Complement System: Innate Defense Mechanisms PDF

The complement system is an essential innate defense system. Although its main role is to kill pathogens immediately when they enter the body, the complement system also alerts the immune systems to the presence of invaders, regulates inflammation, removes damaged or altered cells, and regulates ada...

The complement system is an essential innate defense system. Although its main role is to kill pathogens immediately when they enter the body, the complement system also alerts the immune systems to the presence of invaders, regulates inflammation, removes damaged or altered cells, and regulates adaptive immune responses. It is involved in the clearance of antigen-antibody complexes, blood vessel formation, mobilization of stem cells, tissue regeneration, and lipid metabolism. Protection from infection requires that the body respond to invaders as rapidly as possible. A critical component of this early response is the complement system. It can detect and kill invaders before other defenses have had a chance to respond. The complement system is a network of interacting pattern-recognition proteins, proteases, serum proteins, receptors, and regulators that kills invaders fast (Fig. 4.1). The major complement proteins bind covalently (and hence irreversibly) to the surface of invading microbes and then destroy them. The complement system is activated by the presence of either pathogen-associated molecular patterns (PAMPs) or by antigenbound antibodies. Because the complement system is so potent, it must be tightly regulated and controlled. Disturbances in this system may result in inflammation or autoimmunity. FIG. 4.1 The functions of the complement system. Complement may either alter microbial membranes or trigger inflammation. Either way, it hastens the elimination of microbial invaders and is thus a key component of the innate immune system. It has multiple other functions as well as those noted here. The complement system consists of multiple proteins that are activated in sequence. Once activated, the system generates multiple effector molecules. The first step, complement activation, occurs by three different pathways, referred to as the alternative, the lectin, and the classical pathways (Fig. 4.2). The alternative and lectin pathways are activated by microbial carbohydrates and are thus typical patternrecognition pathways that trigger innate responses. The classical pathway, in contrast, is activated by antibodies and thus works in association with adaptive immune responses. FIG. 4.2 The three pathways by which the complement system can be activated. The alternate and lectin pathways are innate, the classical pathway is adaptive since it is triggered by antibodies. All three activate C3, which in turn initiates the amplification pathway and generates the terminal complement complex. Complement Proteins Complement proteins are either labeled numerically with the prefix C (e.g., C1, C2, C3) or designated as "factors" by letters of the alphabet (FB, FD, FP, and so forth). Some are found free in serum, whereas others are cell-surface receptors. Collectively, complement components account for 5% to 10% of the proteins in blood serum---a reflection of the critical importance of this system. Their size varies from 24 kDa for factor D (FD) to 460 kDa for C1q. Their serum concentrations in humans vary between 20 µg/mL of C2 and 1300 µg/mL of C3 (Table 4.1). Complement proteins are synthesized at multiple sites. C3, C6, C8, and FB are made in the liver, whereas C2, C3, C4, C5, FB, FD, FP, and FI are made by macrophages. C1q is produced by mast cells. Neutrophil granules may store large quantities of C6 and C7. As a result, these proteins are readily available for defense at sites where neutrophils accumulate. TABLE 4.1 Complement Components Name MW (kDa) Serum Concentration (mg/mL) Classical Pathway C1q 460 80 C1r 83 50 C1s 83 50 C4 200 600 C2 102 20 C3 185 1300 Alternate Pathway FD 24 1 FB 90 210 Terminal Components C5 195 70 C6 120 65 C7 120 55 C8 160 55 C9 70 60 Control Proteins C1-INH 105 200 C4BP 550 250 FH 150 480 FI 88 35 Ana INH 310 35 FP 4 × 56 20 S 83 500 Activation Pathways The Alternative Pathway The alternative pathway is an evolutionary ancient innate pathway. It is triggered when microbial cell walls meet complement proteins in the blood. The most important