Complement System

THE COMPLEMENT SYSTEM

INTRODUCTION:The term complement refers to a set of serum proteins that cooperates with both the innate and the adaptive immune systems to eliminate blood and tissue pathogens. Like the components of the blood clotting system, complement proteins interact with one another in catalytic cascades. Various complement components bind and opsonize bacteria, rendering them susceptible to receptor-mediated phagocytosis by macrophages, which express membrane receptors for complement proteins. Other complement proteins elicit inflammatory responses, interface with components of the adaptive immune system, clear immune complexes from the serum, and/or eliminate apoptotic cells. Finally, a Membrane Attack Complex (MAC) assembled from complement proteins directly kills some pathogens by creating pores in microbial membranes. The biological importance of complement is emphasized both by the pathological consequences of mutations in the genes encoding complement proteins as well as by the broad range of strategies that have evolved in microorganisms to evade it.

HISTORY:Research on complement began in the 1890s, when Jules Bordet at the Institute Pasteur in Paris showed that sheep antiserum to the bacterium Vibrio cholera 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.

COMPONENTS OF COMPLEMENT SYSTEM:

Initiator complement components : These proteins initiate their respective complement cascades by binding to particular soluble or membrane-bound molecules. Once bound to their activating ligand, they undergo conformational alterations resulting in changes in their biological activity. The C1q complex,

Mannose Binding Lectin (MBL), and the ficolins are examples of initiator complement components.

Enzymatic mediator : Several complement components, (e.g., C1r, C1s, MASP2, and factor B) are proteolytic enzymes that cleave and activate other members of the complement cascade. Some of these proteases are activated by binding to other macromolecules and undergoing a conformational change. Others are inactive until cleaved by another protease enzyme and are thus termed zymogens: proteins that are activated by proteolytic cleavage. The two enzyme complexes that cleave complement components C3 and C5, respectively, are called the C3 and C5 convertases and occupy places of central importance in the complement cascades.

Membrane-binding components or opsonins : Upon activation of the complement cascade, several proteins are cleaved into two fragments, each of which then takes on a particular role. For C3 and C4, the larger fragments, C3b and C4b, serve as opsonins, enhancing phagocytosis by binding to microbial cells and serving as binding tags for phagocytic cells bearing receptors for C3b or C4b. As a general rule, the larger fragment of a cleaved complement component is designated with the suffi x “b,” and the smaller with the suffi x “a.” However, there is one exception to this rule: the larger, enzymatically active form of the C2 component is named C2a.

Inflammatory mediators : Some small complement fragments act as infl ammatory mediators. These fragments enhance the blood supply to the area in which they are released, by binding to receptors on endothelial cells lining the small blood vessels and inducing an increase in capillary diameter. Th ey also attract other cells to the site of tissue damage. Because such effects can be harmful in excess, these fragments are called anaphylatoxins, meaning substances that cause anaphylaxis (“against protection”). Examples include C3a, C5a, and C4a.

Membrane attack proteins : The proteins of the membrane attack complex (MAC) insert into the cell membranes of invading microorganisms and punch holes that result in lysis of the pathogen. Th e complement components of the MAC are C5b, C6, C7, C8, and multiple copies of C9.

Complement receptor proteins : Receptor molecules on cell surfaces bind complement proteins and signal specific cell functions. For example, some complement receptors such as CR1 bind to complement components such as C3b on the surface of pathogens, triggering phagocytosis of the C3-bound pathogen. Binding of the complement component C5a to C5aR receptors on neutrophils stimulates neutrophil degranulation and inflammation. Complement receptors are named with “R,” such as CR1, CR2, and C5aR.

Regulatory complement components : Host cells are protected from unintended complement-mediated lysis by the presence of membrane-bound as well as soluble regulatory proteins. Th ese regulatory proteins include factor I, which degrades C3b, and Protectin, which inhibits the formation of the MAC on host cells.

FUNCTIONS: 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.

COMPLEMENT ACTIVATION:Complement activation can take place by three major pathways viz., classical pathway, the alternative pathway or the lectin pathway.

