《免疫学》（英文版） Chapter 13 The Complement System.pdf_大学文库

chapter 13 The Complement System HE COMPLEMENT SYSTEM IS THE MAJOR EFFECTOR of the humoral branch of the immune system Research on complement began in the 1890s, when Jules bordet at the institut pasteur in paris showed that sheep antiserum to the bacterium vibrio cholerae caused lysis of the bacteria and that heating the antiserum destroyed its bacteriolytic activity. Surprisingly, the ability to lyse the bacteria was restored to the heated serum by adding fresh serum that contained no antibodies directed a The Functions of Complement against the bacterium and was unable to kill the bacterium by itself. Bordet correctly reasoned that bacteriolytic activ- a The Complement Components y requires two different substances: first, the specific an- n Complement Activation tibacterial antibodies, which survive the heating process, and a second, heat-sensitive component responsible for the a Regulation of the Complement System lytic activity. Bordet devised a simple test for the lytic ac- Biological Consequences of Complement tivity, the easily detected lysis of antibody-coated red blood Activation lls, called hemolysis. Paul Ehrlich in Berlin indepen- dently carried out similar experiments and coined the term a Complement Deficiencies complement, defining it as"the activity of blood serum that completes the action of antibody. In ensuing years, re- searchers discovered that the action of complement wa the result of interactions of a large and complex group of 3 This chapter describes the complement components and receptors with complement proteins controls B-cell activi- heir activation pathways, the regulation of the complement ties gives this system a role in the highly developed acquired system, the effector functions of various complement com- immune system. Thus we have a system that straddles in- ponents, and the consequences of deficiencies in them. a nate and acquired immunity, contributing to each in a vari Clinical Focus section describes consequences of a defect in ety of ways proteins that regulate complement activity. After initial activation, the various complement compo- nents interact, in a highly regulated cascade, to carry out a number of basic functions(Figure 13-1)including a Lysis of cells, bacteria, and viruses The Functions of Complement Opsonization, which promotes phagocytosis of Research on complement now includes more than 30 solu particulate antigens ble and cell-bound proteins. The biological activities of this system affect both innate and acquired immunity a Binding to specific complement receptors on cells of reach far beyond the original observations of antibody he immune system, triggering specific cell functions, mediated lysis of bacteria and red blood cells. Structural nflammation. and secretion of comparisons of the proteins involved in complement path- rays place the origin of this system in primitive organisms Immune clearance, which immune complexes from the circulation and deposits them in the spleen and

receptors with complement proteins controls B-cell activities gives this system a role in the highly developed acquired immune system. Thus we have a system that straddles innate and acquired immunity, contributing to each in a variety of ways. After initial activation, the various complement components interact, in a highly regulated cascade, to carry out a number of basic functions (Figure 13-1) including: ■ Lysis of cells, bacteria, and viruses ■ Opsonization, which promotes phagocytosis of particulate antigens ■ Binding to specific complement receptors on cells of the immune system, triggering specific cell functions, inflammation, and secretion of immunoregulatory molecules ■ Immune clearance, which removes immune complexes from the circulation and deposits them in the spleen and liver chapter 13 ■ The Functions of Complement ■ The Complement Components ■ Complement Activation ■ Regulation of the Complement System ■ Biological Consequences of Complement Activation ■ Complement Deficiencies The Complement System T       of the humoral branch of the immune system. Research on complement began in the 1890s, when Jules Bordet at the Institut Pasteur in Paris showed that sheep antiserum to the bacterium Vibrio cholerae caused lysis of the bacteria and that heating the antiserum destroyed its bacteriolytic activity. Surprisingly, the ability to lyse the bacteria was restored to the heated serum by adding fresh serum that contained no antibodies directed against the bacterium and was unable to kill the bacterium by itself. Bordet correctly reasoned that bacteriolytic activity requires two different substances: first, the specific antibacterial antibodies, which survive the heating process, and a second, heat-sensitive component responsible for the lytic activity. Bordet devised a simple test for the lytic activity, the easily detected lysis of antibody-coated red blood cells, called hemolysis. Paul Ehrlich in Berlin independently carried out similar experiments and coined the term complement, defining it as “the activity of blood serum that completes the action of antibody.” In ensuing years, researchers discovered that the action of complement was the result of interactions of a large and complex group of proteins. This chapter describes the complement components and their activation pathways, the regulation of the complement system, the effector functions of various complement components, and the consequences of deficiencies in them. A Clinical Focus section describes consequences of a defect in proteins that regulate complement activity. The Functions of Complement Research on complement now includes more than 30 soluble and cell-bound proteins. The biological activities of this system affect both innate and acquired immunity and reach far beyond the original observations of antibodymediated lysis of bacteria and red blood cells. Structural comparisons of the proteins involved in complement pathways place the origin of this system in primitive organisms possessing the most rudimentary innate immune systems. By contrast, the realization that interaction of cellular Poly-C9 Complex ART TO COME

