries. Proc. Natl. Acad. Sci. USA 90, 4141-4145.

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RECOMMENDED READINGS Mayforth, R.D. (1993). Designer Antibodies. Academic Press, Inc., San Diego, California. Mayforth, R.D., & Quintans, J.Q. (1991). Designer and Catalytic Antibodies. New England J. Medicine 323, 173-178. Riethmuller, G., Scheider-Gadicke, E., & Johnson, J.P. (1993). Monoclonal antibodies in cancer therapy. Curr. Opinion in Immunology 5, 732-739. Waldmann, T.A. (1991). Monoclonal antibodies in diagnosis and therapy. Science 252, 1657-1662.

Chapter 16

Psychoneuroimmunology RUTH M. BENCA

Introduction Evidence for Immune System-Nervous System Interactions Conditioned Immune Responses Stress and Immune Function Stress Effects on Humans Conclusion Recommended Readings

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INTRODUCTION The nature and extent of the relationship between the mind and body has been a central issue for the practice of medicine since its beginnings. The early Greeks believed that emotions could influence the course of physical illnesses, and emphasized the importance of an holistic approach. However, from the 17th to 19th Principles of Medical Biology, Volume 6 Immunobiology, pages 303-313. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-811-0

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century, a variety of influences led to the view that disease was exclusively a physical disorder. The philosopher Descartes proposed that mind and body were separate and distinct entities; the impact of Cartesian dualism continues to this day. The major medical discoveries of this period—^including Hook's discovery of the cell itself, the connection between diseased cells and bodily illness described by Virchow, the identification of pathogens responsible for disease by Pasteur and others, and the development of vaccination by Jenner—resulted in an increasing disregard for the idea that psychological processes might play a role in disease states. The modem era of psychosomatic medicine began in the early 20th century with the theories of Freud and others which postulated that psychological conflict could be expressed as physical dysfunction, as in the case of conversion disorders. In the 1930s, Alexander hypothesized that certain medical illnesses were the result of unconscious psychological conflicts, and that specific emotional states resulted in specific physical disorders. The impact of Alexander's work had a major influence on the modern practice of psychiatry; the standard psychiatric nosology included a listing of psychosomafic disorders, "characterized by physical symptoms that are caused by emotional factors and involve a single organ system" as recently as 1968. Although the failure to provide empirical support for the specificity hypothesis of psychosomatic illness gradually led to an abandonment of that theory, evidence for the relationship between psychological factors and illness has continued to accumulate. One of the first mechanisfic explanations relating psychological factors to illness was provided by Selye in the 1940s. He described a general adaptation syndrome to stress which involved activation of the hypothalamic-pituitary-adrenal cortical axis and proposed that excessive stress could result in physical illness. Subsequently, a number of studies have demonstrated significant correlations between stressful life events and increased risk for illness. "Psychosomatic medicine" has gradually been replaced by the biopsychosocial model of illness, first proposed by Engel in the 1950s, which emphasizes the importance of psychological and cultural factors in influencing the onset and course of disease states. Disease has muUifactorial etiologies, including causative and contributory factors, all of which need to be addressed in clinical treatment. Although there has been an exponential increase in our understanding of the molecular basis for disease over the past several decades, far less is known about the mechanisms linking psychological factors with illness. The relatively new field of psychoneuroimmunology represents an attempt to explain one possible route by which emotional states may influence disease, and is based on evidence that the immune and nervous systems may regulate each other.

EVIDENCE FOR IMMUNE SYSTEM-NERVOUS SYSTEM INTERACTIONS The concept of nervous system control over bodily fimctions has long been accepted, with the major exception of the immune system. Unlike most other

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physiological systems which consist of sessile organs hard-wired to the brain, the immune system is comprised of lymphocytes which perform their surveillance functions by wandering throughout the body and communicating via secretory substances or cytokines. Furthermore, lymphocytes can carry out their various functions adequately when removed from the body and placed in tissue culture dishes, suggesting that the nervous system is unnecessary for a functional immune system. However, recent work has elucidated potential pathways for communication between the nervous and immune systems. First, the immune system has the capacity to receive direction from the nervous system. Although lymphocytes themselves do not receive direct neural input, the vasculature of lymphoid organs is extensively innervated. Noradrenergic sympathetic fibers are found in all primary and secondary lymphoid organs. The thymus, which is the primary organ responsible for T cell production, and the secondary lymphoid organs, lymph nodes and spleen, also appear to receive input from a variety of peptidergic fibers, including vasoactive intestinal peptide (VIP), substance P (SP), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP). Cholinergic fibers may also be present in the thymus, lymph nodes, and spleen. The immunologic significance of lymphoid innervation is not known definitively, but it is possible that neural input may affect lymphocyte traffic through effects on vasculature, and thus impact on immune function. This idea is supported by sympathetic denervation studies, which have shown variable effects on immune responses in rodents. In addition to the demonstration of nerve input to lymphatic organs, lymphocytes themselves have receptors for and/or respond to a variety of neurotransmitters, neuropeptides, and neuroendocrine factors. Beta-adrenoreceptors are found on lymphocytes, including T cells, B cells, monocytes, and macrophages. Norepinephrine has been reported to exert enhancing effects on the inductive phase of immune responses, but has significant immunosuppressive effects on the effector phase of mitogen responses, cytotoxic responses, antibody production, interleukin2 (IL-2) induced proliferation, and natural killer cell (NK) activity. It is thought that norepinephrine exerts its effects on lymphocytes by increasing cAMP activity and thus decreasing responses to IL-2; conversely, alpha-adrenergic agonists and beta-endorphins, which decrease cAMP within cells and enhance their responses to IL-2, have more consistent stimulatory effects on immune responses. The variable effects of adrenergic compounds may also be related to the unequal distribution of receptors on lymphocyte subpopulations. The neuropeptides SP and VIP also influence lymphocyte function. SP binds to tachykinin receptors on lymphocytes and monocytes. It induces chemotaxis in monocytes, as well as stimulating production of the cytokines IL-1, IL-6, and tumor necrosis factor (TNF-alpha). SP stimulates T cell proliferation and increases antibody production by B cells, probably via IL-6. VIP also appears to bind to most lymphocytes, including T and B cells and monocytes, and tends to show immuno-

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suppressive effects on T cell mitogen responses, mixed lymphocyte responses, and NK activity. It may also inhibit homing of lymphocytes into and prevent their migration out of lymph nodes. It should be less surprising that lymphocytes have hormone receptors since this is a characteristic of most cells in the body. Some hormones released as part of the stress response appear to have specific effects on the immune system; the effects of glucocorticoids on immune responses have been studied the most extensively. Glucocorticoids are used widely in clinical practice for their powerful immunosuppressive and anti-inflammatory properties. They have been shown to inhibit monocyte and macrophage activities, including production and secretion of the cytokines IL-1, IL-2, TNF, and gamma-interferon. Conversely, systemic glucocorticoid depletion through adrenalectomy or Addison's disease results in lymphocytosis and thymic hypertrophy. It has been suggested that one of the primary functions of glucocorticoids during stress responses is to prevent the self-reactivity which might occur if the immune system became hyperactivated. On the other hand, prolactin, which is also released during an acute stress response, appears to have immunoenhancing effects. Increased levels of prolactin have been associated with increased mitogen responses. Conversely, suppression of prolactin with administration of bromocriptine suppresses the ability of rodents to combat infections and decreases T and B cell responses. Other hormones may also have effects on the immune system. Hypophysectomy results in thymic atrophy, decreased antibody function, cytoxic T cell activity, and NK activity, all of which can be reversed at least in part with administration of growth hormone. Not only does the nervous system have the capacity to influence the immune system, but the reverse also appears to be possible. Lymphocytes synthesize a number of compounds similar if not identical to those produced by neurons, including endorphins, enkephalins, SP, VIP, CRH, and ACTH. The nervous system also responds to cytokines produced by lymphocytes and macrophages, particularly IL-1, tumor necrosis factor (TNF), and IL-6. IL-1 stimulates ACTH secretion by the pituitary and thus can affect the functioning of the hypothalamic-pituitary-adrenal axis. Glial cells can produce IL-1, and receptors for IL-1 have been demonstrated in the nervous system. Cytokines also have significant effects on temperature and behavioral state. Both IL-1 and tumor necrosis factor (TNF) can act as endogenous pyrogens. IL-1 administered intracerebroventricularly increases non-rapid eye movement (NREM) sleep in various mammalian species. These data have been interpreted to suggest that an infectious process resulting in a significant immune response sends a signal to the brain to increase sleep amount, perhaps a homeostatic response to infection. In summary, the immune system can be regulated by a number of neurotransmitters, neuropeptides, and hormones as indicated by the presence of receptors on

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lymphocytes, as well as their ability to respond to these substances. The nervous system, in turn, has the ability to receive messages from the immune system through receptors for cytokines. Afferent and efferent nerve pathways between the brain and lymphoid organs have also been identified. The data presented so far, however, only demonstrate the potential for communication between the immune and nervous systems. The existence of physiological regulatory connections between the two systems has been more difficult to establish. Most studies to date have focused primarily on nervous system control of immune function through conditioning or via stress effects.

