Complement

Understand the mechanisms of regulating complement activation and it products

Jules Bordet (1870-1961), discoverer of complement National Library of Medicine

Figure 1
Pathways of complement activation

COMPLEMENT FUNCTIONS

Historically, the term complement (C) was used to refer to a heat-labile serum component that was able to lyse bacteria (activity is destroyed (inactivated) by heating serum at 56 degrees C for 30 minutes). However, complement is now known to contribute to host defenses in other ways as well. Complement can opsonize bacteria for enhanced phagocytosis; it can recruit and activate various cells including polymorphonuclear cells (PMNs) and macrophages; it can participate in regulation of antibody responses and it can aid in the clearance of immune complexes and apoptotic cells. Complement can also have detrimental effects for the host; it contributes to inflammation and tissue damage and it can trigger anaphylaxis.

Complement comprises over 20 different serum proteins (see Table 1) that are produced by a variety of cells including, hepatocytes, macrophages and gut epithelial cells. Some complement proteins bind to immunoglobulins or to membrane components of cells. Others are proenzymes that, when activated, cleave one or more other complement proteins. Upon cleavage some of the complement proteins yield fragments that activate cells, increase vascular permeability or opsonize bacteria.

Complement nomenclature

Table 1. Proteins of the Complement system
Classical Pathway	Lectin Pathway	Alternative Pathway	Lytic Pathway
Activation Proteins: C1qrs, C2, C3, C4 Control Proteins: C1-INH, C4-BP	Mannan binding protein (MBP), mannan-asociated serine protease (MASP, MASP2)	C3, Factors B & D^, Properdin (P) Factors I^ & H, decay accelerating factor (DAF), Complement receptor 1(CR1), etc.	C5, C6, C7, C8, C9 Protein S
Components underlined acquire enzymatic activity when activated. Components marked with an asterisk have enzymatic activity in their native form.

Pathways of complement activation

Complement activation can be divided into four pathways (figure 1): the classical pathway, the lectin pathway, the alternative pathway and the membrane attack (or lytic) pathway. Both classical and alternative pathways lead to the activation of C5 convertase and result in the production of C5b which is essential for the activation of the membrane attack pathway.

CGAP
More detailed complement pathways from CGAP/Biocarta

Classical Pathway (Figure 2)

C1 activation

C1, a multi-subunit protein containing three different proteins (C1q, C1r and C1s), binds to the Fc region of IgG and IgM antibody molecules that have interacted with antigen. C1 binding does not occur to antibodies that have not complexed with antigen and binding requires calcium and magnesium ions. (N.B. In some cases C1 can bind to aggregated immunoglobulin [e.g. aggregated IgG] or to certain pathogen surfaces in the absence of antibody). The binding of C1 to antibody is via C1q and C1q must cross link at least two antibody molecules before it is firmly fixed. The binding of C1q results in the activation of C1r which in turn activates C1s. The result is the formation of an activated “C1qrs”, which is an enzyme that cleaves C4 into two fragments C4a and C4b.

C4 and C2 activation (generation of C3 convertase)

The C4b fragment binds to the membrane and the C4a fragment is released into the microenvironment. Activated “C1qrs” also cleaves C2 into C2a and C2b. C2a binds to the membrane in association with C4b, and C2b is released into the microenvironment. The resulting C4bC2a complex is a C3 convertase, which cleaves C3 into C3a and C3b.

C3 activation (generation of C5 convertase)

C3b binds to the membrane in association with C4b and C2a, and C3a is released into the microenvironment. The resulting C4bC2aC3b is a C5 convertase. The generation of C5 convertase is the end of the classical pathway.

Several of the products of the classical pathway have potent biological activities that contribute to host defenses. Some of these products may also have detrimental effects if produced in an unregulated manner. Table 2 summarizes the biological activities of classical pathway components.