300 paRt I Immune Effector Mechanisms LYSIS OPSONIZATION ACTIVATION OF INFLAMMATORY LEARANCE OF RESPONSE IMMUNE COMPLEXES Bacteria Complement complex Extravasation Blood Target cell P FIGURE 13-1 The multiple activities of the complement system. phagocytes: activation of inflammatory responses; and clearance of Serum complement proteins and membrane-bound complement circulating immune complexes by cells in the liver and spleen receptors partake in a number of immune activities: lysis of foreign Soluble complement proteins are schematically indicated by a trian- cells by antibody-dependent or antibody-independent pathways: gle and receptors by a semi-circle; no attempt is made to differenti opsonization or uptake of particulate antigens, including bacteria, by ate among individual components of the complement system here C5b, can occur by the classical pathway, the alternative The Complement Components pathway, or the lectin pathway. The final steps that lead to a membrane attack are the same in all pathways The proteins and glycoproteins that compose the complement system are synthesized mainly by liver hepatocytes, although signilicant amounts are also produced by blood monocytes, tis. The Classical Pathway Begins with sue macrophages, and epithelial clls of the gastrointestinal and Antigen-Antibody Binding weight) of the serum globulin fraction. Most circulate in the Complement activation by the classical pathway commonly serum in functionally inactive forms as proenzymes, or zymo- begins with the formation of soluble antigen-antibody com gens, which are inactive until proteolytic cleavage, which re- plexes(immune complexes)or with the binding of antibody moves an inhibitory fragment and exposes the active site. The to antigen on a suitable target, such as a bacterial cell IgM and complement-reaction sequence starts with an enzyme cascade. certain subclasses of IgG(human IgG1, IgG2, and igG3)can Complement components are designated by numerals activate the classical complement pathway. The initial stage of (C1-C9), by letter symbols (e.g, factor D), or by trivial activation involves C1, C2, C3, and CA, which are present in names(e.g, homologous restriction factor). Peptide frag- plasma in functionally inactive forms. Because the compo- ments formed by activation of a component are denoted by nents were named in order of their discovery and before their small letters. In most cases, the smaller fragment resulting functional roles had been determined, the numbers in their from cleavage of a component is designated"a"and the larger names do not always reflect the order in which they react. fragment designated"b"(e. g, C3a, C3b: note that C2 is an The formation of an antigen-antibody complex induces exception: C2a is the larger cleavage fragment). The larger conformational changes in the Fc portion of the igM mole fragments bind to the target near the site of activation, and cule that expose a binding site for the cl component of the the smaller fragments diffuse from the site and can initiate complement system. Cl in serum is a macromolecular com localized inflammatory responses by binding to specific re- plex consisting of Clq and two molecules each of Cir and ceptors. The complement fragments interact with one an- Cls, held together in a complex(C1qr2S2)stabilized by Ca other to form functional complexes. Those complexes that ions. The Clq molecule is composed of 18 polypeptide nated by a bar over the num- chains that associate to form six collagen-like triple helical ber or symbol (e.g, C4b2a, arms, the tips of which bind to exposed Clq- binding sites in the Ch2 domain of the antibody molecule(Figure 13-3, page 302). Each Clr and Cls monomer contains a catalytic Complement Activation domain and an interaction domain the latter facilitates in- teraction with Clq or with each other. Figure 13-2 on page 301 outlines the pathways of com Each C1 molecule must bind by its C1q globular heads to ment activation. The early steps, culminating in formation at least two Fc sites for a stable Cl-antibody interaction to

The Complement Components The proteins and glycoproteins that compose the complement system are synthesized mainly by liver hepatocytes, although significant amounts are also produced by blood monocytes, tissue macrophages, and epithelial cells of the gastrointestinal and genitourinary tracts. These components constitute 5% (by weight) of the serum globulin fraction. Most circulate in the serum in functionally inactive forms as proenzymes, or zymogens, which are inactive until proteolytic cleavage, which removes an inhibitory fragment and exposes the active site. The complement-reaction sequence starts with an enzyme cascade. Complement components are designated by numerals (C1–C9), by letter symbols (e.g., factor D), or by trivial names (e.g., homologous restriction factor). Peptide fragments formed by activation of a component are denoted by small letters. In most cases, the smaller fragment resulting from cleavage of a component is designated “a” and the larger fragment designated “b” (e.g., C3a, C3b; note that C2 is an exception: C2a is the larger cleavage fragment). The larger fragments bind to the target near the site of activation, and the smaller fragments diffuse from the site and can initiate localized inflammatory responses by binding to specific receptors. The complement fragments interact with one another to form functional complexes. Those complexes that have enzymatic activity are designated by a bar over the number or symbol (e.g., C4b2a, C3bBb). Complement Activation Figure 13-2 on page 301 outlines the pathways of complement activation. The early steps, culminating in formation of 300 PART III Immune Effector Mechanisms C5b, can occur by the classical pathway, the alternative pathway, or the lectin pathway. The final steps that lead to a membrane attack are the same in all pathways. The Classical Pathway Begins with Antigen-Antibody Binding Complement activation by the classical pathway commonly begins with the formation of soluble antigen-antibody complexes (immune complexes) or with the binding of antibody to antigen on a suitable target, such as a bacterial cell. IgM and certain subclasses of IgG (human IgG1, IgG2, and IgG3) can activate the classical complement pathway. The initial stage of activation involves C1, C2, C3, and C4, which are present in plasma in functionally inactive forms. Because the components were named in order of their discovery and before their functional roles had been determined, the numbers in their names do not always reflect the order in which they react. The formation of an antigen-antibody complex induces conformational changes in the Fc portion of the IgM molecule that expose a binding site for the C1 component of the complement system. C1 in serum is a macromolecular complex consisting of C1q and two molecules each of C1r and C1s, held together in a complex (C1qr2s2) stabilized by Ca2 ions. The C1q molecule is composed of 18 polypeptide chains that associate to form six collagen-like triple helical arms, the tips of which bind to exposed C1q-binding sites in the CH2 domain of the antibody molecule (Figure 13-3, on page 302). Each C1r and C1s monomer contains a catalytic domain and an interaction