CONDITIONED IMMUNE RESPONSES Classical conditioning is an example of associative learning and involves the attachment of a behavioral response to a stimulus previously unrelated to that response. A neutral, conditioned stimulus (CS) is presented immediately prior to an unconditioned stimulus (US), which is known to elicit the desired behavior. After repeated, paired presentation of the CS and US, however, presentation of the CS alone will elicit the behavior as the organism learns to predict the relationship between the CS and US. At a cellular level conditioning is known to depend on presynaptic facilitation of sensory neurons by the CS. The sensory neurons are, in turn, linked to efferent neurons which regulate the behavior. The technique of classical conditioning has until recently been applied only to responses known to be under direct neural control. Thus, the ability to demonstrate classical conditioning of immunological responses has been interpreted to suggest that these responses may be regulated by the nervous system. There is a long history of anecdotal evidence of conditioned immune responses, such as the ability to elicit allergic responses in patients through the presentation of pictures or artificial representations of allergens. One of the first experimental demonstrations of conditioned effects on the immune system was the production of immunosuppression in rats by conditioning by Ader and Cohen in 1975. Saccharin-flavored drinking water (the CS) was paired with injection of the immunosuppressive drug cyclophosphamide (the US). Conditioned animals given saccharin solution at the time of immunization with sheep red blood cells (SRBC) showed a decreased response to SRBC in comparison with non-conditioned animals exposed to the CS or conditioned animals not exposed to the CS. Conditioned immunosuppression using similar CS/US paradigms has been demonstrated subsequently in both rats and mice for a variety of immune parameters, including both T-dependent and T-independent responses to antigen challenges, T-cell proliferation responses to mitogens, graft-vs-host responses, and natural killer cell (NK) activity. However, not all attempts at conditioned immunosuppression have been successful, as some studies have failed to demonstrate significant effects on antibody responses, particularly to T-independent antigens, and delayed-type hypersensitivity. Furthermore, although statistically significant, the magnitude of

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immunosuppression achieved through conditioning was relatively small in comparison to the normal effects of the immunosuppressive drugs. Another problem with the paradigms of conditioned immunosuppression is that they are connected to taste aversion, making it difficult to determine whether the CS-induced immunosuppression is the result of a non-specific stress response or a process involving specific learning and memory of immune function. The ability to demonstrate immunosuppression following exposure to the CS without significant taste aversion in some studies has been taken as evidence against a non-specific stress response mediating the conditioning effects. However, a specific neurological mechanism for conditioned immunosuppression has not yet been demonstrated. Attempts have also been made to condition stimulation or enhancement of immune function, which might further support the idea of neuronal regulation of immune function. To date, there has been no convincing evidence for the ability to condition antigen-specific responses. Perhaps the best example of immunoenhancement has been the CS-induced increase in cytotoxic T cells following conditioning by repeated allogeneic skin grafting; in this paradigm, the grafting procedure represented the CS and the presentation of histoincompatible donor skin the US.

STRESS AND IMMUNE FUNCTION The idea that stress could affect the immune system grew out of attempts to explain the observation that stress was associated with illness. Selye and others demonstrated in animals that stressors induced activation of the hypothalamic-pituitaryadrenal axis and led to pathological changes in various organ systems. In the 1970s, epidemiological studies established a correlation between stressful life events and an increased risk of subsequent illness or death. The term "stress" generally refers to noxious or arousing stimuli, which can be physical or emotional in nature. One major area of research has been to study the effects of stress on in vitro immune parameters in rodents, particularly lymphocyte proliferation responses to mitogens and NK cell activity. These assays can be performed on lymphocytes obtained from peripheral blood samples or spleen, making them relatively easy to perform. Both NK cells and mitogen-induced lymphocyte proliferation represent more primitive components of the immune system; neither involves antigen-specific recognition. The clinical significance of these parameters is not fully understood, although both responses are significantly decreased in severely ill or immunocompromised subjects. Rodents have been subjected to a variety of stressors, including inescapable electric shock, noise, isolation, or crowding. The results have been mixed; both decreased and increased immune responses were observed in early studies. More recent studies have attempted to assess the effects of duration or intensity of the stressor in rodents. Although increased intensity of the stressor was associated with increased suppression of mitogen responses by peripheral blood lymphocytes

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(PBLs), a longer duration of the stressor resulted in return to normal levels of mitogen responses or even enhanced responses in other studies. Lymphocytes derived from spleen appeared to be less susceptible to stress-induced suppression of mitogen responses than PBLs. NK cells obtained from rat spleen, however, showed decreased abilities to lyse target cells following acute or chronic stress. In vivo tests performed in rodents have assessed effects of stressors on tumor growth and response to bacterial/viral challenges. A number of studies have suggested that tumor growth and susceptibility to infection are increased with stress, although opposite effects (i.e., decreased rates of infection or tumor growth in stressed animals) have also been reported. The variability of the results has been interpreted as suggesting that acute stress is immunosuppressive, whereas chronic stress may result in immunoenhancement. The differing natures of the stressors and the challenging agents applied makes it difficult to make generalizations about the epidemiology of stress effects on host responses. Furthermore, most of these studies have not included direct measures of immune function, which makes it difficult to know whether changes in the immune system are primarily responsible for the observed effects. To address the effects of stress on the immune system more specifically, in vivo tests of immune function have been performed on both rodents and humans exposed to stressors. Once again, the results have been mixed. The preponderance of studies has documented immune suppression, including decreased antibody production following antigenic challenge, decreased delayed-type hypersensitivity (DTH), and reduced graft-versus-host responses. A few studies have shown immunoenhancement following stress, however. An alternative view regarding stress and immune function has been proposed recently by Dhabhar and colleagues, who hypothesize that stress may actually have immunoenhancing effects. Their studies have demonstrated decreases in peripheral blood lymphycytes, NK cells and monocytes following acute stress with redistribution of these cells to other compartments such as skin. Given that immune responses do not tend to occur in blood, the stress-induced migration of leukocytes to target organs may enhance the ability of the organism to respond to antigenic challenge. As predicted, in their studies, rats subjected to restraint or shaking stress also showed increased DTH responses. The seemingly contradictory nature of the results should not be particularly surprising. Immune responses in vivo involve sequential steps of antigen recognition, processing, and presentation, as well as the involvement of different interacting lymphocyte and accessory cell populations. Stress effects could interact with the immune system in any number of ways, including altering patterns of lymphocyte release from lymphoid organs and recirculation, impacting on the function of specific cell types through neuroendocrine mechanisms, or modifying the integrity of host barriers. Clearly, stress has demonstrable effects on the immune system

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which cannot be categorized reHably until the mechanisms for these effects are better understood. Stress Effects on Humans Because of the potential clinical importance of stress effects on disease susceptibility and progression, a number of paradigms of stress and immune function are being studied in humans. Most have been confined to measuring mitogen responses and/or NK activity in peripheral blood lymphocytes, or serum antibody titers. Some of the earliest clinical studies performed were assessments of lymphocyte proliferation to mitogens in recently bereaved spouses. There is epidemiological evidence that bereaved spouses have a greater incidence of morbidity and mortality immediately after the death of their spouses than age-matched nonbereaved subjects. In the earliest bereavement study, Bartrop and colleagues found that mitogen responses in widowers were decreased significantly in the weeks following the deaths of their wives in comparison to nonbereaved controls. Prospective studies of both men and women who were about to lose their spouses to cancer showed that mitogen responses immediately following the death of the spouses were decreased in comparison to responses measured in the same subject prior to the spouse's death. Although these studies have received methodological criticisms (i.e., the period immediately prior to the death of the spouse is not particularly stress-free), they nevertheless suggest that changes in some compartments of the immune system predictably occur following bereavement. Since bereavement is associated with increases in depressive symptomatology, it is reasonable to hypothesize that bereavement affects the immune system by causing depression, which can result in a number of neuroendocrine changes that could impact on immune function. A number of investigators have assessed immune parameters in patients with major depression.^ The results for depression have been less consistent than for bereavement, however. Studies of peripheral blood lymphocyte stimulation by mitogens have shown decreased responses in some studies and no changes in others. Similarly, NK activity in depression has been reported as either decreased or unchanged in comparison to normal controls. Counts of white blood cells, lymphocytes, neutrophils, and NK cells have also not been consistently altered in peripheral blood samples obtained from depressed patients. Since depressed patients may not be necessarily homogenous in either their symptomatology or pathophysiology, some studies have attempted to determine whether particular patient characteristics are more likely to be associated with immune system changes. There is some evidence that increased age and severity of depression may be associated with decreased mitogen responsivity. Several studies have assessed the role of HPA axis activation on immune function in depression, since corticosteroids are known to affect immune function. However, no consistent changes in lymphocyte counts or mitogen responses were found in