CLASSICAL PATHWAY: Activation: Interaction of antibody with antigen initiates the classical pathway

of complement activation. Binding of IgM or IgG antibody to antigen causes a conformational change in the Fc region of the immunoglobulin molecule. This conformational change enables binding of the first component of the classic pathway, C1q. Each head of C1q may bind to a CH2 domain (within the Fc portion) of an antibody molecule. Upon binding to antibody, C1q undergoes a conformational change that leads to the sequential binding and activation of the serine proteases C1r and C1s. The C1qrs complex has enzymatic activity

for both C4 and C2, indicated by a horizontal bar as either C1qrs or abbreviated as C1s .

Production of C3 Convertase: Activation of C1qrs leads to the rapid cleavage and activation of components C4, C2, and C3.

Production of C5 Convertase: The binding of C 4 b 2b to C3b leads to the formation of the C 4 b 2b 3b complex. This complex, a C5 convertase initiates the construction of the membrane attack complex on the microbial surface.

Schematic diagram of Classical pathway for Complement activation

ALTERNATIVE PATHWAY: The alternative pathway does not require antigen-antibody complexes and is

initiated in most cases by cell-surface constituents that are foreign to the host.

In this 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.

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 substrate 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 C 3 bBb. The C 3 bBb complex has C3 convertase activity and thus is analogous to the C 4 b 2a complex in the classical pathway.

Schematic diagram of Alternative pathway of Complement activation

The C 3 bBb 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 C 3 bBb generates the C 3 bBb 3 b complex which exhibits C5 convertase activity, analogous to the C 4 b 2b 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 the latter binds to the antigenic surface.

LECTIN PATHWAY: 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.

Initiation of Lectin Pathway

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.

FORMATION OF MEMBRANE ATTACK COMPLEX:

Formation of Membrane Attack Complex (MAC)

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).

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, α and β. After binding of C5 to the nonenzymatic C3b component of the convertase, the amino terminus of the α 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.

The C5b component is extremely labile and becomes inactive within 2 minutes unless C6 binds to it and stabilizes its activity.

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.

Binding of C8 to membrane-bound C5b67 induces a conformational change in C8, so that it too undergoes a hydrophilic amphiphilic structural transition, exposing a hydrophobic region, which interacts with the plasma membrane. The C5b678 complex creates a small pore, 10 Å in diameter.

The final step in formation of 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. 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.

CONSEQUENCES OF COMPLEMENT ACTIVATION: CELL LYSIS:

The membrane-attack complex formed by complement activation can lyse gram-negative bacteria, parasites, viruses, erythrocytes, and nucleated cells.

Antibody and complement do play a role in host defense against viruses and are often crucial in containing viral spread during acute infection and in protecting against reinfection.

The complement system is generally quite effective in lysing gram-negative bacteria. However, some gram-negative bacteria and most gram-positive bacteria have mechanisms for evading complement-mediated damage.

For example, a few gram-negative bacteria can develop resistance to complement-mediated lysis that correlates with the virulence of the organism.

INFLAMMATION: Cleavage Products of Complement Components Mediate Inflammation. The smaller fragments resulting from complement cleavage, C3a, C4a, and

C5a, called anaphylatoxins, bind to receptors on mast cells and blood basophils and induce degranulation, with release of histamine and other pharmacologically active mediators.

The anaphylatoxins also induce smooth-muscle contraction and increased vascular permeability. Activation of the complement system thus results in influxes of fluid that carries antibody and phagocytic cells to the site of antigen entry.

C3a, C5a, and C5b67 can each induce monocytes and neutrophils to adhere to vascular endothelial cells, extravasate through the endothelial lining of the capillary, and migrate toward the site of complement activation in the tissues. C5a is most potent in mediating these processes, effective in picomolar quantities.

OPSONIZATION: C3b is the major opsonin of the complement system, although C4b and iC3b

also have opsonizing activity. The amplification that occurs with C3 activation results in a coating of C3b on immune complexes and particulate antigens.

Phagocytic cells, as well as some other cells, express complement receptors (CR1, CR3, and CR4) that bind C3b, C4b, or iC3b.

Antigen coated with C3b binds to cells bearing CR1. If the cell is a phagocyte (e.g., a neutrophil, monocyte, or macrophage), phagocytosis will be enhanced.