domain; the latter facilitates interaction with C1q or with each other. Each C1 molecule must bind by its C1q globular heads to at least two Fc sites for a stable C1-antibody interaction to FIGURE 13-1 The multiple activities of the complement system. Serum complement proteins and membrane-bound complement receptors partake in a number of immune activities: lysis of foreign cells by antibody-dependent or antibody-independent pathways; opsonization or uptake of particulate antigens, including bacteria, by phagocytes; activation of inflammatory responses; and clearance of circulating immune complexes by cells in the liver and spleen. Soluble complement proteins are schematically indicated by a triangle and receptors by a semi-circle; no attempt is made to differentiate among individual components of the complement system here. Complement receptor Blood Tissue Phagocyte Phagocyte Degranulation Target cell Ag-Ab complex Complement Extravasation Bacteria LYSIS OPSONIZATION ACTIVATION OF INFLAMMATORY RESPONSE CLEARANCE OF IMMUNE COMPLEXES

The Complement System CHAPTER 13 30 Mannose-binding Classical MBL-associated proteases (MASPl+2) bind MBL, Cl-like complex Cl binds gen-antibo Activa OD C3 convertase ①c2a5 C5b step C3 convertase( cabBy c3bBb3b 5 convertase Factor D Alternative (cabl FIGURE 13-2 Overview of the complement activation pathways. brane-attack complex(MAC)by a common sequence of terminal The classical pathway is initiated when C1 binds to antigen-antibody reactions. Hydrolysis of C3 is the major amplification step in all path- complexes. The alternative pathway is initiated by binding of spon- ways, generating large amounts of C3b, which forms part of C5 con- taneously generated C3b to activating surfaces such as microbial cell vertase C3b also can diffuse away from the activating surface and walls. The lectin pathway is initiated by binding of the serum protein bind to immune complexes or foreign cell surfaces, where it func- MBL to the surface of a pathogen. All three pathways generate C3 ons as an opsonin and c5 convertases and bound c5b, which is converted into a mem- occur. When pentameric IgM is bound to antigen on a target providing two attachment sites for Clg. This difference ac- surface it assumes the so-called"staple"configuration, in counts for the observation that a single molecule of igM which at least three binding sites for Clq are exposed. Circu- bound to ared blood cell can activate the classical complement lating IgM, however, exists as a planar configuration in which pathway and lyse the red blood cell while some 1000 mole the Clq-binding sites are not exposed(Figure 13-4, on page cules of igg are required to assure that two lgg molecules are 302)and therefore cannot activate the complement cascade. close enough to each other on the cell surface to initiate Clq An IgG molecule, on the other hand, contains only a single binding Clg-binding site in the Ch2 domain of the Fc, so that firm Clq The intermediates in the classical activation pathway binding is achieved only when two IgG molecules are within depicted schematically in Figure 13-5(page 303). Binding of 30-40 nm of each other on a target surface or in a complex, Clq to Fc binding sites induces a conformational change in (text continues on page 304)

occur. When pentameric IgM is bound to antigen on a target surface it assumes the so-called “staple” configuration, in which at least three binding sites for C1q are exposed. Circulating IgM, however, exists as a planar configuration in which the C1q-binding sites are not exposed (Figure 13-4, on page 302) and therefore cannot activate the complement cascade. An IgG molecule, on the other hand, contains only a single C1q-binding site in the CH2 domain of the Fc, so that firm C1q binding is achieved only when two IgG molecules are within 30–40 nm of each other on a target surface or in a complex, The Complement System CHAPTER 13 301 providing two attachment sites for C1q. This difference accounts for the observation that a single molecule of IgM bound to a red blood cell can activate the classical complement pathway and lyse the red blood cell while some 1000 molecules of IgG are required to assure that two IgG molecules are close enough to each other on the cell surface to initiate C1q binding. The intermediates in the classical activation pathway are depicted schematically in Figure 13-5 (page 303). Binding of C1q to Fc binding sites induces a conformational change in FIGURE 13-2 Overview of the complement activation pathways. The classical pathway is initiated when C1 binds to antigen-antibody complexes. The alternative pathway is initiated by binding of spontaneously generated C3b to activating surfaces such as microbial cell walls. The lectin pathway is initiated by binding of the serum protein MBL to the surface of a pathogen. All three pathways generate C3 and C5 convertases and bound C5b, which is converted into a membrane-attack complex (MAC) by a common sequence of terminal reactions. Hydrolysis of C3 is the major amplification step in all pathways, generating large amounts of C3b, which forms part of C5 convertase. C3b also can diffuse away from the activating surface and bind to immune complexes or foreign cell surfaces, where it functions as an opsonin. + C4 C2 C3b C3 C5 C5b C9 C8 C7 C6 C3 C3b C3bB Factor B Factor D C4b2a C3bBb C3bBb3b C4b2a3b C5 convertase C5 convertase C3 convertase C3 convertase Activated C1 MBL-associated proteases (MASP1 + 2) bind MBL, generate activated C1-like complex Mannose-binding lectin (MBL) binds foreign surface C1 binds antigen-antibody complex Classical pathway Alternative pathway Lectin pathway Major amplification step Membrane attack complex Spontaneous, slow, small amounts (text continues on page 304)