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either depressed patients with increased Cortisol levels or those who failed to show Cortisol suppression following dexamethasone administration. The effects of various situational stressors on immune function have also been studied. Kiecolt-Glaser and colleagues found decreased mitogen responses by peripheral blood lymphocytes, decreased cellular immune responses to herpesvirus, and reduced NK activity in medical students during their final examination period in comparison with levels obtained earlier. Increases in several herpesvirus antibodies were also found, and were interpreted as indicating decreased cellular control of latent infection, although the results could also be interpreted as a non-specific enhancement of antibody production. Loneliness and social isolation have also been correlated with decreased NK activity and mitogen responses in studies of students, psychiatric patients, and divorced subjects. Decreased immune function has also been shown in primates following maternal separation and/or isolation. Exercise has also been postulated to have similar effects to stress on the immune system. Studies on behavioral states and immune function have not all revealed consistent or significant effects on immune parameters. These studies have been criticized for the failure to correlate any of the observed immune system changes with clinical effects. For example, it is not known whether the small decrements in mitogen responses or NK activity observed in some studies can lead to increased susceptibility to infection or malignancy. Furthermore, the epidemiological relationships between bereavement, depression, or acute stress and diseases specifically associated with immune system dysfunction tend to be weak at best. Finally, behavioral states such as depression or bereavement are probably not equivalent amongst afflicted individuals, which may also account for the failure to document robust or consistent effects on the immune system related to these conditions. Yet another approach to studying immune system-nervous system interactions is to assess the relationships between specific behavioral states and immune function. The effects of sleep and sleep deprivation on immune responses have been widely studied. Sleep is a universal behavior in animals, and although the ultimate function of sleep has not been identified, it is known that sleep is necessary for survival; rats deprived of sleep die within 3 to 4 weeks. Furthermore, sleep loss is a common result of stress and is associated with most psychiatric disorders, particularly depression. A number of studies have looked at the effects of sleep deprivation on immune function in animals and humans. Short-term sleep deprivation in humans has been reported to cause decreases in mitogen responses andNK acfivity in several studies, but in a recent study, subjects deprived of sleep for 60 hours showed an increase in NK activity compared to pre-deprivation levels. Rats subjected to several hours of sleep deprivation showed decreases in secondary but not primary antibody responses to a viral challenge. Rats deprived of sleep to the point of near-death have yielded several interesting—^and seemingly contradictory—results. First, they

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showed no decline in mitogen responses, or in vitro or in vivo primary antibody responses to antigens. However, in another study, 5 of 6 sleep-deprived rats showed bacteremia shortly before death. Taken together, these results suggest that prolonged sleep deprivation in rats may lead to a breakdown in innate defense barriers, yet not necessarily affect all acquired immune responses, such as the ability to produce antibodies in response to antigenic challenge.

CONCLUSION In spite of a great deal of research, the nature of the relationship between the nervous and immune systems is not yet fiilly understood. Obviously, they are both highly complex systems which have the potential for interacting with each other in multiple ways. This complexity creates the potential for seemingly contradictory results, as well as the possibility for providing a scientific basis for reunifying behavioral and physiological approaches to health and disease. There are several important caveats which must be considered in evaluating studies of immune-nervous system interactions, however. First, decreases in immune measures, particularly as indicated by in vitro tests of peripheral blood lymphocytes, may not correlate with how an individual responds to a clinical challenge such as an infection. Conversely, increases in immune measures do not necessarily indicate an improved immune status, since they may represent a compensation for a deficit in another compartment of the immune system, or could even predispose the individual to autoimmune disease. Second, changes in one set of immune responses may not necessarily represent the immune system as a whole, since the immune system can simultaneously upregulate some responses while downregulating others. Third, although much of the popular appeal of psychoneuroimmunology has been its potential application for cancer patients, there is little evidence that the immune system plays a significant role in combatting many forms of cancer, particularly established tumors. An accurate understanding of the current limits of our knowledge of psychoneuroimmunology is particularly important for the practice of clinical medicine. It is possible for patients to believe that their illnesses—especially cancer—^have been caused by their inability to "handle stress" or a poor psychological state, thus causing them to blame themselves for their illnesses. Although some studies have suggested that various behavioral and psychosocial interventions such as psychotherapy, relaxation training, or guided imagery techniques (i.e., imagining one's immune system successfully fighting off a tumor) might have small but significant positive effects for some patients, in the majority of cases, cancer survival is most closely associated with tumor pathology rather than psychological state. There is no consistent evidence to date that particular psychological states or psychosocial interventions are predictably and consistently associated with specific changes in the immune system. Thus, although psychological and behavioral treatments may be helpful for improving a cancer patient's sense of well-being and thereby improve

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quality of life, it is important to keep in mind that a failure of these interventions to cure or even significantly retard disease progression does not mean that the individual has "failed" to adhere to the treatments correctly. Although the nervous system does not appear to be the primary regulator of the immune system (and vice versa), it does not mean that immune-nervous system interactions are unimportant or irrelevant to clinical medicine. On the contrary, perturbations of one system can have significant impact on the other. The potential for applications to various areas of clinical medicine—such as infectious diseases, autoimmune diseases, transplantation, and AIDS, for example—^has hardly begun to be explored. Much further work is needed to define the mechanisms of the interactions between the two systems, which in turn should lead to more effective treatment strategies.

NOTE 1. Major depression is defined by the presence of depressed mood or loss of interest or pleasure for a period of two weeks or more, accompanied by at least five of the following symptoms: significant change in appetite or weight, insomnia or hypersomnia, psychomotor agitation or retardation, fatigue, sense of worthlessness or guilt, decreased concentration, and/or suicidal ideation.

RECOMMENDED READINGS Ader, R. (1992). On the clinical relevance of psychoneuroimmunology. Clinical. Immunol. Immunopathol. 64, 6-8. Ader, R., Felten, D.L., & Cohen, N. (eds). (1991). Psychoneuroimmunology (2nd edn.) Academic Press, Inc., New York. Cotman, C.W., Brinton, R.E., Galaburda, A., McEwen, B., & Schneider, D.M. (eds). (1987). The Neuro-Immune-Endocrine Connection. Raven Press, New York. Dhabhar, F.S., & McEwen, B.S. (1996). Stress-induced enhancement of antigen-specific cell-mediated immunity. J. Immunol. 156, 2608-2615. Hoffman-Goetz, L., & Pedersen, B.K. (1994). Exercise and the immune system: A model of the stress response? Immunol. Today 15, 382-387. Husband, A.J. (ed). (1993). Psychoimmunology: CNS-Immune Interactions. CRC Press, Boca Raton. Kiecolt-Glaser, J.K., & Glaser, R. (1995). Psychoneuroimmunology and health consequences: Data and shared mechanisms. Psychosom. Med. 57, 269-274. Kreuger, J.M., Toth, L.A., Floyd, R., Fang, J., Kapas, L., Brendow, S., & Obal, F. (1994). Sleep, Microbes and Cytokines. Neuroimmunomodulation. 1, 100-109. Lloyd, R. (ed). (1994). Explorations in Psychoneuroimmunology. Grune and Stratton, Orlando. Maier, S.F., Watkins, L.R., & Fleshner, M. (1994). Psychoneuroimmunology. Am. Psychol. 49, 1004-1017.

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INDEX

Acquired Immunodeficiency Syndrome (AIDS), 17 Activation induced cell death (AICD), 270 Adenosine deaminase (ADA), 17 Affinity maturation, 35, 54, 284 mimicking, 294 AITD, 243-252 (see also "Autoimmune diseases....") Allelic exclusion, 94 Allografts, 76 rejection, use of antibodies in, 285 Alloresponsiveness, 120 Allotype, 75 Amiodarone, 251 Anaphylactoid reactions, 232 Anaphylatoxins, 200-201 Anaphylaxis, 186, 223, 229-236 causes, classification of, 231-235 anaphylactoid reactions, 232 angioedema, 233 "aspirin triad," 232 blood transfusions, 231-232 catamenial, 233 clinical findings, 233 complement-mediated, 231-232 differential diagnosis, 234-235 direct mast cell activators, 232233

exercise-induced, 233 haptens, 231 hereditary angioedema, 234-235 histamine, 233, 234, 235 idiopathic, 233 IgE mediated, 231 non-immunologically mediated mast cell reactions, 232-233 nonsteroidal antiinflammatory drugs (NSAID), 232-233 pathogenesis, 233-234 radiocontrast media, iodinated, 232 systemic mastocytosis, 234-235 tryptase, 235 urticaria, 233 introduction, 230-231 components, 230 definition, 230 mast cell degranulation, 230 summary, 235 treatment, 235 epinephrine, subcutaneous, 235 Anergy, 70-71, 74-75, 78, 80, 242, 253 Antagonist peptides, 77 Antibodies, designer, 279-300 bispecific, 297 chimeric and humanized, 287-288 conclusion, 298 315