Table 2. Biological Activity of classical pathway products
Component	Biological Activity
C2b	Prokinin; cleaved by plasmin to yield kinin, which results in edema
C3a	Anaphylotoxin; can activate basophils and mast cells to degranulate resulting in increased vascular permeability and contraction of smooth muscle cells, which may lead to anaphylaxis
C3b	Opsonin; promotes phagocytosis by binding to complement receptors Activation of phagocytic cells
C4a	Anaphylotoxin (weaker than C3a)
C4b	Opsonin; promotes phagocytosis by binding to complement receptors

If the classical pathway were not regulated there would be continued production of C2b, C3a, and C4a. Thus, there must be some way to regulate the activity of the classical pathway. Table 3 summarizes the ways in which the classical pathway is regulated.

Table 3. Regulation of the Classical Pathway
Component	Regulation
All	C1-INH; dissociates C1r and C1s from C1q
C3a	C3a inactivator (C3a-INA;Carboxypeptidase B); inactivates C3a
C3b	Factors H and I; Factor H facilitates the degradation of C3b by Factor I
C4a	C3-INA
C4b	C4 binding protein(C4-BP) and Factor I; C4-BP facilitates degradation of C4b by Factor I; C4-BP also prevents association of C2a with C4b thus blocking the formation of C3 convertase

The importance of C1-INH in regulating the classical pathway is demonstrated by the result of a deficiency in this inhibitor. C1-INH deficiencies are associated with the development of hereditary angioedema.

Generation of C3 convertase in the classical pathway

B Generation of C5 convertase in the classical pathway

C
Activation of C3 by the classical pathway

Figure 2

Figure 3 Lectin-initiated pathway

Lectin Pathway
The lectin pathway (figure 3) is very similar to the classical pathway. It is initiated by the binding of mannose-binding lectin (MBL) to bacterial surfaces with mannose-containing polysaccharides (mannans). Binding of MBL to a pathogen results in the association of two serine proteases, MASP-1 and MASP-2 (MBL-associated serine proteases). MASP-1 and MASP-2 are similar to C1r and C1s, respectively and MBL is similar to C1q. Formation of the MBL/MASP-1/MASP-2 tri-molecular complex results in the activation of the MASPs and subsequent cleavage of C4 into C4a and C4b. The C4b fragment binds to the membrane and the C4a fragment is released into the microenvironment. Activated MASPs also cleave C2 into C2a and C2b. C2a binds to the membrane in association with C4b and C2b is released into the microenvironment. The resulting C4bC2a complex is a C3 convertase, which cleaves C3 into C3a and C3b. C3b binds to the membrane in association with C4b and C2a and C3a is released into the microenvironment. The resulting C4bC2aC3b is a C5 convertase. The generation of C5 convertase is the end of the lectin pathway.

The biological activities and the regulatory proteins of the lectin pathway are the same as those of the classical pathway.

Figure 4 Spontaneous activation of C3 (C3 tick-over)

Alternative Pathway

The alternative pathway begins with the activation of C3 and requires Factors B and D and Mg⁺⁺ cation, all present in normal serum.

Amplification loop of C3b formation (Figure 4)

In serum there is low level spontaneous hydrolysis of C3 to produce C3i. Factor B binds to C3i and becomes susceptible to Factor D, which cleaves Factor B into Bb. The C3iBb complex acts as a C3 convertase and cleaves C3 into C3a and C3b. Once C3b is formed, Factor B will bind to it and becomes susceptible to cleavage by Factor D. The resulting C3bBb complex is a C3 convertase that will continue to generate more C3b, thus amplifying C3b production. If this process continues unchecked, the result would be the consumption of all C3 in the serum. Thus, the spontaneous production of C3b is tightly controlled.

Figure 5
Regulation of activated C3 by DAF

Figure 6 Regulation of activated C3 by Cr1

Figure 7 Stabilization of C3 convertase

Figure 8
Stabilized C5 convertase of the alternative pathway

Control of the amplification loop (Figures 5 and 6)

As spontaneously produced C3b binds to autologous host membranes, it interacts with DAF (decay accelerating factor), which blocks the association of Factor B with C3b thereby preventing the formation of additional C3 convertase. In addition, DAF accelerates the dissociation of Bb from C3b in C3 convertase that has already formed, thereby stopping the production of additional C3b. Some cells possess complement receptor 1 (CR1). Binding of C3b to CR1 facilitates the enzymatic degradation of C3b by Factor I. In addition, binding of C3 convertase (C3bBb) to CR1 also dissociates Bb from the complex. Thus, in cells possessing complement receptors, CR1 also plays a role in controlling the amplification loop. Finally, Factor H can bind to C3b bound to a cell or in the in the fluid phase and facilitate the enzymatic degradation of C3b by Factor I. Thus, the amplification loop is controlled by either blocking the formation of C3 convertase, dissociating C3 convertase, or by enzymatically digesting C3b. The importance of controlling this amplification loop is illustrated in patients with genetic deficiencies of Factor H or I. These patients have a C3 deficiency and increased susceptibility to certain infections.