ART III Immune Effector mechanisms Clr that converts Clr to an active serine protease enzyme, The Alternative Pathway ls crr, which then cleaves Cls to a similar active enzyme, C Cis has two substrates, CA and C2. The C4 component is a Antibody-Independent glycoprotein containing three polypeptide chains a, B, and y. The alternative pathway generates bound C5b, the same C4 is activated when Cis hydrolyzes a small fragment( C4a) product that the classical pathway generates, but it does so from the amino terminus of the a chain, exposing a binding without the need for antigen-antibody complexes for initia site on the larger fragment(C4b). The CAb fragment attaches tion. Because no antibody is required, the alternative path to the target surface in the vicinity of Cl, and the C2 proen- way is a component of the innate immune system. This yme then attaches to the exposed binding site on C4b, where major pathway of complement activation involves four the C2 is then cleaved by the neighboring CIs; the smaller serum proteins: C3, factor B, factor D, and properdin. The al- fragment( C2b)diffuses away. The resulting C4b2a complex ternative pathway is initiated in most cases by cell-surface is called C3 convertase, referring to its role in converting the constituents that are foreign to the host(Table 13-1).For ex 3 into an active form. The smaller fragment from C4 cleav- ample both gram-negative and gram-positive bacteria have age, C4a, is an anaphylatoxin, or mediator of inflammation, cell-wall constituents that can activate the alternative pat which does not participate directly in the complement cas- way. The intermediates in the alternative pathway for gener cade; the anaphylatoxins, which include the smaller frag- ating C5b are shown schematically in Figure 13-7(page 306) ments of C4. C3 and C5 are described below In the classical pathway, C3 is rapidly cleaved to C3a and The native C3 component consists of two polypeptide C3b by the enzymatic activity of the C3 convertase. In the al hains, a and B. Hydrolysis of a short fragment( C3a) from ternative pathway, serum C3, which contains an unstable the amino terminus of the a chain by the C3 convertase gen- thioester bond, is subject to slow spontaneous hydrolysis to erates C3b(Figure 13-6). A single C3 convertase molecule can yield C3a and C3b. The C3b component can bind to foreign generate over 200 molecules of C3b, resulting in tremendous surface antigens(such as those on bacterial cells or viral par amplification at this step of the sequence. Some of the C3b ticles) or even to the host's own cells(see Figure 13-6c).The binds to CAb2a to form a trimolecular complex C4b2a3b membranes of most mammalian cells have high levels of called C5 convertase. The C3b component of this complex sialic acid, which contributes to the rapid inactivation of binds C5 and alters its conformation, so that the C4b2a com- bound C3b molecules on host cells; consequently this bind ponent can cleave C5 into C5a, which diffuses away, and C5b, ing rarely leads to further reactions on the host cell mem- which attaches to C6 and initiates formation of the membrane- brane. Because many foreign antigenic surfaces(e. g, bac- attack complex in a sequence described later. Some of the terial cell walls, yeast cell walls, and certain viral envelopes) C3b generated by C3 convertase activity does not associate have only low levels of sialic acid, C3b bound to these sur- with C4b2a; instead it diffuses away and then coats immune faces remains active for a longer time. The C3b present on the complexes and particulate antigens, functioning as an opsonin surface of the foreign cells can bind another serum protein s described in the Clinical Focus C3b may also bind directly called factor b to form a complex stabilized by Mg2+Bind- to cell membranes ing to C3b exposes a site on factor B that serves as the sub 0 Cell membrane C4b2a O=C SH Activated c3b FIGURE 13-6 Hydrolysis of C3 by C3 convertase C4b2a(a)Native fragment to bind to free hydroxyl or amino groups(R)on a cell mem- C3. (b)Activated C3 showing site of cleavage by C4b2a resulting in brane. Bound C3b exhibits various biological activities, including production of the C3a and C3b fragments. (c)A labile intemal binding of C5 and binding to C3b receptors on phagocytic cells thioester bond in C3 is activated as C3b is formed, allowing the C3b

C1r that converts C1r to an active serine protease enzyme, C1r, which then cleaves C1s to a similar active enzyme, C1s. C1s has two substrates, C4 and C2. The C4 component is a glycoprotein containing three polypeptide chains ,, and . C4 is activated when C1s hydrolyzes a small fragment (C4a) from the amino terminus of the chain, exposing a binding site on the larger fragment (C4b). The C4b fragment attaches to the target surface in the vicinity of C1, and the C2 proenzyme then attaches to the exposed binding site on C4b, where the C2 is then cleaved by the neighboring C1s; the smaller fragment (C2b) diffuses away. The resulting C4b2a complex is called C3 convertase, referring to its role in converting the C3 into an active form. The smaller fragment from C4 cleavage, C4a, is an anaphylatoxin, or mediator of inflammation, which does not participate directly in the complement cascade; the anaphylatoxins, which include the smaller fragments of C4, C3, and C5 are described below. The native C3 component consists of two polypeptide chains, and . Hydrolysis of a short fragment (C3a) from the amino terminus of the chain by the C3 convertase generates C3b (Figure 13-6). A single C3 convertase molecule can generate over 200 molecules of C3b, resulting in tremendous amplification at this step of the sequence. Some of the C3b binds to C4b2a to form a trimolecular complex C4b2a3b, called C5 convertase. The C3b component of this complex binds C5 and alters its conformation, so that the C4b2a component can cleave C5 into C5a, which diffuses away, and C5b, which attaches to C6 and initiates formation of the membraneattack complex in a sequence described later. Some of the C3b generated by C3 convertase activity does not associate with C4b2a ; instead it diffuses away and then coats immune complexes and particulate antigens, functioning as an opsonin as described in the Clinical Focus. C3b may also bind directly to cell membranes. The Alternative Pathway Is Antibody-Independent The alternative pathway generates bound C5b, the same product that the classical pathway generates, but it does so without the need for antigen-antibody complexes for initiation. Because no antibody is required, the alternative pathway is a component of the innate immune system. This major pathway of complement activation involves four serum proteins: C3, factor B, factor D, and properdin. The alternative pathway is initiated in most cases by cell-surface constituents that are foreign to the host (Table 13-1). For example, both gram-negative and gram-positive bacteria have cell-wall constituents that can activate the alternative pathway. The intermediates in the alternative pathway for generating C5b are shown schematically in Figure 13-7 (page 306). In the classical pathway, C3 is rapidly cleaved to C3a and C3b by the enzymatic activity of the C3 convertase. In the alternative pathway, serum C3, which contains an unstable thioester bond, is subject to slow spontaneous hydrolysis to yield C3a and C3b. The C3b component can bind to foreign surface antigens (such as those on bacterial cells or viral particles) or even to the host’s own cells (see Figure 13-6c). The membranes of most mammalian cells have high levels of sialic acid, which contributes to the rapid inactivation of bound C3b molecules on host cells; consequently