complement protein is called C3. C3 is a disulfide-linked heterodimer with α and β chains. It is synthesized by liver cells and macrophages and is the most abundant complement component in serum. C3 has a reactive thioester side-chain, that, when activated, binds to microbes and marks them for destruction by immune cells. Activation of this thioester side-chain must be carefully regulated to ensure that C3 does not bind to normal cells. To prevent such accidents, the thioester group in inactive C3 is hidden within the folded molecule like a pocket knife. In healthy normal animals, C3 spontaneously breaks down into two fragments called C3a and C3b (Fig. 4.3). This breakdown exposes the reactive thioester group in C3b. The thioester then generates a carbonyl group that covalently binds the C3b to carbohydrates and proteins on nearby cell surfaces (Fig. 4.4). The breakdown of C3 also exposes binding sites for a protein called factor H (FH). When FH binds to these sites, a protease called factor I (FI) can then cleave the C3b, preventing further activation and generating two fragments, iC3b and C3c. iC3b binds receptors on circulating leukocytes (Fig. 4.5). It stimulates these cells to engulf pathogens and it activates inflammatory cells. The final breakdown product of C3, C3dg, targets pathogens to surface receptors on B cells and so promotes antibody production (Chapter 15). FIG. 4.3 The alternative complement pathway. Surface-bound C3b may either be destroyed, as normally happens, or activated by the presence of an activating surface. FIG. 4.4 Activation of C3 involves its cleavage by C3 convertase. This exposes a thioester bond between a cysteine and a glutamine. This bond breaks to form a reactive carbonyl group that enables the molecule to bind covalently (and hence irreversibly) to target cell surfaces. Removal of C3a also reveals the binding sites for FH and FB. FIG. 4.5 Activated C3 binds to a cell surfaces. This C3b is normally inactivated by the actions of FH and FI. However, FH must first be activated by binding to the surface. In the absence of FH, FI will not work. In this case, C3b persists and activates the terminal complement pathway. The consequences of the breakdown of cell-bound C3b depend on FH binding. This binding depends in turn on the nature of the target surface. When FH interacts with normal cells, glycoproteins rich in sialic acid (N-acetylneuraminic acid) and other neutral or anionic polysaccharides enhance FH binding to C3b. As a result, FI is activated, and the C3b is destroyed. In a healthy animal, therefore, there is a continuous low-level activation of C3, but FH and FI destroy C3b as fast as it is generated. In contrast, bacterial cell walls lack sialic acid. When C3b binds to bacteria, FH cannot bind, FI is inactivated, and the C3b remains attached to the microbial surface. This bound C3b exposes a binding site for another complement protein called factor B (FB) and as a result, a complex called C3bB forms. The bound FB is then cleaved by a protease called factor D (FD), releasing a soluble fragment called Ba and leaving C3bBb attached to the bacteria. This C3bBb complex is a protease whose preferred substrate is C3. (It is therefore called the alternative C3 convertase.) FD can act only on FB after it has bound to C3b but not before. This constraint is called substrate modulation and regulates several reactions in the complement pathways. It ensures that the activities of enzymes such as FD are confined to the correct molecules. The alternative C3 convertase, C3bBb, cleaves bound C3 to generate more C3b. C3bBb is, however, very unstable, with a half-life of only 5 minutes. If a protein called factor P (FP or properdin) binds to the complex, it forms a stable C3bBbP complex with a half-life of 30 minutes. Since C3b generates more C3bBbP, the net effect of all this is that a positive feedback loop is established where increasing amounts of C3b are produced and irreversibly bound to the surface of the invading organism. Despite its name, the alternative complement pathway accounts for 80% to 90% of all complement activation. The Lectin Pathway A second method of activating complement is triggered by the binding of soluble pattern-recognition molecules (lectins) to microbial carbohydrates. When these lectins bind to microbes, they activate proteases that activate complement. Like the alternative