316

effector functions, antibodytargeted, 295-298 antibody-effector molecule conjugates, 296-297 cancer vaccines, implications for, 297 GM-CSF, fusion of, 297 immunotoxins, 295-296 radioisotopes, 296 ricin, 296 toxicity, 296 Escherichia Coli, expression of fragments in, 288-289 advantages, 288 Fv fragments, 289 single-chain Fv fragments, 289 technical overview, 288-289 transfectoma, 288 in gene therapy, 298 immunoglobulin fragment combinatorial libraries, 289-295 affinity maturation, mimicking, 294 antibody secretion vs. cell surface expression, mimicking, 291 antigen-driven selection, mimicking, 291 bypassing immunization, 291294 chain shuffling, 294 filamentous bacteriophage, 290 fragment expression library, 290-291 murine expression systems, 294295 phage library, 294 Polymerase Chain Reaction (PCR), 294 SCID mice and transgenic mice, using, 295 intracellular, 298 HIV, 298

INDEX

introduction, 280-281 monoclonal antibodies in therapeutics, 284-287 in allograft rejection, 285 Campath-1 antibody, 287 in cancer, 285 concerns and limitations, 286287 costimulation, 285 diagnostic imaging, 286 HAMA response, 286, 287 hybridoma technology, 286 immunotoxin, 285 quantity, 286 radioisotopes, 285 transplanatation, 285 uses, other, 285-286 structure, general, and biosynthesis, 281-284 affinity maturation, 284 biosynthesis, 284 complementarity determining regions (CDR), 281 constant domains, 281, 282-284 effector functions, 282-284 fragments, 282 hypervariable regions, 281, 283 immunoglobulin domains, 281 introduction, 281-282 variable domain, 281, 283 Antiidiotypy, 117 Antigen-antibody complex, 105-118 antibody structure, 106-111 Complementarity Determining Regions of variable domains (CDR), 107-108, 117 constant domains, 107-108 Fv fragments, 109-110 IgG, 106 immunoglobulin domain, 107, 108 quaternary arrangement, 106

Index

VL-VH pairing, 110-111, 118 variable domains, 107-108 antibody-antigen-complexes, 114117 antiidiotypy, 117 conformational changes, 116 Fv fragments, 116 hemoglobin, 117 somatic hypermutation, 116 antigen structure, 111-114 hemagglutinin, 111-112 influenza viruses, 111,113 monoclonal antibodies, 112 neuraminidase, 111-112 introduction, 105-106 summary, 117-118 Antigens, 84 Antigen-presenting cells, 65, 67, 87, 199-200 (see also "Immunological tolerance") Apoptosis, 88, 98, 240, 263-278 {see afco "Cell death ") Arachidonic acid, 222-223 Arthus reaction, 201 "Aspirin triad," 232 Autocrine proliferation, 53 Autoimmune diseases, 17-18, 237261 {see also "Thymus...") autoimmunity, 238 disease characteristics, 238 "horror autotoxicus," 238 immunological unresponsiveness, 238 self-tolerance, 238 thyroglobulin (Tg), 238 Witebsky's postulates, 238 disease, 242-243 diabetes, Type I, 242 effector mechanisms, 243 multiple sclerosis, 242 introduction, 238

317

negative selection and selftolerance, 240-242 affinity theory, 241 clonal anergy, 242, 253 clonal balance, 242, 253 clonal deletion, 241-242, 253 thymus, 242 positive selection, mechanisms of, 239-240 CD4 and CD8, 239 tyrosine kinase, 239-240 self and non-self, 238-239 negative selection, 239, 240-242 positive selection, 239-240 T cell receptor, 239 summary, 253-254 thyroglobulin, 252-253 thyroxine, as prohormone for, 252 thyroid disease, autoimmune (AITD), 243-252 amiodarone, 251 animal models, 245 autoantigens, 244 cytokines, role of, 247 drugs, 251 endocrine abnormality, 249-250 environmental factors, 250-251 Epstein-Barr virus infection, 250 genetic control of susceptibility, 249 genetic predisposition, 248-249 Graves' disease, 243 Hashimoto's thyroiditis (HT), 243 histopathological changes, 243244 HLA genes, 248 hormones in regulation of, 250 immunological response, 246248 incidence, 243 infectious agents, 250

318

iodine, 250-251 long-acting thyroid stimulator (LATS), 243 multifactorial etiology, 245-252 prolactin, 250 stress, 251 suppressor cell defect, 247-248 thyroglobulin (Tg), 244 thyroid peroxidase (TPO), 244 thyroid-stimulating hormone receptor (TSHR), 244 toxins, 251-252 Azurophil granules, 218 B cell in immunity, 5-6, 21-44, 65 antigen, responses to, 31-33 early stages, 32 T-dependent and T-independent responses, 32-33 in bone marrow, 23-30 anergy, 28 autoreactivity, 28 CDIO, 26, 27 CD19, 26 CD23, 29 CD40, 29 clonal deletion, 28 HLA-DR, 27 Ig-transgenic, 28 IgD, 29 IgE, 29 IgM, 29 IL7, 26 immature cells, 25 MHC class II antigens, 27 pre-B cells, 25, 26, 27-28 pro-B cells, 25, 26 scid mutation, 26-27, 40 stem cell factor (SCF), 25 stem cells, 24 tolerance, 28 virgin cells, 25, 26

INDEX

cell migration, 30-31 bursa of Fabricius, 31 dendritic cells, 30 follicles, 30 follicular dendritic cell (FDC), 30,32 IgM'igD'', 30 interdigitating dendritic cells (IDCs), 30, 32 marginal sinus, 30 marginal zone, 30 periarteriolar lymphocytic sheath (PALS),30 deficiencies, 39-42 bacterial meningitis, 42 btK 41 common variable immunodeficiency (CVID), 42 IgA deficiency, 41 IgM and IgC deficiencies, 42 immunoglobulin isotype deficiencies, selective, 41-42 X-LA, 39-41 X-LA with hyper-IgM, 39, 4142 {see also "X-LA with hyper-IgM") introduction, 22-23 antibody, 22, 23 antigen, 22, 23 in bone marrow, 22, 23-30 germinal center, 22, 23 immunoglobulin gene recombination, 32 isotype switching, 22 memory B cells, 22 plasma cell, 22, 26 repertoire, 22 somatic mutation, 22, 23 schematic, 23, 37 secondary response, 37-38 summary, 42 and T cells, interaction between, 5356 {see also "Cell-to-cell....")

Index

T-cell dependent responses, 33-37 affinity maturation, 35 B7,33 centroblasts, 35 centrocytes, 35 complementarity determining regions (CDRs), 36, 37 germinal center, 34, 35 {see also "Germinal....") IgM, 33, 34 IL4, 34 interdigitating dendritic cells (IDCs), 33 isotype switching, 34 Langerhans cells, 33 memory B cell, 36-37 PALS, 33 somatic mutation, 35, 37 Tingible bodies, 34-35 tolerogenic, 33 T cell independent responses, 38 lipopolysaccharide (LPS), 38 type 1 and type 2 antigens, 38 xW, 38,41 types, other, 39 B-CLLs, 39 CD5, 39 Ly-IB cell, 39 B cell signaling and T cell signaling at molecular level, 135-147 BCR and TCR receptors, 136138 CD3, 136 CD4 and CD8, 137 major histocompatibility complex (MHC), 137 structure, 137 inositol phospholipid hydrolysis and calcium mobilization, 138-139 phospholipase C(PLC), 138-139 protein kinase C (PKC), 138

319

introduction, 136 B cell antigen receptor complex (BCR), 136-138 T cell antigen receptor complex (TCR), 136 lymphocytes, other PTKs in, 142 nucleus, toward, 145-147 Grb2, 146 c-Raf, 146 mitogen activated protein (MAP) kinases, 146 Ras protein, 146 She, 146 Sos, 146 TCR-induced tyrosine phosphorylation cascade, 145-146 Vav, 146 Src family of nonreceptor PTKs, 141-142 Blk, 142 CD45 PTPase, 144 Csk PTK, 144 Fyn, 141, 142 Lck, 141 Lyn, 142 phosphatidylinositol 3-kinase (PI3-K), 143 regulation of, 142-144 SH2 and SH3, 143 summary, 147 pathways, three, of intracellular signal transduction, 147 SYK family of nonreceptor PTKs, 142 Zap, 142 tyrosine phosphorylation, 139-141 phosphotyrosine phosphatases (PTPases), 140 protein tyrosine kinases (PTKs), 139-140 Src family kinases, 140-142 {see afao "...Src family....")