this binding rarely leads to further reactions on the host cell membrane. Because many foreign antigenic surfaces (e.g., bacterial cell walls, yeast cell walls, and certain viral envelopes) have only low levels of sialic acid, C3b bound to these surfaces remains active for a longer time. The C3b present on the surface of the foreign cells can bind another serum protein called factor B to form a complex stabilized by Mg2. Binding to C3b exposes a site on factor B that serves as the sub- 304 PART III Immune Effector Mechanisms FIGURE 13-6 Hydrolysis of C3 by C3 convertase C4b2a (a) Native C3. (b) Activated C3 showing site of cleavage by C4b2a resulting in production of the C3a and C3b fragments. (c) A labile internal thioester bond in C3 is activated as C3b is formed, allowing the C3b fragment to bind to free hydroxyl or amino groups (R) on a cell membrane. Bound C3b exhibits various biological activities, including binding of C5 and binding to C3b receptors on phagocytic cells. (a) (c) (b) C O S S S S S S S S S S S S S α β C3 C4b2a C3a C + O – S Activated C3b C O SH R O Bound C3b Cell membrane

The Complement System cHAPTER 13 305 Initiators of the alternative pathway ment binds to mannose residues, some authors designate this TABLE 13-1 of complement activation the MBLectin pathway or mannan-binding lectin pathway. The lectin pathway, like the alternative pathway, does not de- PATHOGENS AND PARTICLES OF MICROBIAL ORIGIN pend on antibody for its activation. However, the mechanism is more like that of the classical pathway, because after initia Many strains of gram-negative bacteria tion, it proceeds, through the action of C4 and C2, to pro- Lipopolysaccharides from gram-negative bacteria duce a C5 convertase(see Figure 13-2) Many strains of gram-positive bacteria The lectin pathway is activated by the binding of man- nose-binding lectin (MBL) to mannose residues on glyce proteins or carbohydrates on the surface of microorganisms Fungal and yeast cell walls(zymosa including certain Salmonella, Listeria, and Neisseria strains, Some viruses and virus-infected cells ell as Cryptococcus neoformans and Candida albicans. Some tumor cells(Raji) MBL is an acute phase protein produced in inflammatory Its function in the way is sin Parasites(trypanosomes to that of C1q, which it resembles in structure. After MBL NONPATHOGENS binds to the surface of a cell or pathogen, MBL-associated serine proteases, MASP-1 and MASP-2, bind to MBL. The ac Human igG, IgA and ige in complexes tive complex formed by this association causes cleavage and Rabbit and guinea pig IgG in complexes activation of C4 and C2. The MASP-1 and-2 proteins have Cobra venom factor structural similarity to CIr and Cls and mimic their activi- ties. This means of activating the C2-C4 components to Heterologous erythrocytes(rabbit, mouse, chicken) form a C5 convertase without need for specific antibody Anionic polymers(dextran sulfate) binding represents an important innate defense mechanism Pure carbohydrates(agarose, inulin) able to the alternative pathway but utilizing the ele- ments of the classical pathway except for the Cl proteins SOURCE: Adapted from M.K. Pangburn, 1986, in Immunobiology of the Complement System, Academic Press The Three Complement Pathways Converge at the Membrane-Attack Complex The terminal sequence of complement activation involves strate for an enzymatically active serum protein called factor C5b, C6, C7, C8, and C9, which interact sequentially to form D Factor d cleaves the C3b-bound factor B, releasing a small a macromolecular structure called the membrane-attack fragment(Ba)that diffuses away and generating C3bBb. The complex (MAC). This complex forms a large channel C3bBb complex has C3 convertase activity and thus is analo- through the membrane of the target cell, enabling ions and gous to the C4b2a complex in the classical pathway. The C3 small molecules to diffuse freely across the membrane convertase activity of C3b Bb has a half-life of only 5 minutes The end result of activating the classical, alternative, or unless the serum protein properdin binds to it, stabilizing lectin pathways is production of an active C5 convertase it and extending the half-life of this convertase activity to his enzyme cleaves C5, which contains two protein chains, 30 minutes a and B After binding of C5 to the nonenzymatic C3b com The C3bBb generated in the alternative pathway can acti- ponent of the convertase, the amino terminus of the a chain te unhydrolyzed C3 to generate more C3b autocatalytically. is cleaved. This generates the small C5a fragment, which dif- As a result, the initial steps are repeated and amplified, so fuses away, and the large C5b fragment, which binds to the that more than 2 x 106 molecules of C3b can be deposited surface of the target cell and provides a binding site for the on an antigenic surface in less than 5 minutes. The C3 con- subsequent components of the membrane-attack complex vertase activity of c3bbb generates the C3bBb36 complex, see Figure 13-5, step 5). The C5b component is extremely la which exhibits C5 convertase activity, analogous to the bile and becomes inactive within 2 minutes unless c6 binds C4b2a3b col to it and stabilizes its activity matic C3b component binds C5, and the Bb component Up to this point, all the complement reactions take place C5b(see Figure 13-7); the latter binds to the antigenic surface. complexes in the fluid phase. As C5b6 binds to C7, the result ing complex undergoes a hydrophilic-amphiphilic structural The Lectin Pathway Originates With Host transition that exposes hydrophobic regions, which serve as Proteins Binding Microbial Surfaces binding sites for membrane phospholipids. If the occurs on a target-cell membrane, the hydrophobic Lectins are proteins that recognize and bind to specific car- sites enable the C5b67 complex to insert into the phospho- bohydrate targets. (Because the lectin that activates comple- lipid bilayer. If, however, the reaction occurs on an immune