Stabilization of C convertase by activator (protector) surfaces (Figure 7)

            When bound to an appropriate activator of the alternative pathway, C3b will bind Factor B, which is enzymatically cleaved by Factor D to produce C3 convertase (C3bBb). However, C3b is resistant to degradation by Factor I and the C3 convertase is not rapidly degraded, since it is stabilized by the activator surface. The complex is further stabilized by properdin binding to C3bBb. Activators of the alternate pathway are components on the surface of pathogens and include: LPS of Gram-negative bacteria and the cell walls of some bacteria and yeasts. Thus, when C3b binds to an activator surface, the C3 convertase formed will be stable and continue to generate additional C3a and C3b by cleavage of C3.

            Generation of C5 convertase (Figure 10)

            Some of the C3b generated by the stabilized C3 convertase on the activator surface associates with the C3bBb complex to form a C3bBbC3b complex. This is the C5 convertase of the alternative pathway. The generation of C5 convertase is the end of the alternative pathway. The alternative pathway can be activated by many Gram-negative (most significantly, Neisseria meningitidis and N. gonorrhoea), some Gram-positive bacteria and certain viruses and parasites, and results in the lysis of these organisms. Thus, the alternative pathway of C activation provides another means of protection against certain pathogens before an antibody response is mounted. A deficiency of C3 results in an increased susceptibility to these organisms. The alternate pathway may be the more primitive pathway and the classical and lectin pathways probably developed from it.

Remember that the alternative pathway provides a means of non-specific resistance against infection without the participation of antibodies and hence provides a first line of defense against a number of infectious agents.

Many gram negative and some gram positive bacteria, certain viruses, parasites, heterologous red cells, aggregated immunoglobulins (particularly, IgA) and some other proteins (e.g. proteases, clotting pathway products) can activate the alternative pathway. One protein, cobra venom factor (CVF), has been extensively studied for its ability to activate this pathway.

Figure 9 The lytic pathway

Membrane Attack (Lytic) Pathway (figure 9)

C5 convertase from the classical (C4b2a3b), lectin (C4b2a3b) or alternative (C3bBb3b) pathway cleaves C5 into C5a and C5b. C5a remains in the fluid phase and the C5b rapidly associates with C6 and C7 and inserts into the membrane. Subsequently C8 binds, followed by several molecules of C9. The C9 molecules form a pore in the membrane through which the cellular contents leak and lysis occurs. Lysis is not an enzymatic process; it is thought to be due to physical damage to the membrane. The complex consisting of C5bC6C7C8C9 is referred to as the membrane attack complex (MAC).

C5a generated in the lytic pathway has several potent biological activities. It is the most potent anaphylotoxin. In addition, it is a chemotactic factor for neutrophils and stimulates the respiratory burst in them and it stimulates inflammatory cytokine production by macrophages. Its activities are controlled by inactivation by carboxypeptidase B (C3-INA).

Some of the C5b67 complex formed can dissociate from the membrane and enter the fluid phase. If this were to occur it could then bind to other nearby cells and lead to their lysis. The damage to bystander cells is prevented by Protein S (vitronectin). Protein S binds to soluble C5b67 and prevents its binding to other cells.

Figure 10 Regulation of C1rs (C4 convertase) by C1-INH

Biologically active products of Complement activation

Activation of complement results in the production of several biologically active molecules which contribute to resistance, anaphylaxis and inflammation.