320

Syk family kinases, 140-141 TCR/CD3 complex, 139 Bacterial killing by complement, IS3-IU (see also "Complement...") Bacterial meningitis, 42 Basophils, 204, 206-209, 218 Bat (B Associated Transcript) series of genes, 123 BCR, 136-138 (see also "B cell signaling....") bel-2, 266 Bereavement, effect of, 307-308 Bispecific antibodies, 297 (see also "Antibodies...") "Bjorkman's groove," 125, 126 Blood transfusion, anaphylaxis and, 231-232 Bombay blood group, 215 Bone marrow, 86 (see also "Diversity ") transplantation, 129 graft versus host disease, 129 Booster vaccination, 90 btk, 41 Burnet, Sir MacFarlane, 86 Bursa, 4-5 of Fabricius, 31, 150 Bystander killing, 274 Bystander lysis, 179 c-myc, 266 Cancer, cell death and, 275-276 antibodies, use of, 285 Cardiac transplantation, 129 Catamenial anaphylaxis, 233 CD5, 39 CD45 PTPase, 144 (see also "B cell signaling....") CDRs, 36, 84 Cell adhesion superfamilies, 46-47 immunoglobulin, 46 "barrel," 46

INDEX

integrins, 47 Leukocyte Function Antigen-1 (LFA-1), 47 selectins, 47 carbohydrate, binding, 47 transferase enzyme, 47 Cell-to-cell interactions in immune system, 45-60 antigen presentation to lymphocytes, 49-53 alpha helices, 51 autocrine proliferation, 53 to B cells, 50 CD3, 52 CD28, 52 clonal selection, 49 costimulation, 52 on dendritic cells, 51 endogenous peptides, 51-52 endoplasmic reticulum, 52 epitopes, 50 follicular dendritic cells (FDC), 50 ICAM family, 52 IL-2 and IL-2 receptors, 53 immunological memory, 50 introduction, 49 Langerhans cells, 51 LFA-1, 52 MHC molecules, 50-53 peptides, 51-52 to T cells, 50-53 T cell receptor complex, 52 leukocytes and endothelium, 58-59 E-selectin, 58, 59 high endotheUal venules (HEV), 58 L-selectin, 58, 59 lymphoid organs, 58 memory cells vs. naive cells, 58 model, three-step, 58-59 molecular, basis of, 46-47 adhesion, 46

Index

cell adhesion superfamilies, 4647 (see also "Cell adhesion ") cell surface molecules, 46-47 cytokines, 48-49 (see also "Cytokines") extracellular matrix, 46 as receptors for soluble factors, 46 T cell, 46 T cell antigen receptor, 46 summary, 59 in secondary lymphoid tissue, 59 T cells, effector mechanisms of, 53-58 affinity maturation, 54 CD40L, 54-55 class switching, 54 cytokines, 54-55 cytotoxic cells, 56-58 interferon-gamma (IFN-a), 5455 Leishmania infections, 56 and macrophages, 56 natural killer cells, 57 phagocytosis, 56 plasma cells, 54 T and B cells, interaction between, 53-56 T independent antigens, 54 Thl and Th2 cells, 56 tetanus toxoid, 54 transforming growth factor-beta (TGF-/?), 54-55 Cell death and immune system, 263278 biochemistry of apoptosis, 266-268 bel-2, 266, 270 c-myc, 266 DNA cleavage, 266 oxidative stress as mediator, 268

321

p53, 266 phagocytosis, 268 characteristics of apoptosis and necrosis, 265 clinical immunology, apoptosis in, 274-276 autoimmune disease, 276 cancer, 275-276 Epstein-Barr virus, 275 HIV, 275 host-graft interactions, 276 infection, 275 inflammation, 275 definitions, 264 apoptosis, 264 inflammation, 264 morphology of apoptosis and/ or necrosis, 264, 265, 267 necrosis, 264 programmed cell death, 264 of immune cells, 268-272 activation induced cell death (AICD), 270, 272 B cell selection, 270-271 cell types, other, 271 during development and maturation, 268-271 of mature cells, 271-272 negative selection, 270 T cell selection in thymus, 269270 introduction, 264 apoptosis, 264 summary, 276-277 target cell death, 272-274 bystander killing, 274 cell-mediated cytotoxicity, 273 Cell-mediated immunity, 155-157 Chain shuffling, 294 Charcot-Leyden crystal, 206 Chemoattractants, 195-227 (see also "Phagocytes....")

322

Chemokines, 155, 159-160 C-C branch, 159 C-X-C branch, 159 chemotaxis, 159 monokines TNF-a and IL-1, 160 stem cell growth inhibitors, 159 in vivo, 160 Chemokinesis, 209 Chemotaxis, 87, 159, 184, 199, 209 Chimerism, 75-76, 80 Chronic Granulomatous Disease (CGD), 221 Class switching, 54 Classical conditioning, 305-306 Clonal abortion, 66, 78 Clonal anergy, 67, 70-71, 74-75, 78, 80 Clonal deletion, 66, 70-71, 72-74, 241-242 intra-thymic, 68-69, 270 Clonal exhaustion, 76-77 Clonal ignorance, 66 Clonal selection, 49 Clonal Selection Theory, 100 Clusterin, 182 Collaborative Transplant Study, 129 Collagenase, 212-213 Colony-stimulating factors (CSFs), 150 (see also "Cytokines") Common variable immunodeficiency (CVID), 42 Complement system, activation and control of, 169-193 activation, 173-180 alternative pathway, 177-178 amplification on activator surfaces, 178 bystander lysis, 179 CI, 173-175 C2, 176-177 C3, 176, 177 C4, 175 C5, 177, 178-180

INDEX

C5b67, 179 C6, 179 C7, 179 C8, 179 C9, 179 clasical pathway, 173-177 factor B and factor D, 178 IgG, 174 IgM, 174 mannose binding protein (MBP), 175 MBP-associated serine protease (MASP), 175 membrane attack complexes (MACs), 179-180, 181, 185 membrane attack pathway, 179180 "tickover" phenomenon, 178 control, 180-183 in alternative pathway, 182 CI-inhibitor, 180 C4b-binding protein, 180-181 CD59, 183 in classical pathway, 180-182 clusterin, 182 complement receptor 1 (CRl), 181, 182 decay accelerating factor (DAF), 181 Factor 1, 180, 182 Factor H, 180-181,182 homologous restriction factor (HRF), 183 in membrane attack pathway, 182-183 membrane cofactor protein (MCP), 181, 182 properdin (P), 182 regulation of, 181 Regulators of Complement Activation (RCA), 182 S protein, 182

Index

serine protease inhibitor (Serpin) family, 180 vitronectin, 182 deficiencies, 185-189 (see also "....pathology") introduction and basic concepts, 170-173 innate immune defense, central role in, 173 pathways, three, 170, 173 purpose, 173 pathology, 185-189 in autoimmune diseases, 187 C3, 186 control of activation in vivo, 188-189 CRl, 189 CVF, 188 deficiencies, 185-187 Echovirus, 188 Epstein-Barr virus, 188 evasion of by microorganisms, 187-188 in hemodialysis, 188 hereditary angioedema (HAE), 186 HIV, 187 hyperacute rejection, 188 iatrogenic activation, 188 measles, 188 meningococcal meningitis, 186 Neisseria inkciions, 186 opsonization, 183-189 overactivation, 189 paroxysmal nocturnal hemoglobinuria (PNH), 1816-187 properdin deficiency, 186 systemic lupus erythematosus (SLE), 186 in vivo, control of activation in, 188-189 xenotransplantation, 188

323

physiological roles, 183-185 anaphylatoxin inactivator, 184 anaphylaxis, 184 bacterial killing, 183-184 C5a,184 cell activation, 184-185 chemotaxis, 184 coating with complement, 184 CR3, 183 erythrocytes, 184 immune complex solubilization. 184 and immune response, 185 Neisseria, exception of, 183 opsonization, 183-189 summary, 189 Complementarity determining regions (CDRs), 36, 84, 107108,117,281 Conditioned immune responses, 305306 Congenic partners, 65-66 Cortex of thymus, 2, 3 Crossmatch, 130 CSFs, 150 (see also "Cytokines") Csk PTK, 144 (see also "B cell signaling..") CVID (common variable immunodeficiency), 42 Cyclic neutropenia, 203 Cytokines, 15, 48-49, 149-167 autocrine, 48 in autoimmune thyroid disease (AITD), 247 clinical applications, 162-164 G-CSF, 163 IL-2, 162-163 families and their cellular sources, 150-160 chemokines, 159-160 (see also "Chemokines") colony-stimulating factors (CSFs), 150

324

interleukins, 150 lymphokines, 150, 152-157 (see also "Lymphokines") lymphotoxin, 150 monokines, 157-158 (see also "Monokines") transforming growth factors, 150 tumor necrosis factor a, 150 IL-lra, 48 IL-6, 48 in immune system, 49 interleukin-1, 48, 150, 152 (see also "Interleukins") introduction, 149-150 bursa of Fabricius, 150 lymphocytes, 149-150 T cells and B cells, 149-150 macrophages, 48 mRNA, 48 paracrine, 48 proinflammatory, 158 receptors and signaling pathways, 161-162 Duffy antigen, 162 IL-2, 161 tumor necrosis factor (TNF), 48, 150, 157, 158, 160 Cytotoxic cells, 56-58, 87 Darier's sign, 235 Dendritic cells, 3, 51, 68, 87 Depression, 308 Designer antibodies, 279-300 (see also "Antibodies....") Desmosomes, 3 Diabetes, type I, 242 Diapedesis, 199, 212-213 diGeorge syndrome, 17 (see also "Thymus...") Directed mutagenesis, 100-101 (see also "Diversity ") Disease, human, phagocytes and, 198