strate for an enzymatically active serum protein called factor D. Factor D cleaves the C3b-bound factor B, releasing a small fragment (Ba) that diffuses away and generating C3bBb . The C3bBb complex has C3 convertase activity and thus is analogous to the C4b2a complex in the classical pathway. The C3 convertase activity of C3bBb has a half-life of only 5 minutes unless the serum protein properdin binds to it, stabilizing it and extending the half-life of this convertase activity to 30 minutes. The C3bBb generated in the alternative pathway can activate unhydrolyzed C3 to generate more C3b autocatalytically. As a result, the initial steps are repeated and amplified, so that more than 2 106 molecules of C3b can be deposited on an antigenic surface in less than 5 minutes. The C3 convertase activity of C3bBb generates the C3b Bb3b complex, which exhibits C5 convertase activity, analogous to the C4b2a 3b complex in the classical pathway. The nonenzymatic C3b component binds C5, and the Bb component subsequently hydrolyzes the bound C5 to generate C5a and C5b (see Figure 13-7); the latter binds to the antigenic surface. The Lectin Pathway Originates With Host Proteins Binding Microbial Surfaces Lectins are proteins that recognize and bind to specific carbohydrate targets. (Because the lectin that activates complement binds to mannose residues, some authors designate this the MBLectin pathway or mannan-binding lectin pathway.) The lectin pathway, like the alternative pathway, does not depend on antibody for its activation. However, the mechanism is more like that of the classical pathway, because after initiation, it proceeds, through the action of C4 and C2, to produce a C5 convertase (see Figure 13-2). The lectin pathway is activated by the binding of mannose-binding lectin (MBL) to mannose residues on glycoproteins or carbohydrates on the surface of microorganisms including certain Salmonella, Listeria, and Neisseria strains, as well as Cryptococcus neoformans and Candida albicans. MBL is an acute phase protein produced in inflammatory responses. Its function in the complement pathway is similar to that of C1q, which it resembles in structure. After MBL binds to the surface of a cell or pathogen, MBL-associated serine proteases, MASP-1 and MASP-2, bind to MBL. The active complex formed by this association causes cleavage and activation of C4 and C2. The MASP-1 and -2 proteins have structural similarity to C1r and C1s and mimic their activities. This means of activating the C2–C4 components to form a C5 convertase without need for specific antibody binding represents an important innate defense mechanism comparable to the alternative pathway, but utilizing the elements of the classical pathway except for the C1 proteins. The Three Complement Pathways Converge at the Membrane-Attack Complex The terminal sequence of complement activation involves C5b, C6, C7, C8, and C9, which interact sequentially to form a macromolecular structure called the membrane-attack complex (MAC). This complex forms a large channel through the membrane of the target cell, enabling ions and small molecules to diffuse freely across the membrane. The end result of activating the classical, alternative, or lectin pathways is production of an active C5 convertase. This enzyme cleaves C5, which contains two protein chains, and . After binding of C5 to the nonenzymatic C3b component of the convertase, the amino terminus of the chain is cleaved. This generates the small C5a fragment, which diffuses away, and the large C5b fragment, which binds to the surface of the target cell and provides a binding site for the subsequent components of the membrane-attack complex (see Figure 13-5, step 5). The C5b component is extremely labile and becomes inactive within 2 minutes unless C6 binds to it and stabilizes its activity. Up to this point, all the complement reactions take place on the hydrophilic surface of membranes or on immune complexes in the fluid phase. As C5b6 binds to C7, the resulting complex undergoes a hydrophilic-amphiphilic structural transition that exposes hydrophobic regions, which serve as binding sites for membrane phospholipids. If the reaction occurs on a target-cell membrane, the hydrophobic binding sites enable the C5b67 complex to insert into the phospholipid bilayer. If, however, the reaction occurs on an immune The Complement System CHAPTER 13 305 TABLE 13-1 Initiators of the alternative pathway of complement activation PATHOGENS AND PARTICLES OF MICROBIAL ORIGIN Many strains of gram-negative bacteria Lipopolysaccharides from gram-negative bacteria Many strains of gram-positive bacteria Teichoic acid from gram-positive cell walls Fungal and yeast cell walls (zymosan) Some viruses and virus-infected cells Some tumor cells (Raji) Parasites (trypanosomes) NONPATHOGENS Human IgG, IgA, and IgE in complexes Rabbit and guinea pig IgG in complexes Cobra venom factor Heterologous erythrocytes (rabbit, mouse, chicken) Anionic polymers (dextran sulfate) Pure carbohydrates (agarose, inulin) SOURCE: Adapted from M. K. Pangburn, 1986, in Immunobiology of the Complement System, Academic Press.

The Complement System cHAPTER 13 the MAc is the binding and polymerization of C9, a per forin-like molecule, to the C5b678 complex. As many as Regulation of the Complement 10-17 molecules of C9 can be bound and polymerized by System a single C5b678 complex During polymerization, the CS molecules undergo a hydrophilic-amphiphilic transition, so Because many elements of the complement system are capa that they too can insert into the membrane. The completed ble of attacking host cells as well as foreign cells and microor- MAC, which has a tubular form and functional pore size of ganisms, elaborate regulatory mechanisms have evolved to 70-100 A, consists of a C5b678 complex surrounded by a restrict complement activity to designated targets. a general poly-C9 complex(Figure 13-8). Since ions and small mole- mechanism of regulation in all complement pathways is the cules can diffuse freely through the central channel of the inclusion of highly labile components that undergo sponta- MAC, the cell cannot maintain its osmotic stability and is neous inactivation if they are not stabilized by reaction with killed by an influx of water and loss of electrolytes other components. In addition, a series of regulatory pro- teins can inactivate various complement components(Table 2).Fo r examI he glycoprotein CI inhibitor(ClInh can form a complex with CIr2S2, causing it to dissociate from Clq and preventing further activation of C4 or C2(Figure 139a(1) The reaction catalyzed by the C3 convertase enzymes of the lassical, lectin, and alternative pathways is the major amplifi cation step in complement activation, generating hundreds of molecules of C3b. The C3b generated by these enzymes has the potential to bind to nearby cells, mediating damage to the healthy cells by causing their opsonization by phagocytic cells bearing C3b receptors or by induction of the membrane- attack complex. Damage to normal host cells is prevented be cause C3b undergoes spontaneous hydrolysis by the time it has diffused 40 nm away from the C4b2a or C3bBb convertase en- zymes, so that it can no longer bind to its target site. The po- tential destruction of healthy host cells by C3b is further limited by a family of related proteins that regulate C3 conver- tase activity in the classical and alternative pathways. These