Activation of phagocytic cells by various agents, including C5a anaphylatoxin, has been shown to increase the number of CR1s from 5000 on resting phagocytes to 50,000 on activated cells, greatly facilitating their phagocytosis of C3b-coated antigen.

C3b targets the antigen directly to the phagocyte, enhancing the initiation of antigen processing and accelerating specific antibody production.

VIRAL NEUTRALIZTION:

For most viruses, the binding of serum antibody to the repeating subunits of the viral structural proteins creates particulate immune complexes ideally suited for complement activation by the classical pathway.

Some viruses (e.g., retroviruses, Epstein-Barr virus, Newcastle disease virus, and rubella virus) can activate the alternative, lectin, or even the classical pathway in the absence of antibody.

The complement system mediates viral neutralization by a number of mechanisms. Some degree of neutralization is achieved through the formation of larger viral aggregates, simply because these aggregates reduce the net number of infectious viral particles.

The binding of antibody and/or complement to the surface of a viral particle creates a thick protein coating that can be visualized by electron microscopy. This coating neutralizes viral infectivity by blocking attachment to susceptible host cells. The deposits of antibody and complement on viral particles also facilitate binding of the viral particle to cells possessing Fc or type 1 complement receptors (CR1).

In the case of phagocytic cells, such binding can be followed by phagocytosis and intracellular destruction of the ingested viral particle. Finally, complement is effective in lysing most, if not all, enveloped viruses, resulting in fragmentation of the envelope and disintegration of the nucleocapsid.

SOLUBILISATION OF IMMUNE COMPLEXES:

The importance of the complement system in clearing immune complexes is seen in patients with the autoimmune disease systemic lupus erythematosus (SLE).

These individuals produce large quantities of immune complexes and suffer tissue damage as a result of complement-mediated lysis and the induction of type II or type III hypersensitivity.

Although complement plays a significant role in the development of tissue damage in SLE, the paradoxical finding is that deficiencies in C1, C2, C4, and CR1 predispose an individual to SLE; indeed, 90% of individuals who completely lack C4 develop SLE.

Clearance of circulating Immune complexes

COMPLEMENT DEFICIENCIES: CLASSICAL PATHWAY DEFICIENCIES:

Deficiency of any of the components of the classical pathway (C1, C4, and C2) predisposes to a condition that closely resembles the autoimmune disease systemic lupus erythematosus (SLE), in which immune complexes become deposited in the kidney, skin, and blood vessels.

Deficiency of any of the C1 subunits (C1q, C1r, or C1s) invariably causes severe disease with typical SLE features including skin lesions and kidney damage. The disease usually manifests early in childhood and few patients reach adulthood.

C4 deficiency also causes severe SLE. Total deficiency of C4 is extremely rare because C4 is encoded by two separate genes (C4A and C4B), but partial deficiencies of C4 are relatively common and are associated with an increased incidence of SLE.

C2 deficiency is the commonest complement deficiency in Caucasians. Although it predisposes to SLE, the majority of C2-deficient individuals are healthy.

ALTERNATIVE PATHWAY DEFICIENCIES: Individuals with fB or fD deficiencies are susceptible to bacterial infections

and present with a history of severe recurrent infections with a variety of pyogenic (pus-forming) bacteria.

C3 is the cornerstone of complement, essential for all activation pathways and for MAC assembly, and is also the source of the major opsonic fragments C3b and iC3b. Individuals with C3 deficiency present early in childhood with a history of severe, recurrent bacterial infections affecting the respiratory system, gut, skin, and other organs. Untreated, all die before adulthood. When given broad-spectrum antibiotic prophylaxis, patients do reasonably well and survival into adulthood becomes the norm.

Properdin deficiency is inherited in an X-linked manner and is therefore seen exclusively in males. Boys deficient in properdin present with severe meningococcal meningitis, often with septicemia. The first attack is often fatal and survivors do not usually have recurrent infections because the acquisition

of anti-meningococcal antibody enables a response via the classical pathway in the next encounter.

TERMINAL PATHWAY DEFICIENCIES: Deficiencies of any of the terminal complement components (C5, C6, C7, C8,

or C9) predisposes to infections with Gram-negative bacteria, particularly those of the genus Neisseria. This genus includes the meningococci responsible for meningococcal meningitis and the gonococci responsible for gonorrhea.