pathway, this is an innate pathway triggered simply by the presence of bacterial PAMPs (Fig. 4.6). FIG. 4.6 Complement activation by the lectin pathway. Complement-activating lectins include mannose-binding lectin (MBL) and a family of proteins called ficolins. MBL attaches to mannose or N-acetylglucosamine on the walls of bacteria, fungi, and protozoa. It does not bind to mammalian glycoproteins. Once bound, MBL activates a serum protease called MASP-2. (MASP stands for MBL-associated serine protease.) Activated MASP-2, in turn, acts on the complement component C4, splitting it into C4a and C4b. This exposes a thioester group on the C4b that generates a reactive carbonyl group that binds the C4b to the microbial surface (Fig. 4.7). Another complement component, C2, then binds to the C4b to form a complex, C4b2. The bound C2 is then cleaved by MASP-2 to generate C4b2b (Box 4.1). FIG. 4.7 The two C3 convertases, C4b2b and C3bBb, act on C5 when it is linked to C3b and cleave off a small peptide called C5a. In so doing they reveal a site that binds C6 and C7. Box 4.1 The Defense Collagens C1q and mannose-binding lectin (MBL) are members of a unique protein family called the defense collagens. Other members of this family include surfactant protein A, adiponectin, conglutinin, and ficolin. These are soluble lectins characterized as containing a conserved collagen-like region as well as a carbohydrate recognition domain. Like C1q, they commonly polymerize. These proteins serve as soluble pattern-recognition receptors. They can bind to foreign pathogens and subsequently interact with phagocytic cells or complement. Thus MBL recognizes mannosecontaining carbohydrates. On binding to their ligands, they trigger an immediate protective response such as activation of complement or promotion of phagocytosis. Cell-bound C4b2b is a protease that acts on C3 to generate C3a and C3b and exposes the thioester group on the C3b. The activation of C3b by C4b2b is a major step because each C4b2b complex can generate as many as 200 C3b molecules. Since these reactions are confined to the microenvironment close to microbial surfaces, newly formed C3b will bind to nearby microbes. The bound C3b can also bind C5 and cleave it to generate C5a and C5b. The complement pathway then can proceed to completion and the killing of the organism by terminal complement complexes. The lectin pathway is ancient, having existed for at least 300 million years (It is present in many invertebrates (see Chapter 43). Although in many ways it duplicates the alternative pathway, it is an example of the way the body uses redundant mechanisms to ensure protection. The Classical Pathway The classical complement pathway (Fig. 4.8) is triggered when the complement component C1q encounters antibody molecules bound to an invading microorganism. FIG. 4.8 The basic features of the classical complement pathway. Unlike the alternate and lectin pathways, the classical pathway cannot be activated until antibodies are made and immune complexes form, which may take 7 to 10 days after onset of infection. When antibody molecules bind to an invader, active sites on their Fc regions are exposed. When multiple antibody molecules bind to an organism, these active sites collectively trigger the classical complement pathway. The first component of the classical pathway is a protein complex called C1. C1 consists of three subunits (C1q, C1r, C1s) bound together by calcium. The completely assembled C1q looks like a six-stranded whip when viewed by electron microscopy (Fig. 4.9). Two molecules each of C1r and C1s form a complex located between the C1q strands. C1q is activated when its strands bind to activating sites on clustered antibody molecules. This binding triggers a conformational change in C1q that permits C1r to interact with C1s, and C1s is converted into an active protease. FIG. 4.9 The structure of C1 and its role in interacting with antibodies to initiate the classical complement pathway. Single, antigen-bound molecules of immunoglobulin (Ig) M or paired, antigen-bound molecules of IgG are required to activate C1 (Chapter 16). The polymeric IgM structure readily provides several closely spaced complement-activating sites. In contrast, two IgG molecules must be located close to each other to have the same effect. Thus IgG is much less efficient than IgM in activating the