regulatory proteins all contain repeating amino acid sequences (or motifs)of about 60 residues, termed short consensus repeats (SCRs). All these proteins are encoded at a single location on chromosome 1 in humans, known as the regulators of comple- ment activation(RCa) gene cluster. In the classical and lectin pathways, three structurally dis- nct RCa proteins act similarly to prevent assembly of C3 convertase(Figure 13-9a(2). These regulatory proteins in- clude soluble C4b-binding protein( C4bBP) and two mem- brane-bound proteins, complement receptor type 1(Cr1) rane cofactor protein(MCP). Each of these latory proteins binds to CAb and prevents its association with C2a. Once cbBp crl or mcp is bound to c4b. another regulatory protein, factor l, cleaves the C4b into bound C4d and soluble CAc(Figure 13-9a(3)). A similar regulatory se- quence operates to prevent assembly of the C3 convertase C3b Bb in the alternative pathway. In this case CRl, MCP,ora regulatory component called factor H binds to C3b and pre- IGURE 13-8(a)Photomicrograph of poly-C9 complex formed by vents its association with factor B(Figure 13-9a(4). Once in vitro polymerization of C9.(b)Photomicrograph of complement- CRl, MCP, or factor H is bound to C3b, factor I cleaves the induced lesions on the membrane of a red blood cell. These lesions C3b into a bound iC3b fragment and a soluble C3f fragment result from formation of membrane- attack complexes. /Part (a)from Further cleavage of iC3b by factor I releases C3c and leaves E.R. Podack, 1986, in Immunobiology of the Complement System, C3dg bound to the membrane(figure 13-9a(5). The mole cademic Press: part(b)from J. Humphrey and R. Dourmashkin, cular events involved in regulation of cell-bound C4b and 1969, Adv. Immunol. 11: 75. C3b are depicted in Figure 13-10(page 310)

the MAC is the binding and polymerization of C9, a perforin-like molecule, to the C5b678 complex. As many as 10–17 molecules of C9 can be bound and polymerized by a single C5b678 complex. During polymerization, the C9 molecules undergo a hydrophilic-amphiphilic transition, so that they too can insert into the membrane. The completed MAC, which has a tubular form and functional pore size of 70–100 Å, consists of a C5b678 complex surrounded by a poly-C9 complex (Figure 13-8). Since ions and small molecules can diffuse freely through the central channel of the MAC, the cell cannot maintain its osmotic stability and is killed by an influx of water and loss of electrolytes. Regulation of the Complement System Because many elements of the complement system are capable of attacking host cells as well as foreign cells and microorganisms, elaborate regulatory mechanisms have evolved to restrict complement activity to designated targets. A general mechanism of regulation in all complement pathways is the inclusion of highly labile components that undergo spontaneous inactivation if they are not stabilized by reaction with other components. In addition, a series of regulatory proteins can inactivate various complement components (Table 13-2). For example, the glycoprotein C1 inhibitor (C1Inh) can form a complex with C1r2s2, causing it to dissociate from C1q and preventing further activation of C4 or C2 (Figure 13-9a(1)). The reaction catalyzed by the C3 convertase enzymes of the classical, lectin, and alternative pathways is the major amplification step in complement activation, generating hundreds of molecules of C3b. The C3b generated by these enzymes has the potential to bind to nearby cells, mediating damage to the healthy cells by causing their opsonization by phagocytic cells bearing C3b receptors or by induction of the membraneattack complex. Damage to normal host cells is prevented because C3b undergoes spontaneous hydrolysis by the time it has diffused 40 nm away from the C4b2a or C3bBb convertase enzymes, so that it can no longer bind to its target site. The potential destruction of healthy host cells by C3b is further limited by a family of related proteins that regulate C3 convertase activity in the classical and alternative pathways. These regulatory proteins all contain repeating amino acid sequences (or motifs) of about 60 residues, termed short consensus repeats (SCRs). All these proteins are encoded at a single location on chromosome 1 in humans, known as the regulators of complement activation (RCA) gene cluster. In the classical and lectin pathways, three structurally distinct RCA proteins act similarly to prevent assembly of C3 convertase (Figure 13-9a(2)). These regulatory proteins include soluble C4b-binding protein (C4bBP) and two membrane-bound proteins, complement receptor type 1 (CR1) and membrane cofactor protein (MCP). Each of these regulatory proteins binds to C4b and prevents its association with C2a. Once C4bBP, CR1, or MCP is bound to C4b, another regulatory protein, factor I, cleaves the C4b into bound C4d and soluble C4c (Figure 13-9a(3)). A similar regulatory sequence operates to prevent assembly of the C3 convertase C3bBb in the alternative pathway. In this case CR1, MCP, or a regulatory component called factor H binds to C3b and prevents its association with factor B (Figure 13-9a(4)). Once CR1, MCP, or factor H is bound to C3b, factor I cleaves the C3b into a bound iC3b fragment and a soluble C3f fragment. Further cleavage of iC3b by factor I releases C3c and leaves C3dg bound to the membrane (Figure 13-9a(5)). The molecular events involved in regulation of cell-bound C4b and C3b are depicted in Figure 13-10 (page 310). The Complement System CHAPTER 13 307 (a) FIGURE 13-8 (a) Photomicrograph of poly-C9 complex formed by in vitro polymerization of C9. (b) Photomicrograph of complementinduced lesions on the membrane of a red blood cell. These lesions result from formation of membrane-attack complexes. [Part (a) from E. R. Podack, 1986, in Immunobiology of the Complement System, Academic Press; part (b) from J. Humphrey and R. Dourmashkin, 1969, Adv. Immunol. 11:75.] (b)