INDEX

Diversity in immune system, generation of, 83-104 clonal selection, 85-90 affinity maturation, 88-89 antibody molecules, 86 antigen-presenting cells, 87 bone marrow, 86 chemotaxis, 87 Clonal Selection Theory, 86 cytotoxic T cells, 87 follicular dendritic cell (FDC), 88 germinal centers, 88, 90, 101 helper T cells, 87, 90 human immunodeficiency viruses (HIV), 90 immunoglobulin (Ig), 86 lymphocytes, 85-86 medullary cords, 88 MHC antigens, 87 opsonization, 87 phagocytosis, 87 somatic hypermutation, 88 T cell receptor (TcR), 86 target cell lysis, 87 directed mutagenesis, 100-101 Clonal Selection Theory, 100 isotypes of immunoglobulins, functional and structural, 90-100 allelic exclusion, 94 diversity genes, 93-95 germline repertoire, 92-93 Human Genome Project (HUGO), 92-93 IgA, 91-92 IgD, 91-92 IgE, 91 IgG, 91-92 IgM, 91 isotype switch, 91-92 joining gene, 93-95 junctional diversity, 95-96

Index

Mannik Phenomenon, 95 N regions, 95-96 palindromes, 97 rearrangements, 91, 93-95 receptor editing, 97 secondary rearrangements, 97 somatic hypermutation of rearranged V(D)J genes, 98-100 somatic mutations, 97, 98-100 somatic repertoire, generation of, 93-100 systemic lupus erythematosus, rearrangement from, 95-96 terminal deoxynucleotidyl transferase, 95 V-DJ rearrangement, 96-97, 99 variable gene, 93-95 Wu-Kabat structures, 98-99 introduction, 84-85 antigen receptors, diagrams of, 84 antigens, 84 B cell and T cell receptors, 85 complementarity determining regions (CDR), 84 constant region, 84 epitopes, 85 framework regions (FR), 84 heavy (H) chains, 84 light (L) chains, 84 self antigens, 84 variable region, 84 summary, 101 Duffy antigen, 162, 212 (see also "Cytokines") E-selectin, 58, 213 Echovirus, 188 Ehrlich, Paul, 62-63, 212-213, 217, 238, 298, 299 Elastase, 212-213 Endocrine abnormality in AITD, 249-250

325

Endogenous peptides, 52 Endothelium and leukocytes, interaction between, 58-59 (see afao "Cell-to-cell....") Environmental factors in autoimmune thyroid disease (AITD), 250-251 Eosinophils, 204, 205-206, 218, 219 cationic protein (ECP), 219 -derived neurotoxin (EDN), 219 peroxidase (EPO), 219 Epidermal growth factor (EGF) receptor, 146-147 Epitopes, 50, 62 Epstein-Barr virus, 188, 250, 275 Erythrocytes, 184 Exercise-induced anaphylaxis, 233 Extracellular matrix, 46 Fenton reaction, 221 Follicles, 30 Follicular dendritic cell (FDC), 30, 50,88 Framework regions (FR), 84 Fv fragments, 109-110 single-chain, 289 Fyn family PTK, 141 GALT, 34 Gelsolin, 218 Genetic predisposition to AITD, 248-249 Germinal center, 22, 34, 35-37 centroblasts, 35-36 centrocytes, 35-36 functioning, model for, 35-36 complementarity determining regions (CDRs), 36 as site of cell death, 35 "Germlike genes," 10 Glucocorticoids, 304 Graft-versus-host disease (GVHD), 76, 129

326

Granule contents, 217-19 (see also "Phagocytes...") Graves' disease (GD), 243 Gut associated lymphoid tissues (GALT), 34 HAE, 186 HAMA response, 286 Haptens, 231 Haptotaxis, 209 Haplotype, 120 Hashimoto's thyroiditis (HT), 243,253 Hassall's corpuscles, 3 Heat shock protein 70, 123 Helminths, defense against, 206, 218 Helper T cells (Th), 87, 90 Hereditary angioedema (HAE), 186, 234-235 Hewson, William, 197 High endothelial venules (HEV), 58 Histiocytes, 207 HIV, 90, 275, 298 and complement, 187 HLA genes and AITD, 248 HLA-ABCgtms, 120-122 HLA system in transplantation, 128130 (see also "Major Histocompatibility ") Homologous restriction factor (HRF), 183 Hormones, 304-305 in regulation of autoimmune process, 250 "Horror autotoxicus," 62-63, 238 (see also "Immunological tolerance") HRF, 183 HTLV-1, 17 Human Genome Project (HUGO), 92-93 Human immunodeficiency viruses (HIV), 90, 275, 298 (see also "HIV")

INDEX

H-Y, 68-69 (see also "Immunological tolerance") Hybridoma technology, 286 Hyperacute rejection, 188 Hypermutation, 35 Hypothalamic-pituitary-adrenalcortical axis, 302, 306 ICAM family, 52, 214 Idiopathic anaphylaxis, 233 Idiotype antigen, 77 IgA deficiency, 41-42 IgE, mediated anaphylaxis and, 231 IL-1, 303 IL-2, 161-164 IL-6, 48, 303 Immune deviation, 77-78 (see also "Immunological tolerance") Immune system, cell-to-cell interactions in,45-60 (see also "Cell-to-cell ") Immune system, cell death and, 263278 (5^^flfao "Cell death...") Immune system-nervous system interactions, 302-305 (see also "Psychoneuroimmunology") Immunity: B cell in, 21-44 (see also "B cell....") definition, 199 phagocytes, 195-227 (see also "Phagocytes....") Immunoglobulin (Ig), 86 (see also "Diversity....") Immunoglobulin superfamily, 46-47 (see also "Cell adhesion superfamily") Immunological memory, 50 (see also "Cell-to-cell....") Immunological tolerance, 61-82 artificially induced, 75-79 antagonist peptides, 77 anti-idiotypic responses, generation of, 77

Index

chimerism, 75-76 clonal abortion, 78 clonal exhaustion, 76-77 graft-versus-host disease, 76 idiotype, 77 immune deviation, 77-78 oral tolerance, 76 persistence of antigen essential, 78 pre-B cells, 78 soluble antigens, 76 T cell accessory molecules, antibodies to, 76 T cell independent antigens, 77 targeting antigen to naive B cells, 76 thymectomy, 78 in tissue culture, 78-79 veto and suppression, 77 in vivo, 75-78 autoimmunity and breakdown of immunological self tolerance, 79-80 immunoregulatory T cells, 79-80 molecular mimicry, 79 Thl and Th2 cells, 80 historical background, 62-66 allografts, 63 antigen-presenting cells (APC), 65,67 clonal selection theory, 64 co-stimulator signal, 65 congenic partners, 65-66 "horror autotoxicus," 62 immune repertoire, 64 LCMV, 63 T and B cells, 65 thymus, role of, 65 transgenic technology, introduction of, 65-66 introduction, 62 co-stimulatory molecules, 62, 67 epitopes, 62

327

"self," 62-66 self-reactivity, 62 self-reacting lymphocytes, possible fate of, 66-67 affinity, 67 B7 molecule, 67 CD28, 67 clonal abortion, 66 clonal anergy, 67, 70-71 clonal deletion, 66, 70-71 clonal ignorance, 66 lymphokines, 67 suppression, 67, 71 self-tolerance in B cells, 72-75 absence of T cell help, 72 allotype, 75 anti-idiotypic B cells, 73 clonal anergy, 74-75 clonal deletion, 72-74 hypermutation, 72-73 IgC class as T-cell dependent, 72 IgM antibodies, 72 interleukins, 72 self-tolerance in T cells, 68-71 binding, 68 CD4^ and CD^^ cells, death of, 68 at cortico-meduUary junction, 68 cytotoxic, 68 dendritic cells, 68 "forbidden clones" of Burnet, 69 H-Y, 68-69 helper, 68 intra-thymic clonal deletion, 6869 macrophages, 68, 71 major histocompatibility complex (MHC), 68 positive selection, role of, 68 post-thymic tolerance, 70-71 programmed cell death, 68

INDEX

328

TCR gene rearrangement, 68 TCR genes, 68 TGF-/?, 71 Thl and Th2 cells, 71,80 "veto" cells, 68 summary, 82 therapeutic applications, potential, 80 non-depleting CD4 antibodies, 80 Immunoregulation, 223 Immunoregulatory T cells, 79-80 Immunosuppression, 305-306 Immunotoxins, 295-296 Infection: apoptosis and, 275 phagocytes and, 197-199 Infectious agents, role of in autoimmune disease, 250 Inflammation, 196, 275 definition, 264 {see also "Cell death....") phagocytes and, 200-201 Influenza viruses. 111, 113 Insulin, receptor for, 142 Integrins, 47, 214 {see also "Cell adhesion superfamily") Intercellular adhesion molecules, 214 Interdigitating dendritic cells (IDCs), 30,33,51 Interferons, 154-155 a, 158 )8 1, 158 -7(IFN-7), 11,54-55 receptors, 146-147 Interleukins: definition, 152 -1 (IL-1), 48, 72, 160 -la, 157-158, 160 -1)8, 157-158, 160 -2, 11, 15,72,303 -3, 156 -4, 154

