582
Complement PHCL System
Jan 25, 2006
The Complement System
A system of > 34 proteins interacting in a cascade (similar to the clotting system) that leads to a variety of biologic processes. Many complement proteins are enzymes that exist in serum as inactive precursors (zymogens); many others reside on cell surfaces. Complement proteins make up about 10% of the serum proteins, with the third component (C3) present in the highest concentration (about 1.5 mg/mL). For components of the complement system, see Tables 1 and 2 The three pathways of complement activation are called the classical, the alternative, and the mannan binding lectin (MBL) pathway (see Fig. 1). All are directed at the single most important step in activation, the cleavage of C3. A common final pathway is called the terminal pathway, or the membrane attack complex (MAC). FIG. 1. Complement pathways. Components are listed in parentheses; regulatory proteins are bracketed in italics. MBL = Mannan binding lectin.
Nomenclature: Classical pathway components are labeled with a C and a number (eg, C1, C3). Because of the sequence in which they were identified, the first four components are numbered C1, C4, C2, and C3. Alternative pathway components are lettered (eg, B, P, D). Some components are called factors (eg, factor B, factor D). Activated components or complexes have a bar over them to indicate activation (eg, C1, C1r, C3b,Bb). Cleavage fragments are designated with a small letter after the component (eg, C3a and C3b are fragments of C3). Inactive C3b is designated iC3b. Polypeptide chains of complement proteins are designated with a Greek letter after the component (eg, C3 and C3 are the and chains of C3). Cell membrane receptors for C3 are abbreviated CR1, CR2, CR3, and CR4.
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PHCL 582
Complement System
Jan 25, 2006
The Classical Pathway
FIG. 2. Activation of the classical pathway.
Activation: The classical pathway (see Fig. 2) is activated normally by complement fixing Abs (Abs that bind to complement), which are in Ag-Ab complexes or in which the Ab (IgG or IgM) is aggregated. Thus, the classical pathway serves specific immunity because only Ab of specific classes, formed in response to Ag stimulation, is able to activate this pathway. The C1 macromolecule is a Ca++-dependent complex of one C1q, two C1r, and two C1s molecules. The C1 macromolecule remains intact only when Ca++ is present; otherwise the individual subunits dissociate from each other. Activation occurs when two of C1q's six monomers bind to the Fc regions of two IgG molecules or to one pentameric IgM molecule. Two IgG molecules must be properly spaced to cause activation, whereas a single pentameric IgM has that proximity built into its structure. Therefore, IgM is much more efficient at activating complement than IgG. Activity of IgG is in the order IgG3 > IgG1 > IgG2. IgG4 does not fix complement. Once Ig is bound to C1q, the C1q molecule undergoes change in tertiary structure, causing autocatalytic activation of C1r to C1r. C1r then cleaves a bond in C1s to produce C1s. No cleavage fragment is released when either C1r or C1s is cleaved.
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PHCL 582
Complement System
Jan 25, 2006
C1s is also called C1 esterase. C1s may cleave C4 into C4a and C4b. C4b, the major cleavage fragment, binds to membrane if membrane is present. C1s can then either cleave free C2 to produce C2a and C2b, which is an inefficient process, or cleave C2 in a C4b,C2 complex to produce C4b,C2a and free C2b, which is a very efficient process. C2a is the major cleavage fragment of C2. If free C2 has been cleaved, then C2a must bind to C4b to form a C4b,2a complex, or the C2a will decay and be inactive. C4b,2a is the classical pathway C3 convertase, which may cleave C3 into C3a and C3b. C2a contains the enzymatic site for cleavage of C3. C4b,2a requires the presence of magnesium and decays over time at physiologic temperatures. The classical pathway can also be activated by mechanisms independent of Ab. Heparin (a polyanionic anticoagulant) and protamine (a polycation that is used to block heparin), when present in equimolar concentrations, can activate the classical pathway. Various other polyanions (eg, DNA and RNA) are thought to be able to react directly with C1q to activate the classical pathway. C-reactive protein is capable of leading to classical pathway activation without the presence of Abs. C1 bypass pathways have also been described, which do not use components of the classical pathway but result in C3 cleavage. One of these has been characterized as the MBL pathway. Regulation: The classical pathway is regulated by C1 esterase inhibitor (C1INH), which binds stoichiometrically (1:1) to C1r and C1s and to C1r and C1s to inactivate these proteins permanently. C1INH also binds stoichiometrically to plasmin, kallikrein, activated Hageman factor, and coagulation factor XIa. Its absence leads to hereditary angioedema. Factor J is a cationic glycoprotein that also inhibits C1 activity. C4-binding protein (C4BP) disassembles the C4b,2a complex, allowing factor I to inactivate C4b.