classical pathway. Active C1s cleaves C4 into C4a and C4b. C2 then binds to C4b to form the complex C4b2. Activated C1s then splits the bound C2, generating a small peptide fragment C2a and the C4b2b complex. C1s cannot act on soluble C2; the C2 must first be bound to C4b before it can be split (another example of substrate modulation). The C4b2b complex is a potent protease whose target is C3, and it is therefore called classical C3 convertase. The newly generated C3b binds and activates C5. Subsequent reactions lead to formation of the terminal complement complex and microbial killing. In addition to binding immune complexes, C1 can also be activated directly by some viruses, or by bacteria such as Escherichia coli and Klebsiella pneumoniae. C1q can also bind to apoptotic and necrotic cells, extracellular matrix proteins, pentraxins such as C-reactive protein, amyloid and prion proteins, and DNA. However, all these substances (with the exception of immune complexes) can also bind the complement inhibitors C1-BP and FH so that full complement activation does not occur. If these inhibitory processes are blocked, uncontrolled complement activation may lead to unwanted inflammation. The Amplification Pathway All surface-bound C3 convertases, regardless of their origin, can induce the next steps in complement activation, the amplification pathway (Fig. 4.10). Once C5 binds to C3b, substrate modulation occurs, and the C5 is then cleaved by C3bBb (Fig. 4.11). The convertases break C5 (195 kDa) into a small fragment called C5a, leaving a large fragment C5b attached to the C3b. This cleavage also exposes a site on C5b that can bind two new proteins, C6 and C7, to form a multimolecular complex called C5b67 (Fig. 4.12). The C5b67 complex can then insert itself into the microbial cell wall. Once inserted in the surface of an organism, the complex first binds a molecule of C8 to form C5b678. Twelve to 18 C9 molecules then polymerize with the C5b678 complex to form a tubular structure called the terminal complement complex (TCC), (also called the membrane attack complex \[MAC\] or C5b6789). The TCC inserts into microbial cell membranes and punches a hole in the invader. If sufficient TCCs are formed on an organism, it will be killed by osmotic lysis. These TCCs can be seen by electron microscopy as ringshaped structures on the microbial surface with a central electrondense area surrounded by a lighter ring of poly C9 (see Fig. 4.12). FIG. 4.10 The amplification pathway. The progressive aggregation of the terminal complement components eventually leads to the polymerization of C9 and the assembly of a membrane attack complex. FIG. 4.11 Substrate modulation is one way in which the complement system is regulated. The target for a protease cannot be cleaved unless it is first bound to another protein. Examples of substrate modulation include the cleavage of factors C2, B, and C5 only after they have bound to C4, C3, and C3, respectively. FIG. 4.12 Formation of poly C9 by the amplification pathway and an electron micrograph of poly C9-complement lesions on an erythrocyte membrane. The insert shows a mouse complement lesion. The arrow points to a possible C5b678 complex. Compare these lesions to the T cell polyperforins in Fig. 18.9. (From Podack ER, Dennert G: Assembly of two types of tubules with putative cytolytic function by cloned natural killer cells, Nature 307:442, 1983.) More important than direct TCC-mediated lysis are the potent inflammatory effects of the small released peptide C5a. C5a can degranulate mast cells and stimulate platelets to release histamine and serotonin. It also triggers inflammation through its cell surface receptor. C5a is a powerful attractant for neutrophils and macrophages. It increases vascular permeability, causes lysosomal enzyme release from neutrophils and thromboxane release from macrophages, and regulates some T cell responses (Fig. 4.13). The other small complement peptide, C3a, can kill bacteria such as E. coli, Pseudomonas aeruginosa, Enterococcus faecalis, and Streptococcus pyogenes. C3a acts like other antimicrobial peptides by disrupting bacterial membranes. (C3a and C5a are also called anaphylatoxins since, when injected in sufficient amounts, they can kill an animal in a manner similar to anaphylaxis \[Chapter 30\].) FIG. 4.13 Some of the biological consequences of complement activation.