-5, 156 -6,157,158 -12, 156-157 receptors, 146-147 Iodine and autoimmune thyroid disease, 250-251 Jerne, Niels, 86 Koch's Postulates, 196 Kostmann's syndrome, 205 Kupffer cells, 207 L-selectin, 58, 213 Lactoferrin, 219 LAD-1 and LAD-2, 214-215 Langerhans cells, 33, 51, 207 LATS, 243 LBP, 215 Lck family PTKs, 141 Leishmania infections, 56, 250 Leukocyte adhesion deficiency 1 and 2 (LAD-1 and LAD-2), 214215 Leukocyte Function Antigen-1 (LFA-1), 47, 52 Leukocyte integrins, 214 Leukocytes, 197 and endothelium, interaction between, 58-59 Leukotriene B4, 223 LFA-1, 47, 52,214 Lipopolysaccharide-binding protein (LBP), 215 Lithium therapy and autoimmune thyroid disease, 251 Liver transplantation, 129, 130 Ly-IB cell, 39 Lymph nodes, 51 Lymphoblastic leukemia, 17 Lymphocytes, 2, 61-82, 149-150 {see also "Cytokines" and "Immunological tolerance")

Index

Lymphokines, 150, 152-157 (see also "Cytokines") in B cell responses, 153-155 CD4 and CD8 T cells, 152-153 CD40, 156 in cell-mediated immunity, 155157 GM-CSF production, 155-156 IL-2, 152 IL-3, 156 IL-4, 154 IL-5, 156 IL-12, 156-157 interferon-7, 154, 155 interleukins, 152 interleukin-3, 156 in T cell responses, 152-153 source, 152 Lymphoma, 17 Lymphotoxin (TNF ^8), 150 MACS, 179-180, 181, 185 Mac-1, 214 Macrophages, 3, 68, 71, 87, 207-209 and T cells, interaction between, 56 Major basic protein (MBP), 219 Major Histocompatibility Complex (MHC), 119-134 disease associations, HLA and, 130-131 antigen presentation, restriction of, 130-131 of autoimmune nature, 130 class III association, 131 molecular mimicry, 130 Systemic Lupus Erythematosus (SLE), 131 function of Class I and Class II molecules, 128 endoplasmic reticulum (ER), synthesized in, 128 LMP2andLMP7, 128 Tap 1 and Tap 2, 128

329 genetic structure of, 120-123 C2 and C4, 123 class I region, 120-122 class II region, 122-123 class III region, 123 DO/DN sub-region, 123 DP sub-region, 122-123 DQ sub-region, 122 DR sub-region, 122 haplotype, 120 heat shock protein 70, 123 HLA-ABC gQuts, 120-122 microlymphocytotoxicity assay, 122, 127 Nomenclature Committee reports, 123 one dimensional isoelectric focusing (lEF), 122 primed lymphocyte test (PLT), 123 properdin factor (Bf), 123 tumor necrosis factor A and B, 123 21-OHA and 21-OHB, 123 HLA and disease associations, 130-131 {see also "...disease ") HLA system in transplantation, \2^-U0 (see also ".. ..transplanation...") introduction, 120 alloresponsiveness, 120 polymorphism, 120 molecules, 50-53 nomenclature and polymorphism, 126-127 allele sequences, 126-127 class I, 126-127 class II, 127 protein structure of molecules, 124-126 "Bjorkman's groove," 125, 126 class I molecules, 124-125

330

class II molecules, 125-126 peptide binding groove, 125 summary, 131 techniques for detecting polymorphism, 127-128 molecular techniques, 127-128 Polymerase Chain Reaction— Sequence Specific Oligonucleotide technique (PCRSSO), 128, 129 serology, 127 transplantation, HLA system in, 128-130 bone marrow, 129 cardiac, 129 Collaborative Transplant Study, 129 crossmatch, 130 graft versus host disease (GVHD), 129 influences, two, 128 liver, 129, 130 matching, 129 renal transplants, 129 sensitization, 129-130 solid organs, 129 Mannik Phenomenon, 95 (see also "Diversity....") Mannose binding protein (MBP), 175,215 Mast cell degranulation, 230 MBP, 219 MBP-associated serine protease (MASP), 175 MCP, 181 Measles, 188 Medulla of thymus, 2, 3 Membrane attack complexes (MACs), 179-180, 181, 185 Membrane cofactor protein (MCP), 181 Memory B cell, 22, 36-37 (see also "Bcell...")

INDEX

Meningitis, bacterial, 42 Metachromasia, 206 Metchnikoff, Elie, 197 MHC molecules, 50-53 Microlymphocytotoxicity assay, 122, 127 Mitogen activated protein (MAP) kinases, 146 "Molecular mimicry," 79, 87, 130, 212, 242 Monocytes, 207 Monokines, 157-158 interferons a and )8-l, 158 interleukins-la and -l/J, 157-158 interleukin-6, 157, 158 proinflammatory cytokines, 158 TNF-a, 157, 158 Multiple sclerosis, 242 Mycobacteria, 56 Mycoplasma, 250 Myelomas, 280 Myeloperoxidase, 218, 221 Natural killer (NK) cells, 57, 303 Necrosis, 263-278 (see also "Cell death....") Necrotaxis, 207 Nervous system-immune system interactions, 302-305 (see also "Psychoneuroimmunology") Neuropeptides, 304-305 Neurotransmitters, 304-305 Neutropenia, 205 Neutrophil-Specific Granule Deficiency (SGD), 219 Neutrophils, 204-205, 217 Nomenclature Committee reports, 123 Nonsteroidal antiinflammatory drugs (NSAID), anaphylaxis and, 232-233 Norepinephrine, 303

Index

NSAID, 232-233 "Nurse cells," 2 One dimensional isoelectric focusing (lEF), 122 Opelz, Professor G., 129 Opsonization, 87, 155, 183-189, 199 phagocytosis, 215-217 P-selectin, 213 p 2 r ^ 146 p53, 266 p 7 y ' , 146 FAF, 223 PALS, 30 Paroxysmal nocturnal hemoglobinuria (PNH), 186-187 Pasteur, Louis, 196 Penicillins, 231 Peptide binding groove, 125, 126 Periarteriolar lymphocytic sheath (PALS), 30 Phagocytes in immunity and inflammation, 195-227 development, 201-209 basophils, 204, 206-209 bone marrow, differentiation from stem cells in, 201-203 Charcot-Leyden crystal, 206 colony forming unit, 203 colony-stimulating factors (CSF), 203 cyclic neutropenia, 203 eosinophils, 204, 205-206 glucocorticoids, 205 GMCSF and GCSF, 203 helminths, defense against, 206 hematopoietic inductive microenvironment, 203 histamine, 206 histiocytes, 207 IL-5, 205-206 Kostmann's syndrome, 205

331

Kupffer cells, 207 Langerhans cells, 207 lipopolysaccharide, 205 macrophages, 207-209 marginated pool, 205 mature, 203-209 metachromasia, 206 microglial cells, 207 monocytes, 207 multinucleated giant cells, 207 necrotaxis, 207 neutropenia, 205 neutrophils, 204-205 progenitor cell compartment, 201 promonocyte, 207 pulmonary alveolar macrophages, 207 stem cell compartment, 201-203 functions of, specialized, 209-223 antimicrobial oxidants, production of, 219-222 arachidonic acid, 222-223 azurophil granules, 218 C3b and C3bi, 215 cell migration, 209-215 chemoattractants, 210-212 chemokinesis, 209 chemotaxis, 209 Chronic Granulomatous Disease (CGD), 221 coUagenase, 212-213 complement receptors, 215, 216 CR3, 216 DAG, 212 degranulation, 217-219 Duffy red cell antigen, 212 elastase, 212-213 Fenton reaction, 221 flavocytochrome, 221-222 G proteins, 211-212 gelatinase, 218 gelsolin, 218