Kinin production

C2b generated during the classical pathway of C activation is a prokinin which becomes biologically active following enzymatic alteration by plasmin. Excess C2b production is prevented by limiting C2 activation by C1 inhibitor (C1-INH) also known as serpin which displaces C1rs from the C1qrs complex (Figure 10). A genetic deficiency of C1-INH results in an overproduction of C2b and is the cause of hereditary angioneurotic edema. This condition can be treated with Danazol which promotes C1-INH production or with ε-amino caproic acid which decreases plasmin activity.

Figure 11
Complement proteins bind to the surface of microorganisms and promote phagocytosis via complement receptors

Figure 12
Biological effects of C5a

Anaphylotoxins

C4a, C3a and C5a (in increasing order of activity) are all anaphylotoxins which cause basophil/mast cell degranulation and smooth muscle contraction. Undesirable effects of these peptides are controlled by carboxypeptidase B (C3a-INA).

Chemotactic Factors

C5a and MAC (C5b67) are both chemotactic. C5a is also a potent activator of neutrophils, basophils and macrophages and causes induction of adhesion molecules on vascular endothelial cells (figure 12).

Opsonins

C3b and C4b in the surface of microorganisms attach to C-receptor (CR1) on phagocytic cells and promote phagocytosis (figure 11).

Other Biologically active products of C activation

Degradation products of C3 (iC3b, C3d and C3e) also bind to different cells by distinct receptors and modulate their functions.

In summary, the complement system takes part in both specific and non-specific resistance and generates a number of products of biological and pathophysiological significance (Table 4).

There are known genetic deficiencies of most individual C complement components, but C3 deficiency is most serious and fatal. Complement deficiencies also occur in immune complex diseases (e.g., SLE) and acute and chronic bacterial, viral and parasitic infections.

Table 4. Activities of Complement Activation Products and their Control Factors
Fragment	Activity	Effect	Control Factor (s)
C2a	Prokinin, accumulation of fluids	Edema	C1-INH
C3a	Basophil and mast cells degranulation; enhanced vascular permeability, smooth muscle contraction	Anaphylaxis	C3a-INA
C3b	Opsonin, phagocyte activation	Phagocytosis	Factors H and I
C4a	Basophil and mast cells degranulation; enhanced vascular permeability, smooth muscle contraction	Anaphylaxis (least potent)	C3a-INA
C4b	Opsonin	Phagocytosis	C4-BP and Factor I
C5a	Basophil and mast cells degranulation; enhanced vascular permeability, smooth muscle contraction	Anaphylaxis (most potent)	C3a-INA
C5a	Chemotaxis, stimulation of respiratory burst, activation of phagocytes, stimulation of inflammatory cytokines	Inflammation	C3a-INA
C5bC6C7	Chemotaxis	Inflammation	Protein S (vitronectin)
C5bC6C7	Attaches to other membranes	Tissue damage	Protein S (vitronectin)

You have learned

The proteins of the complement system

The differences and similarities among the different pathways of C3 activation

The significance of the different pathways in specific and nonspecific immunity

The role of different complement activation products in amplification of nonspecific and specific immunity and inflammation

Table 5. Complement deficiencies and disease
Pathway/Component	Disease	Mechanism
Classical Pathway
C1INH	Hereditary angioedema	Overproduction of C2b (prokinin)
C1, C2, C4	Predisposition to SLE	Opsonization of immune complexes help keep them soluble, deficiency results in increased precipitation in tissues and inflammation
Lectin Pathway
MBL	Susceptibility to bacterial infections in infants or immunosuppressed	Inability to initiate the lectin pathway
Alternative Pathway
Factors B or D	Susceptibility to pyogenic (pus-forming) bacterial infections	Lack of sufficient opsonization of bacteria
C3	Susceptibility to bacterial infections	Lack of opsonization and inability to utilize the membrane attack pathway
C5, C6, C7 C8, and C9	Susceptibility to Gram-negative infections	Inability to attack the outer membrane of Gram-negative bacteria
Properdin (X-linked)	Susceptibility meningococcal meningitis	Lack of opsonization of bacteria
Factors H or I	C3 deficiency and susceptibility to bacterial infections	Uncontrolled activation of C3 via alternative pathway resulting in depletion of C3

Return to the Immunology Section of Microbiology and Immunology On-line

This page last changed on Saturday, December 09, 2017
Page maintained by Richard Hunt