The Alternative Pathway
FIG. 3. Activation and regulation of the alternative pathway.
Activation: The alternative pathway (see Fig. 3) is activated by natural substances (eg, yeast walls, cobra venom factor, nephritic factor, bacterial cell wall [endotoxin], rabbit RBCs [in vitro]) and by aggregated IgA as a nonspecific (innate) immune response, ie, one not requiring previous sensitization. The alternative pathway does not involve C1,
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PHCL 582
Complement System
Jan 25, 2006
C4, or C2 but leads to the cleavage of C3. This pathway depends on the constant cleavage of small amounts of C3 into C3a and C3b. This natural cleavage of C3 is poorly understood and is thought to occur through the nonspecific action of enzymes on C3 or by low-level activity of the other two pathways. C3b then serves as a substrate for factor B to produce the complex C3b,B. Factor D (an activated enzyme in plasma) cleaves factor B to produce C3b,Bb. Properdin (P) stabilizes this C3b,Bb complex to retard its decay. C3b,Bb and C3b,Bb,P are the alternative pathway C3 convertases, the enzymes that cleave C3 into C3a and C3b. Bb contains the enzymatic site for cleaving C3. C3b,Bb requires the presence of magnesium and decays over time. The alternative pathway is viewed as an amplification pathway because one C3b,Bb complex can cleave many C3 molecules. However, amplification also occurs when C1s is produced and when C4b,2a is formed. Each of these enzymes may cleave hundreds of molecules, leading to rapid complement activation. Regulation: The alternative pathway C3b,Bb complex is regulated by several factors. Properdin retards the decay of the C3b,Bb complex, increasing its half-life from about 4 min to 40 min. Decay accelerator substances (eg, factor H or decay accelerating factor [DAF]) compete with B for binding to C3b (eg, to produce C3b,H), decreasing the halflife of the C3b,Bb complex and causing dissociation of the complex into C3b and Bb. Factor I acts on C3b,H to degrade C3b (leading to production of iC3b, C3c, C3d, C3f, and C3dg). Circumstances under which the C3b,Bb complex forms will determine whether the alternative pathway is activated. Surfaces onto which the C3b,Bb complex may attach are either activating surfaces (eg, yeast walls, rabbit RBCs) or nonactivating surfaces (eg, sheep RBCs). Activating surfaces prevent factor H from binding to C3b, whereas nonactivating surfaces allow factor H to bind to C3b and dissociate C3b,Bb. Therefore, the C3b,Bb complex remains active much longer on an activating surface than on a nonactivating surface. The mechanisms described above explain how the alternative pathway activated is in vivo. Cobra venom factor (CoVF) is like cobra C3b; the CoVF,Bb complex is very stable and not susceptible to the decay action of factor H. Therefore, CoVF,Bb may lead to brisk and near-total cleavage of C3. C3 nephritic factor (C3NeF) is found in the serum of about 10% of patients with membranoproliferative glomerulonephritis and is an Ig directed to the C3b,Bb complex. C3NeF acts like properdin, except that the C3b,Bb,C3NeF complex is relatively resistant to the decay activity of factor H. Yeast walls (zymosan) and certain membranes (eg, rabbit RBCs) are activating surfaces onto which a C3b,Bb complex is protected from the decay activity of factor H.