332

INDEX

glutathione, 222 haptotaxis, 209 hexose monophosphate shunt, 221 IL-1, 222 IL-6, 222 IL-8,211 immunoregulation, 223 integrins, 214 intercellular adhesion molecules (ICAM-1 and ICAM-2), 214 lactoferrin, 219, 221 lamellipodium, 212 leukocyte adhesion deficiency 1 and 2 (LAD-1 and LAD-2), 214-215 leukotriene B4, 223 lipopolysaccharide-binding protein (LBP), 215 major basic protein (MBP), 219 mannose binding protein (MBP), 215 molecular mimicry, 212 myeloperoxidase, 218, 221 NADPH oxidase, 220-222 Neutrophil-Specific Granule Deficiency (SGD), 219 nitric oxide synthase, 222 opsonins, 215 oxidative killing mechanisms of, 220 phagocytosis / opsonization, 215217 platelet-activating factor (PAF), 223 pro-inflammatory mediators, production of, 222-223 prostaglandins, 222 protein kinase C, 212 pseudopodium, 212 respiratory burst, 219-220 selectins, 213

sialyl Lewis "^ blood group antigen, 213 specific granules, 218-219 superoxide dismutase, 221 synexins, 218 tertiary granule, 218 thromboxanes, 222 tumor necrosis factor, 213 uropod, 212 introduction, 196-201 anaphylatoxins, 200-201 antigen presentation, 199-200 Arthus reaction, 201 chemoattractants, 199, 210-212 chemotaxis, 199 complement system, 200 delayed type hypersensitivity reactions, 201 diapedesis, 199, 212-213 germ theory of disease, 196 granuloma, 201 historical background, 196-197 homeostasis, general role in, 196-201 and human disease, 198 immediate type hypersensitivity reactions, 201 and immunity, 199-200 and infection, 197-199 and inflammation, 200-201 leukocytes, 197 NADPH oxidase, 199 opsonization, 199 phagosome, 199 post-streptococcal glomerulonephritis, 200 smallpox, 196-197 vaccination, 196-197 white corpuscles, 197 summary, 223-224 Phagocytosis, 56, 87 opsonization, 215-217 Phagosome, 199

333

Index

Phosphatidylinositol 3-kinase (PI3K), 143 Phospholipase C (PLC), 138-139 Phosphotyrosine phosphatases (PTPases), 140 Plasma cell, 22 Platelet-activating factor (PAF), 223 Pneumococcus, polysaccharide, 38 PNH, 186-187 Polymerase Chain Reaction (PCR), 294 —Sequence Specific Oligonucleotide technique (PCR-SS), 128, 129 Post-streptococcal glomerulonephritis, 200 Primed lymphocyte test (PLT), 123 Programmed cell death, 98, 240, 263-278 (see also "Cell death....") Prolactin, 250, 304 Promonocyte, 207 Properdin, 182-186 deficiency, 186 factor (Bf), 123 Protein kinase C, 138, 212 Protein tyrosine kinases (PTKs), 139-140 Psychoneuroimmunology, 301-311 conclusion, 309-311 conditioned immune responses, 305-306 classical conditioning, 305-306 immunosuppression, 305 immune system-nervous system interactions, evidence for, 302-305 cytokines, 303 glucocorticoids, 304 hormones, 304-305 interleukin-2, 303 lymphocytes, 303

natural killer (NK) cell activity, 303 neuropeptides, 304-305 neurotransmitters, 304-305 norepinephrine, 303 during sleep, 304 vasoactive intestinal peptide (VIP), 303 introduction, 301-302 biopsychosocial model of illness, 302 hypothalamic-pituitary-adrenal cortical axis, 302, 306 psychosomatic disorders, 302 stress and immune function, 306309 bereavement, effect of, 307-308 definition, 306 depression, 308 humans, effects of on, 307-309 infection, increased susceptibility to, 307 sleep and sleep deprivation, 309 studies' results mixed, 307 Receptor editing, 97 Renal transplants, 129 S protein, 182 Salt wasting, 123 SCID mice,using, 295 Scid mutations, 40 Selectins, 47, 213 (see also "Cell adhesion superfamily") Self-antigens, 84 Self-tolerance, 238 Serine protease inhibitor (Serpin) family, 180 Serology, 127 SGD, 219 Sialyl Lewis "" blood group antigen, 213 SLE, 131

334

Sleep and sleep deprivation, 304,309 Smallpox, 196-197 Soluble antigens, 76 Somatic hypermutation, 88, 284 of rearranged V(D)J genes, 98-100 Somatic mutation, 22, 35, 37, 97, 98100 Southern blotting, 66 {see also "Cellto-cell...") Spleen, 13-14 periarteriolar lymphocyte sheath (PALS), 13-14 red and white pulps, 13 Stem cells, 24, 201-203 Stress and autoimmune thyroid disease, 251 Stress and immune function, 306-309 {see also "Psychoneuroimmunology") Stromal cells, 2 Suppression, 67, 71 Suppressor T cells, 77 defect in as basis for AITD, 247248 Syk family PTKs, 142 Synexins, 218 Systemic Lupus Erythematosus (SLE), 131, 186,276 Systemic mastocytosis, 234-235 T cell independent antigens, 77 T cell receptor (TcR), 86 and autoimmunity, 239 T cell signaling at molecular level, 135-147 (^eea&o"B cell signaling....") T cells, 6-7, 65 antigen presentation to, 50-53 and B cells, interaction between, 53-56 receptor complex, 52 Talmage, David, 86 Target cell lysis, 87

INDEX

TCR, 9-10 Tetanus toxoid, 54 Thymectomy, 4-5, 78 {see also "Thymus....") neo-natal, 242 Thymus, discovery of role of, 65 intra-thymic clonal deletion, 68-69 Thymus in immunity, 1-20 antigen recognition and major histocompatibility complex (MHC), 7-10 antigen-presenting cell (APC), 8 CD3 complex, 9-10 endocytosis, 8 endogenous pathway, 8 endoplasmic reticulum, 8 exogenous pathway, 8 "germlike genes," 10 MNC restriction, 8 restriction elements, 8 superantigens, 7 TCR, 9-10, 12, 18 in disease states, 17-18 Acquired Immunodeficiency Syndrome (AIDS), 17-18 adenosine deaminase (ADA), 17 autoimmune diseases, 18 diGeorge syndrome, 17 HTLV-1, 17 immune deficiency states, 17 lymphoblastic leukemia, 17 lymphoma, 17 thymomas, 17 historical background, 4-7 B cells, 5-6 in birds, 5-6 bursa, 4 cell-mediated immune responses, 5 humoral immune responses, 5 immunoglobulin, 6 obscure prior to 1960, 4 T cells, 6-7

335

Index

intrathymic events, 15-17 cytokines, 15 double-negative (DN) cells, 15 double-positive cells, 15 MHC polymorphism, 15 negative selection, 16-17 positive selection, 16-17 programmed cell death, 16 introduction, 2-4 cortex, 2,3 dendritic cells, 3, 51 desmosomes, 3 as epitheUal organ, 3-4 HassalFs corpuscles, 3 layers, three, 2 location, 2 lymphocytes, 2 macrophages, 3 medulla, 2,3 "nurse cells," 2 as primary or central lymphoid organ, 4 self-reactive lymphocytes, 3 stem cells, 4 stromal cells, 2 structure, 3 peripheral T cell subsets, 10-12 CD4+ and CD8+ cells, 10-12, 18 interferon-7, 11 interleukin-2, 11, 15 MEL 14, 11 "naive" or "virgin" cells, 11 summary, 18 T cell migration, 12-15 B cell dependent area, 13 high endothelial venules (HEV), 13 integrin, 14 L-selectin, 13 memory-type T cells, 14-15 recirculation of naive T cells, 13-14

spleen, 13-14 {see also "Spleen") T cell dependent area, 13 tissue-selective homing of activated and memory T cells, 14-15 Thyroglobulin (Tg), 244 Thyroid disease, autoimmune, 243252 (see also "Autoimmune diseases....") Thyroid peroxidase (TPO), 244 Thyroid-stimulating hormone receptor (TSHR), 244 Thyroiditis, 242 Thyroxine, thyroglobulin as prohormone for, 252 Tingible bodies, 34-35 Tolerance, 61-82 (see also "Immunological tolerance") Toxins and autoimmune thyroid disease, 251-252 TPO, 244 Transfectoma, 288 Transforming growth factor (TGF), 150 -)8 (TGF-jS), 54-55 Transgenic mice, using, 295 Transgenic technology, introduction of, 65-66 Transplant rejection, cytotoxic cells and,57 Tumor cells, lysis of, cytotoxic cells and,57 Tumor necrosis factor (TNF), 48, 125, 213, 272, 274, 303 A and B, 123, 150, 157, 158, 160 Tyrosine kinase, 239-240 Tyrosine phosphorylation, 139-141 Urticaria, 233 Vaccination, 196-197, 199 Vasoactive intestinal peptide (VIP), 303

INDEX

336

"Veto" cells, 68-69 Viral infection, apoptosis and, 275 Virus infection of thyroid cells, AITD and, 250 Vitronectin, 182 Wheal, 233 White corpuscles, 197 Witebsky"s postulates, 238 Wu-Kabat structures, 98-99 X-LA (X-linked agammaglobulinemia), 39-41

X-LA with hyper-IgM, 41-42 common variable immunodeficiency (CVID), 42 meningitis, bacterial, 42 gp39, 41 IgA deficiency, 41-42 IgM and IgC deficiencies, 42 immunoglobulin isotype deficiencies, 41-42 symptoms, 41 Xenotransplantation, 188 Zap, 142