Individuals with terminal pathway deficiencies usually present with meningitis, which is often recurrent and often accompanied by septicemia.

Deficiency of the classical pathway regulator C1inh is responsible for the syndrome hereditary angioedema (HAE). HAE is relatively common because the disease presents even in those heterozygous for the deficiency (i.e. it is an autosomal dominant disease).

C1inh regulates C1 and MBL in the complement system and also controls activation in the kinin pathway that leads to the generation of bradykinin and other active kinins.

Episodes of angioedema are often triggered in the skin or mucous membranes by minor trauma – occasionally stress may be sufficient to induce an attack. Swelling, which may be remarkable in severity, rapidly ensues as unregulated activation of the kinin and complement systems occurs in the affected area, inducing vascular leakiness. Swelling of mucous membranes in the mouth and throat may block the airways, leading to asphyxia.

COMPLEMENT RECEPTORS, OPSONINS AND ANAPHYLATOXINS:Complement activation is a danger signal that alerts the host to infection or injury and initiates appropriate responses. Complement activation products flag danger by physically binding to pathogens, immune complexes, and other toxic bodies, and by their release into the surrounding background; subsequent events require the interaction of these fragments with specific receptors present on a variety of cell types.

RECEPTORS FOR THE OPSONIC FRAGMENTS OF C3 AND C4: C3 is the most abundant complement component and the most important source

of complement fragments. C3b and its degradation products iC3b and C3d coat the target, a process called opsonization, thereby tagging the target for recognition by cells bearing complement receptors. CRl, described as a

complement regulator, is an atypical receptor in that it modifies its own ligand. CRl binds C3b (and C4b) coating immune complexes (ICs) via its binding sites in LHRs 1, 2, and 3, and catalyzes its cleavage by fl. Erythrocyte CR1 plays a critical role in IC handling; the C3b-coated IC binds CRI on the erythrocyte and is held transiently until the ligated C3b is cleaved by fI to iC3b/C3dg; binding affinity is lost and the complex is released, only to bind again via another C3b4. This dynamic binding permits the efficient sequestration of ICs on erythrocytes but without immobilizing them in a way that inhibits their efficient transfer to complement receptor-bearing tissue macrophages in the spleen and liver for final disposal. CRI on other cell types also operates by binding and processing C3b-coated ICs. On dendritic cells, CRI localizes opsonized ICs for presentation of antigen to T cells, while on B cells, C3b coating the IC binds CR1 and is cleaved to C3d, a ligand for another complement receptor, complement receptor 2 (CR2; CD21), clustered with CRI and the B-cell antigen receptor (BCR). Simultaneous engagement of CR2 and the BCR markedly lowers the threshold for antibody response.

CR2 is made up of either 15 or 16 SCR domains (alternative splicing removes SCRII in the smaller isoform), a transmembrane region, and a cytoplasmic tail with important roles in signalling. It is expressed on B cells, T-cell precursors and some mature T cells, dendritic cells, and some other cells involved in antigen presentation, basophils and epithelia. CR2 is a rather promiscuous receptor with numerous binding partners, including the IgE receptor (CD23), interferon-a, and the Epstein-Barr virus; its complement ligands are C3d or iC3b, binding at a single site in SCRI and SCR2. The key role of CR2 is to enhance the immune response to antigens contained within the IC. On B cells, CR2 is clustered with the B cell-specific signalling molecule CD19 in a complex held together by the tetraspanin CD81; this tri-molecular complex interacts with the BCR to modulate the B cell response to antigen. CR2 on dendritic cells contributes to IC trapping in lymph node germinal centres.