The Mannan Binding Lectin Pathway
The mannan binding lectin (MBL) pathway depends on innate recognition of foreign substances (ie, carbohydrates) for activation. This pathway has structural and functional similarities to the classical pathway. MBL is similar to C1q, and MASP-1 and MASP-2
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PHCL 582
Complement System
Jan 25, 2006
appear to be similar to C1r and C1s in the classical pathway, respectively. Thus, MASP-2 may cleave C4 and lead to formation of MBL pathway-derived C3 convertase.
C3 Cleavage and Its Consequences
C3 convertases cleave C3 into C3a and C3b, which generates a metastable binding site in C3b for membranes. If a surface or membrane is available immediately after C3 is acted on by C3 convertase, C3b may bind covalently. If a membrane or surface is not available, then C3b becomes fluid-phase C3b and is unable to bind covalently to cell surfaces. C3 can also become C3b-like if treated with methylamine. Once C3b has bound to membrane via the labile metastable binding site, it can participate in biologic activities by binding to a variety of C3 receptors, serve as an efficient binding site for B to cause more cleavage of C3 via the alternative pathway, participate in the formation of a C5 convertase, or be acted on by factor I and a cofactor to form iC3b. Thus, C3b can bind covalently to membranes by its metastable thiolester binding site and, once bound, can interact with a variety of receptors depending on the availability of C3 receptors on cells and on the decay status of the C3. Binding to membranes through the covalent metastable binding site must not be confused with noncovalent binding to the receptors.
Membrane Attack Complex--The Terminal Pathway
FIG. 4. Formation of C5 convertases.
C3 convertase (eg, C3b,Bb) can become C5 convertase (eg, C3b,Bb,3b) by the addition of a C3b into the complex (see Fig. 4). C5 convertase cleaves C5 into C5a and C5b, 5
PHCL 582
Complement System
Jan 25, 2006
beginning the formation of the membrane attack complex (MAC). C6 may then bind to C5b to produce C5b,6. Next, C7 may bind to form C5b,6,7, which can attach itself to membranes and lipid bilayers. When this occurs on a cell that does not otherwise have any complement products on it, this is called the innocent bystander phenomenon (and may cause hemolysis of the innocent cell). C8 may then bind to the C5b,6,7 complex to form C5b,6,7,8, which can cause slow, inefficient lysis of the cell. Finally, C9 binds to the complex to produce C5b,6,7,8,9, which initiates substantial lysis of the cell. As additional C9 molecules are added to the C5b-9 complex, lysis increases. The MAC is regulated by S protein, also called vitronectin (which controls the activity of C5b-7), by homologous restriction factor (HRF), by SP40,40, and by CD59 (which regulates C8,9 activity).
Biologic Activities Associated With Complement Activation
Cell lysis is only one of many biologic activities associated with complement activation and may not be the most important. Lysis is seen clinically in patients with paroxysmal nocturnal hemoglobinuria, a rare disease involving deficiencies of the membrane proteins DAF (decay accelerating factor), HRF (homologous restriction factor), and CD59. Complement receptors are present on a variety of cells. CR1, membrane cofactor protein (MCP, CD46), and DAF (CD55) regulate breakdown of C3b. HRF and CD59 prevent formation of the membrane attack complex on homologous cells. CR1 (CD35) also plays a role in the clearance of immune complexes. CR2 (CD21) regulates B-cell functions (Ab production) and is the Epstein-Barr virus receptor. CR3 (CD11b/CD18) plays a role in phagocytosis, mediating adherence of iC3b-coated particles for phagocytosis. CR4 appears on platelets and has been less well studied than the other C3 receptors. gp150,95 plays a role in monocyte migration. C3a and C4a receptors bind C3a and C4a, respectively. C5a receptor binds C5a and C5adesarg (C5a without the t...