Activity	Responsible Complement Protein
Host Defense Against Infections
Opsonization	Covalently bonded fragments of C3 and C4
Chemotaxis and leukocyte activation	C5a, C3a, and C4a; anaphylatoxin leukocyte receptors
Lysis of bacterial and mammalian cells	C5-C9 membrane attack complex
Interface Between Innate and Adaptive Immunity
Augmentation of antibody	C3b and C4b bound to immune complexes and to antigen
Responses	C3 receptors on B cells and antigen-presenting cells
Enhancement of immunologic memory	C3b and C4b bound to immune complexes and to antigen; C3 receptors on follicular dendritic cells
Disposal of Waste
Clearance of immune complexes from tissues	C1q; covalently bonded fragments of C3 and C4
Clearance of apoptotic cells

Adapted from Walport MJ: Complement, N Engl J Med 344:1058–1065, 2001.

Table 5-2

Initiators of Three Complement Activation Pathways

Pathway	Initiators
Classic	Immune complexes Apoptotic cells Certain viruses and gram-negative bacteria C-reactive protein bound to ligand
Alternate	Various bacteria, fungi, viruses, or tumor cells
Mannose-binding lectin	Microbes with terminal mannose groups

Adapted from Walport MJ: Complement, N Engl J Med 344:1058–1065, 2001.

1. Classic pathway

2. Alternative pathway

3. Mannose-binding lectin pathway

The three pathways (Fig. 5-1) converge at the point of cleavage of C3 to C3b, the central event of the common final pathway, which in turn leads to the activation of the lytic complement sequence, C5 through C9, and cell destruction (Fig. 5-2).

Figure 5-1 Early steps of complement activation.
The steps in the activation of the alternative, classical, and lectin pathways are shown. Note the sequence of events is similar in all three pathways, although they differ in their requirement for antibody and in the proteins used. (From Abbas AK, Lichtman AH: Basic immunology: functions and disorders of the immune system, updated edition, ed 3, Philadelphia, 2011, Saunders.)

Figure 5-2 Late steps of complement activation.
A, The late steps of complement activation start after the formation of the C5 convertase and are identical in the alternative and classical pathways. Products generated in the late steps induce inflammation (C5a) and cell lysis (the membrane attack complex [MAC]). B, The properties of the proteins of the late steps of complement activation are listed. (From Abbas AK, Lichtman AH: Basic immunology: functions and disorders of the immune system, updated edition, ed 3, Philadelphia, 2011, Saunders.)

Classic Pathway

The classic complement pathway is one of the major effector mechanisms of antibody-mediated immunity. The principal components of the classic pathway are C1 through C9. The sequence of component activation—C1, 4, 2, 3, 5, 6, 7, 8, and 9—does not follow the expected numeric order.

C3 is present in the plasma in the largest quantities; fixation of C3 is the major quantitative reaction of the complement cascade. Although the principal source of synthesis of complement in vivo is debatable, the majority of the plasma complement components are made in hepatic parenchymal cells, except for C1 (a calcium-dependent complex of the three glycoproteins C1q, C1r, and C1s), which is primarily synthesized in the epithelium of the gastrointestinal and urogenital tracts.

The classic pathway has three major stages:

1. Recognition

2. Amplification of proteolytic complement cascade

3. Membrane attack complex (MAC)

Recognition

The recognition unit of the complement system is the C1 complex—C1q, C1r, and C1s, an interlocking enzyme system. In the classic pathway, the first step is initiation of the pathway triggered by recognition by complement factor C1 of antigen-antibody complexes on the cell surface. When C1 complex interacts with aggregates of immunoglobulin G (IgG) with antigen on a cell’s surface, two C1-associated proteases, C1r and C1s, are activated. A single IgM molecule is potentially able to fix C1, but at least two IgG molecules are required for this purpose. The amount of C1 fixed is directly proportional to the concentration of IgM antibodies, although this is not true of IgG molecules. C1s is weakly proteolytic for free intact C2, but is highly active against C2 that has complexed with C4b molecules in the presence of magnesium (Mg²⁺) ions. This reaction will occur only if the C4bC2 complex forms close to the C1s.

The resultant C2a fragment joins with C4b to form the new C4bC2a enzyme, or classic pathway C3 convertase. The catalytic site of the C4bC2a complex is probably in the C2a peptide. A smaller C2b fragment from the C2 component is lost to the surrounding environment.