Complement receptor 3 (CR3; CD11b/CD18) is a heterodimer comprising a 165 kDa α-chain (CDllb) and a 95 kDa β-chain (CD18); it is a member of the β2-integrin family of leukocyte surface heterodimeric proteins sharing the common β-chain. CR3 is expressed on monocytes, neutrophils, mast cells, natural killer cells, dendritic cells, and some T cells. It is notable for its promiscuity, binding adhesion molecules (intercellular adhesion molecule [ICAM]-1 and -2), coagulation proteins, microbial products, and carbohydrate antigens; its principal complement ligand is iC3b, although it binds weakly to C3d. Binding of an iC3b-coated particle to CR3 on phagocytic cells triggers the phagocytic process, leading to the elimination of the opsonized particle. Other phagocytic receptors, including the Ig Fc receptors and CD14, certainly contribute to the phagocytic process, and there is continuing debate around which of these triggers are required for and most important in the phagocytic process.

Complement receptor 4 (CR4; CD11c/CD18), another β2-integrin expressed on myeloid cells, resembles CR3 with regard to distribution and complement ligand binding properties. It is possible that CR4 plays important roles in some dendritic cell processes, although it remains something of an enigma.

Complement receptor of the immunoglobulin superfamily is a recently described receptor for C3b/iC3b expressed exclusively on tissue-resident macrophages, including Kupffer cells in the liver. It plays important roles in the capture and clearance of opsonized ICs and pathogens from the circulation.

RECEPTORS FOR THE ANAPHYLACTIC FRAGMENTS OF C3AND C5:Receptors for the small anaphylatoxin (AT) fragments released from C3 and C5 during activation are extremely important players in the recruitment of inflammatory cells and in a growing list of other events. C3, C4, and C5 are structurally similar molecules and are all activated in similar ways: a single cut by the activating enzyme that releases a peptide, C3a, C4a, and CSa, respectively, from the amino terminus of the ex-chain. Current opinion is that there are no receptors for C4a and that, as a consequence, it has no biological function. In contrast, specific receptors exist for the AT molecules C3a and C5a, which mediate important biological roles. Three AT receptors have been described to date, the C3a receptor (C3aR), the CSa receptor (C5aR; CD88), and the C5a receptor-like 2 (C5L2). They are homologous molecules, members of the G-protein-coupled receptor family of heptaspan membrane proteins. C3aR is expressed on all myeloid cells and some nonmyeloid, including activated T cells, astrocytes, endothelia, epithelia, and smooth muscle. C3aR binds C3a with nanomolar affinity but does not bind C3adesArg, C5a, or CSadesArg. Upon binding of C3a, intracellular signaling cascades are triggered through activation of heterotrimeric G-proteins that in turn cause increased intracellular free calcium and other downstream events. CSaR binds C5a with nanomolar affinity and CSadesArg with tenfold lower affinity; it does not bind C3a/C3adesArg. C5aR is expressed on all myeloid cells and numerous nonmyeloid including endothelia, neurones, and astrocytes; expression on lymphoid cells has been suggested but current evidence does not support this.

RECEPTORS FOR C1q:

The most convincing candidate receptor is the Clq receptor for phagocytic enhancement (ClqRp; CD93), a heavily glycosylated 120 kDa transmembrane

sialoglycoprotein expressed on myeloid cells and endothelia. Its extracellular region comprises a collagen recognition domain followed by five epidermal growth factor (EGF) domains; the collagen recognition domain binds the collagenous regions of C1q, MBL, and another lectin molecule, surfactant protein A (SpA).It is suggested that this interaction is important in the clearance of apoptotic cells; these spontaneously bind C1q, MBL, and likely SpA, tagging them for binding C1qRp and phagocytic clearance. Antibodies against C1qRp inhibit apoptotic cell clearance in vitro, and mice deficient in ClqRp or Clq display delayed apoptotic cell clearance.Several members of the integrin family of cell adhesion molecules have been implicated as C1q binders; of the several collagen-binding integrins, a α 2 β1stands out for its capacity to bind C1q and MBL. A 60 kDa receptor for the collagenous tail of C1q, MBL, and other collectins, termed cClqR, was shown to be identical to calreticulin, a cytoplasmic chaperone protein, raising questions about how this molecule could associate with membranes and act as a C1q receptor. It is now suggested that calreticulin binds the extracellular domains of CD91 to create a receptor. Other molecules suggested to act as receptors for C1q (± MBL) include CR1 (binding the collagenous region through its membrane-proximal SCRs) and a 33 kDa protein that binds the C1q globular heads (gClqR). The physiological roles of these interactions are uncertain.