art 1 Immune effector mechanisms TABLE 13.2 Proteins that requlate the complement system Type of Protein Immunologic function Cl inhibitor(ClInh) Soluble Classical Serine protease inhibitor: causes Clr.S2 to dissociate from C1q C4b-binding pro Soluble Classical and lectin Blocks formation of C3 convertase by (C4bBP) binding C4b: cofactor for cleavage of C4b by factor I Factor H Soluble Alternative Blocks formation of C3 convertase by binding C3b; cofactor for cleavage Complement-receptor Block formation of C3 convertase by type 1(CRT) Membrane Classical, alternative binding C4b or C3b: cofactor for Membrane-cofactor and lectin factor I-catalyzed cleavage of C4b protein(MCP) Decay-accelerating Membrane Classical, alternative Accelerates dissociation of c b2a and factor(DAE or CD55) bound and lectin C3bBb(classical and alternative C3 Factor-I Soluble Serine protease: cleaves C4b or C3b lectin using C4bBP, CRl, factor H, DAE Sprotein Soluble Terminal luble C5b67 and prevents its insertion into cell membrane Homologous restriction factor(HRF) Termina Bind to C5b678 on autologous cells, Membrane inhibitor of blocking binding of C9 reactive lysis( MIRL or CD59 Anaphylatoxin inactivate Soluble Effector Inactivates anaphylatoxin activity of C3a C4a, and C5a by carboxypeptidase N removal of C-terminal Arg An RCA (regulator of complement activation) protein In humans, all RCA proteins are encoded on chromosome 1 and contain short consensus repeats. Several rCa proteins also act on the assembled C3 con- tein can bind to C5b67, inducing a hydrophilic transition vertase, causing it to dissociate; these include the previously and thereby preventing insertion of C5b67 into the mem- mentioned C4bBP, CRl, and factor H. In addition, decay- brane of nearby cells(Figure 13-9c(1)) accelerating factor(DAF or CD55), which is a glycoprotein an- Complement-mediated lysis of cells is more effective if chored covalently to a glycophospholipid membrane protein, the complement is from a species different from that of the has the ability to dissociate C3 convertase. The consequences cells being lysed. This phenomenon depends on two mem- of daF deficiency are described in the Clinical Focus section. brane proteins that block MAC formation. These two pro- Each of these RCa proteins accelerates decay(dissociation) of teins, present on the membrane of many cell types, are C3 convertase by releasing the component with enzymatic ac- homologous restriction factor(HRF)and membrane inhibitor tivity( C2a or Bb)from the cell-bound component( CAb or of reactive lysis(MIRl or CD59). Both HRF and mirl pro- C3b). Once dissociation of the C3 convertase occurs, factor I tect cells from nonspecific complement-mediated lysis by cleaves the remaining membrane-bound C4b or C3b compo- binding to C8, preventing assembly of poly-C9 and its inser nent, irreversibly inactivating the convertase(Figure 13-9b). tion into the plasma membrane(Figure 13-9c(2)).However, Regulatory proteins also operate at the level of the mem- this inhibition occurs only if the complement components brane-attack complex. The potential release of the C5b67 are from the same species as the target cells. For this reason, complex poses a threat of innocent-bystander lysis to healthy MIRL and hrf are said to display homologous restriction, cells. A number of serum proteins counter this threat by for which the latter was named. As discussed in Chapter 21 binding to released C5b67 and preventing its insertion into homologous restriction poses a barrier to the use of organs the membrane of nearby cells. A serum protein called S pro- from other species for clinical transplantation

Several RCA proteins also act on the assembled C3 convertase, causing it to dissociate; these include the previously mentioned C4bBP, CR1, and factor H. In addition, decayaccelerating factor (DAF or CD55), which is a glycoprotein anchored covalently to a glycophospholipid membrane protein, has the ability to dissociate C3 convertase. The consequences of DAF deficiency are described in the Clinical Focus section. Each of these RCA proteins accelerates decay (dissociation) of C3 convertase by releasing the component with enzymatic activity (C2a or Bb) from the cell-bound component (C4b or C3b). Once dissociation of the C3 convertase occurs, factor I cleaves the remaining membrane-bound C4b or C3b component, irreversibly inactivating the convertase (Figure 13-9b). Regulatory proteins also operate at the level of the membrane-attack complex. The potential release of the C5b67 complex poses a threat of innocent-bystander lysis to healthy cells. A number of serum proteins counter this threat by binding to released C5b67 and preventing its insertion into the membrane of nearby cells. A serum protein called S protein can bind to C5b67, inducing a hydrophilic transition and thereby preventing insertion of C5b67 into the membrane of nearby cells (Figure 13-9c(1)). Complement-mediated lysis of cells is more effective if the complement is from a species different from that of the cells being lysed. This phenomenon depends on two membrane proteins that block MAC formation. These two proteins, present on the membrane of many cell types, are homologous restriction factor (HRF) and membrane inhibitor of reactive lysis (MIRL or CD59). Both HRF and MIRL protect cells from nonspecific complement-mediated lysis by binding to C8, preventing assembly of poly-C9 and its insertion into the plasma membrane (Figure 13-9c(2)). However, this inhibition occurs only if the complement components are from the same species as the target cells. For this reason, MIRL and HRF are said to display homologous restriction, for which the latter was named. As discussed in Chapter 21, homologous restriction poses a barrier to the use of organs from other species for clinical transplantation. 308 PART III Immune Effector Mechanisms TABLE 13-2 Proteins that regulate the complement system Type of Pathway Protein protein affected Immunologic function C1 inhibitor (C1Inh) Soluble Classical Serine protease inhibitor: causes C1r2s2 to dissociate from C1q C4b-binding protein Soluble Classical and lectin Blocks formation of C3 convertase by (C4bBP)* binding C4b; cofactor for cleavage of C4b by factor I Factor H* Soluble Alternative Blocks formation of C3 convertase by binding C3b; cofactor for cleavage of C3b by factor I Complement-receptor Block formation of C3 convertase by type 1 (CR1)* Membrane Classical, alternative, binding C4b or C3b; cofactor for Membrane-cofactor bound and lectin factor I-catalyzed cleavage of C4b protein (MCP)* or C3b C3bBb Decay-accelerating Membrane Classical, alternative, Accelerates dissociation of C4b2a and factor (DAE or CD55)* bound and lectin C3bBb (classical and alternative C3 convertases) Factor-I Soluble Classical, alternative, Serine protease: cleaves C4b or C3b and lectin using C4bBP, CR1, factor H, DAE, or MCP as cofactor S protein Soluble Terminal Binds soluble C5b67 and prevents its insertion into cell membrane Homologous restriction factor (HRF) Membrane Terminal Bind to C5b678 on autologous cells, Membrane inhibitor of bound blocking binding of C9 reactive lysis (MIRL or CD59)* Anaphylatoxin inactivator Soluble Effector Inactivates anaphylatoxin activity of C3a, C4a, and C5a by carboxypeptidase N removal of C-terminal Arg *An RCA (regulator of complement activation) protein. In humans, all RCA proteins are encoded on chromosome 1 and contain short consensus repeats. } }