Amplification of Proteolytic Complement Cascade

Once C1s is activated, the proteolytic complement cascade is amplified on the cell membrane through sequential cleavage of complement factors and recruitment of new factors until a cell surface complex containing C5b, C6, C7, and C8 is formed.

The complement cascade reaches its full amplitude at the C3 stage, which represents the heart of the system. The C4bC2a complex, the classic pathway C3 convertase, activates C3 molecules by splitting the peptide, C3 anaphylatoxin, from the N-terminal end of the peptide of C3. This exposes a reactive binding site on the larger fragment, C3b. Consequently, clusters of C3b molecules are activated and bound near the C4bC2a complex. Each catalytic site can bind several hundred C3b molecules, even though the reaction is very efficient because C3 is present in high concentration. Only one C3b molecule combines with C4bC2a to form the final proteolytic complex of the complement cascade.

Membrane Attack Complex

The membrane attack complex (MAC) is a unique system that builds up a lipophilic complex in cell membranes from several plasma proteins. To initiate C5b fixation and the MAC, C3b splits C5a from the alpha chain of C5. No further proteinases are generated in the classic complement sequence. Other bound C3b molecules not involved in the C4b2a3b complex form an opsonic macromolecular coat on the erythrocyte or other target, which renders it susceptible to immune adherence by C3b receptors on phagocytic cells.

When fully assembled in the correct proportions, C7, C6, C5b, and C8 form the MAC (see Fig. 5-2, inset). The C5bC6 complex is hydrophilic but, with the addition of C7, it has additional detergent and phospholipid-binding properties as well. The presence of hydrophobic and hydrophilic groups within the same complex may account for its tendency to polymerize and form small protein micelles (a packet of chain molecules in parallel arrangement). It can attach to any lipid bilayer within its effective diffusion radius, which produces the phenomenon of reactive lysis on innocent so-called bystander cells. Once membrane bound, C5bC6C7 is relatively stable and can interact with C8 and C9.

The C5bC6C7C8 complex polymerizes C9 to form a tubule (pore), which spans the membrane of the cell being attacked, allowing ions to flow freely between the cellular interior and exterior. By complexing with C9, the osmotic cytolytic reaction is accelerated. This tubule is a hollow cylinder with one end inserted into the lipid bilayer and the other projecting from the membrane. A structure of this form can be assumed to disturb the lipid bilayer sufficiently to allow the free exchange of ions and water molecules across the membrane. Ions flow out, but large molecules stay in, causing water to flood into the cell. The consequence in a living cell is that the influx of sodium (Na⁺) ions and H₂O leads to disruption of osmotic balance, which produces cell lysis.

Alternative Pathway

The alternative pathway shows points of similarity with the classic sequence. Both pathways generate a C3 convertase that activates C3 to provide the pivotal event in the final common pathway of both systems. However, in contrast to the classic pathway, which is initiated by the formation of antigen-antibody reactions, the alternate complement pathway is predominantly a non–antibody-initiated pathway.

Microbial and mammalian cell surfaces can activate the alternative pathway in the absence of specific antigen-antibody complexes. Factors capable of activating the alternative pathway include inulin, zymosan (polysaccharide complex from surface of yeast cells), bacterial polysaccharides and endotoxins, and the aggregated IgG2, IgA, and IgE. In paroxysmal nocturnal hemoglobinuria (PNH), the patient’s erythrocytes act as an activator and result in excessive lysis of these erythrocytes. This nonspecific activation is a major physiologic advantage because host protection can be generated before the induction of a humoral immune response.

A key feature of the alternative pathway is that the first three proteins of the classic activation pathway—C1, C4, and C2—do not participate in the cascade sequence. The C3a component is considered to be the counterpart of C2a in the classic pathway. C2 of the classic pathway structurally resembles factor B of the alternative pathway. The omission of C1, C4, and C2 is possible because activators of the alternative pathway catalyze the conversion of another series of normal serum proteins, which leads to the activation of C3. It was previously believed that properdin, a normal protein of human serum, was the first protein to function in the alternative pathway; thus, the pathway was originally named after this protein.

The uptake of factor B onto C3b occurs when C3b is bound to an activator surface. However, C3b in the fluid phase or attached to a nonactivator surface will preferentially bind to and therefore prevent C3b,B formation. C3b and factor B combine to form C3b,B, which is converted into an active C3 convertase, C3b,Bb. This results from the loss of a small fragment, Ba (glycine-rich α₂-globulin believed to be physiologically inert), through the action of the enzyme, factor D. The C3b,Bb complex is able to convert more C3 to C3b, which binds more factor B and the feedback cycle continues.

The major controlling event of the alternative pathway is factor H, which prevents the association between C3b and factor B. Factor H blocks the formation of C3b,Bb, the catalytically active C3 convertase of the feedback loop. Factor H (formerly β₁-H) competes with factor B for its combining site on C3b, eventually leading to C3 inactivation. Factors B and H apparently occupy a common site on C3b. The factor that is preferentially bound to C3b depends on the nature of the surface to which C3b is attached. Polysaccharides are called activator surfaces and favor the uptake of factor B on the chain of C3b, with the corresponding displacement of factor H. In this situation, binding of factor H is inhibited, and consequently factor B will replace H at the common binding site. When factor H is excluded, C3b is thought to be formed continuously in small amounts. Another controlling point in the amplification loop depends on the stability of the C3b,Bb convertase. Ordinarily, C3b,Bb decays because of the loss of Bb, with a half-life of approximately 5 minutes. However, if properdin (P) binds to C3b,Bb, forming C3b,BbP, the half-life is extended to 30 minutes.

The association of numerous C3b units, factor Bb, and properdin on the surface of an aggregate of protein or the surface of a microorganism has potent activity as a C5 convertase. With the cleavage of C5, the remainder of the complement cascade continues as in the classic pathway.

Mannose-Binding Lectin Pathway

Mannose-binding lectin is a member of a family of calcium-dependent lectins, the collectins (collagenous lectins), and is homologous in structure to C1q. Mannose-binding lectin, a pattern recognition molecule of the innate immune system, binds to arrays of terminal mannose groups on a variety of bacteria.

A deficiency of mannose-binding lectin is caused by one of three point mutations in its gene, each of which reduces levels of the lectin. After the discovery that the binding of mannose-binding lectin to mannose residues can initiate complement activation, the mannose-binding lectin–associated serine protease (MASP) enzymes were discovered. MASP activates complement by interacting with two serine proteases called MASP1 and MASP2. These components make up the mannose-binding lectin pathway.

BiologicAl Functions of Complement Proteins

The biological functions of the complement system fall into the following two general categories:

1. Cell lysis by the membrane attack complex (MAC)

2. Biological effects of proteolytic fragments of complement

The first category is the situation in which the MAC leads to osmotic lysis of a cell. The second category encompasses other effects of complement in immunity and inflammation that are mediated by the proteolytic fragments generated during complement activation. These fragments may remain bound to the same cell surfaces at which complement has been activated or may be released into the blood or extracellular fluid. In either situation, active fragments mediate their effects by binding to specific receptors expressed on various types of cells, including phagocytic leukocytes and the endothelium (Table 5-3).

Table 5-3

Selected Complement Components and Functions

Complement Component(s)	Function
C5-C9	Lysis of cells
C3B, IC3B	Opsonization in phagocytosis
C5A >C3A >>C4A	Anaphylatoxins/inflammation (vascular responses)
C5A	Polymorphonuclear leukocyte activation
Classic complement pathway, C3B, ?iC3b, C3dg	Immune complex removal B-lymphocyte activation

In contrast, the absence of an integral component of the classic, alternative, or terminal lytic pathways can lead to decreased complement activation and a lack of complement-mediated biological functions.

Alterations in Complement Levels

The complement system can cause significant tissue damage in response to abnormal stimuli. Biological effects of complement activation can occur as a reaction to persistent infection or an autoantibody response to self antigens. In these infectious or autoimmune conditions, the inflammatory or lytic effects of complement may contribute significantly to the pathology of the disease.

Complement activation is also associated with intravascular thrombosis, which leads to ischemic injury to tissues. Complement levels may be abnormal in certain disease states (e.g., rheumatoid arthritis, systemic lupus erythematosus [SLE]) and in some genetic disorders.

Elevated Complement Levels

The complement level can be elevated in many inflammatory conditions. Increased complement levels are often associated with inflammatory conditions, trauma, or acute illness such as myocardial infarction because separate complement components (e.g., C3) are acute-phase proteins. However, these elevations are common and nonspecific. Therefore, increased levels are of limited clinical significance.

Decreased Complement Levels

Low levels of complement suggest one of the following biological effects:

• Complement has been excessively activated recently.

• Complement is currently being consumed.

• A single complement component is absent because of a genetic defect.

Specific component deficiencies are associated with a variety of disorders (Table 5-4). Deficiencies of complement account for a small percentage of primary immunodeficiencies (<2%), but depression of complement levels frequently coexists with SLE and other disorders associated with an immunopathologic process (Box 5-1).

Table 5-4

Complement Deficiency in Human Beings

Deficiency	Associated Disease
C1q	SLE-like syndrome; decreased secondary to agammaglobulinemia
C1r	SLE-like syndrome; dermatomyositis, vasculitis, recurrent infections and chronic glomerulo-nephritis, necrotizing skin lesions, arthritis
C1s	SLE, SLE-like syndrome
C1 INH	Hereditary angioedema, lupus nephritis
C2	Recurrent pyogenic infections, SLE, SLE-like syndrome, discoid lupus, membranoproliferative glomerulonephritis, dermatomyositis, synovitis, purpura, Henoch-Schönlein purpura, hypertension, Hodgkin’s disease, chronic lymphocytic leukemia, dermatitis herpetiformis, polymyositis
C3	Recurrent pyogenic infections, SLE-like syndrome, arthralgias, skin rash
C3 inactivator	Recurrent pyogenic infections, urticaria
C4	SLE-like syndrome, SLE, dermatomyositis-like syndrome, vasculitis
C5	Neisseria infections, SLE
C5 dysfunction	Leiner’s disease, gram-negative skin and bowel infection
C6	Neisseria infections, SLE, Raynaud’s phenomenon, scleroderma-like syndrome, vasculitis
C7	Neisseria infections, SLE, Raynaud’s phenomenon, scleroderma-like syndrome, vasculitis
C8	Neisseria infections, xeroderma pigmentosa, SLE-like syndrome

Modified from Colten HR, Rosen FS: Complement deficiencies annual review of immunology, 10:809-834, 1992 and Nusinow SR, Zuraw BL, Curd JG: The hereditary and acquired deficiencies of complement, Med Clin North Am 69:487-504, 1985.

Deficiencies in any of the protein components of complement are usually caused by a genetic defect that leads to abnormal patterns of complement activation. If regulatory components are absent, excess activation may occur at the wrong time or at the wrong site. The potential consequences of increased activation are excess inflammation and cell lysis and consumption of complement components.

Hypocomplementemia can result from the complexing of IgG or IgM antibodies capable of activating complement. Depressed values of complement are associated with diseases that give rise to circulating immune complexes. Because of the rapid normal turnover of the complement proteins—within 1 or 2 days of the cessation of complement activation by immune complexes—complement levels return to normal rapidly.

The following three types of complement deficiency can cause increased susceptibility to pyogenic infections:

1. Deficiency of the opsonic activities of complement

2. Any deficiency that compromises the lytic activity of complement

3. Deficient function of the mannose-binding lectin pathway

Increased susceptibility to pyogenic bacteria (e.g., Haemophilus influenzae, Streptococcus pneumoniae) occurs in patients with defects of antibody production, complement proteins of the classic pathway, or phagocyte function. The sole clinical association between inherited deficiency of MAC components and infection is with neisserial infection, particularly Neisseria meningitidis. Low levels of mannose-binding lectin in young children with recurrent infections suggest that the mannose-binding lectin pathway is important during the interval between the loss of passively acquired maternal antibody and the acquisition of a mature immunologic repertoire of antigen exposure.

Diagnostic Evaluation

During immune complex reactions, certain complement proteins become physically bound to the tissue in which the immunologic reaction is occurring. These proteins can be demonstrated in tissue by appropriate immunopathologic stains. The most frequent evaluation of complement is by serum or plasma assay (Table 5-5). Complement components (e.g., C3 and C4) can be assessed by nephelometry. These assays are useful for the diagnosis and monitoring of patients.

Table 5-5

Interpretation of Complement Activation by Individual Components

Complement Determination	Classic Pathway	Alternative Pathway	Improper Specimen^∗	Inflammation
C3	Decreased	Decreased	Normal	Increased
C4	Decreased	Normal	Decreased	Increased