The complement system

The complement system consists of a tightly regulated network of proteins that play an important role in host defense and inflammation. Complement activation results in opsonization of pathogens and their removal by phagocytes, as well as cell lysis. Inappropriate complement activation and complement deficiencies are the underlying cause of the pathophysiology of many diseases such as systemic lupus erythematosus and asthma. This review represents an overview of the complement system in an effort to understand the beneficial as well as harmful roles it plays during inflammatory responses.     

Quantitative complement fixation

Summary In agreement with the investigations ofKent et al. (4), it was found that blood collected in a modifiedAlsevers solution remains constant for a period of at least 14 days.We also succeeded in proving the same with regard to complement, stored according toRichardson (9) and with regard to amboceptor prepared according to the directions of the New York State Department of Health and stored in an equal volume of glycerol at 4° C.The cheap Eel colorimeter proved very suitable for the standardization of the erythrocyte-suspension and the measurement of the percentage of haemolysis.Success was not obtained by means of the method, used in this investigation, to keep the amount of complement, which under the conditions of the test causes haemolysis of 50% of erythrocytes, so constant, as to make daily complement titration unnecessary.     

Inherited complement deficiencies

Isolated genetic deficiencies of individual components of the complementary system have been described in man for all the components of the classical pathway and the membrane attack complex as well as for Factor I, Factor H and properdin. It is only for Factor B and Factor D of the alternative pathway that homozygous deficiency states are not so far known. Complement deficiency states provide the most direct way of looking at the role of the complement system in vivo and emphasize the importance of complement in resistance to bacterial infection and in particular to infection with Neisseria. This association is not unexpected since in vitro studies have shown complement to be an efficient enhancer of phagocytosis and inflammation. The particularly frequent occurrence of neisserial infection may be ascribed to the ability of these organisms to survive in phagocytic cells so that the plasma cytolytic activity provided by complement is needed to kill them. On the other hand the strong association between complement deficiencies and immune-complex diseases--especially systemic lupus erythematosus--was unexpected and seems paradoxical in view of the large part played by complement in the pathogenesis of immune complex mediated tissue damage. The paradox can be explained in part by the necessity for an intact complement system in the solubilization and the proper handling of immune complexes. It is also likely that complement deficiency can allow the persistence of low virulence organisms that produce disease solely by an immune complex mechanism. Recently described deficiencies of complement receptors and their effects in vivo are described.     

Bone marrow determination of complement dependent and complement independent cellular cytotoxicity

Adoptive transfer of both bone marrow and thymus cells is needed in lethally irradiated syngeneic mice to elicit CICC and CDCC responses against SRBC as assayed by the ⁵¹Cr-release cytotoxic test. The contribution of thymocytes to both CDCC and CICC was assessed by limiting dilution assays in BDF₁ and CDF₁ mice. Irradiated mice were reconstituted with a large number of bone marrow cells and graded limiting numbers of thymocytes, and were then immunized with SRBC. The limiting dilution plots conformed to the Poisson Model for both types of responses and both strains of mice. The numbers of thymocytes required for about 63% of the recipient spleens to be positive (i.e., the inoculum sizes containing one detectable antigen reactive unit) were similar for both CDCC and CICC ($\text{1}\text{1.8}\text{× 10}{}^7$ thymocytes) in both strains. Chi-square tests, at the 0.01 level of significance, were incompatible with the hypothesis that CICC and CDCC are independent of each other. Limiting dilutions of bone marrow cells with larger numbers of thymus cells, using CDF₁ mice, showed a non-Poisson curve for both CICC and CDCC. Chi-square values were compatible with the hypothesis of independent assortment of responses, as if the potential of the majority of precursors were restricted to either CICC or CDCC, but not to both. In contrast, BDF₁ mice showed a Poisson curve for CDCC and a non-Poisson curve for CICC. Chi-square values were also compatible with independent assortment of responses.     

Identification of complement regulatory domains in vaccinia virus complement control protein

Vaccinia virus encodes a homolog of the human complement regulators named vaccinia virus complement control protein (VCP). It is composed of four contiguous complement control protein (CCP) domains. Previously, VCP has been shown to bind to C3b and C4b and to inactivate the classical and alternative pathway C3 convertases by accelerating the decay of the classical pathway C3 convertase and (to a limited extent) the alternative pathway C3 convertase, as well as by supporting the factor I-mediated inactivation of C3b and C4b (the subunits of C3 convertases). In this study, we have mapped the CCP domains of VCP important for its cofactor activities, decay-accelerating activities, and binding to the target proteins by utilizing a series of deletion mutants. Our data indicate the following. (i) CCPs 1 to 3 are essential for cofactor activity for C3b and C4b; however, CCP 4 also contributes to the optimal activity. (ii) CCPs 1 to 2 are enough to mediate the classical pathway decay-accelerating activity but show very minimal activity, and all the four CCPs are necessary for its efficient activity. (iii) CCPs 2 to 4 mediate the alternative pathway decay-accelerating activity. (iv) CCPs 1 to 3 are required for binding to C3b and C4b, but the presence of CCP 4 enhances the affinity for both the target proteins. These results together demonstrate that the entire length of the protein is required for VCP's various functional activities and suggests why the four-domain structure of viral CCP is conserved in poxviruses.     

Intravascular schistosomes and complement

Schistosomes are exposed to a variety of immunological effectors, such as host complement, in the bloodstream of their definitive hosts. The parasites are reported to possess a plethora of regulatory proteins, including molecules acquired from the host, which impede the complement cascade. Evidence for the presence of a surface C2-binding protein, a C3-binding protein and a C8- and C9-binding protein has been reported. In addition, a surface Fc receptor might bind immunoglobulin and limit its ability to fix complement. However, the actual protective role of these proteins in vivo remains unresolved.     

Comparison between complement and melittin hemolysis: anti-melittin antibodies inhibit complement lysis

A comparison is made between the hemolytic actions of melittin and the ninth component of complement (C9). Melittin and C9 produce "pores" of similar effective radius in erythrocytes under standardized conditions, and their hemolytic action is suppressed by metal ions at similar concentrations, suggesting a common mechanism. Polyclonal anti-melittin immunoglobulin G (IgG) produced in rabbits retards hemolysis mediated by human C9 in a specific manner. Such antibodies react in several immunoassays with human and monkey C9 but not with C9 from lower animals, and no inhibition of lysis mediated by C9 molecules from these animals is observed. Thus, it is unlikely that anti-melittin IgG reacts with a structural element, such as an amphipathic helix, on human C9 since such structures are also predicted to exist in other C9 molecules. Human C9 and melittin block cross-reactivity in a dose-dependent manner, and anti-melittin IgG recognizes an epitope located between amino acid residues 245 and 390 of human C9 on "Western" blots. Comparison of the melittin and human C9 sequences indicates two regions of complete homology, a tetrapeptide at positions 292-295, and a pentapeptide at positions 527-531 in human C9, corresponding to residues 8-16 in melittin. Inhibition of hemolysis is not caused by blocking of C9 binding to the C5b-8 complex; rather the antibody must dissociate from the bound C9 before lysis ensues, indicating that it interferes with a postbinding event. It is proposed that anti-melittin binds to a conformational epitope on native, folded human C9 and thereby retards unfolding of the molecule, which is required for membrane insertion and hemolysis.     

Role of complement and complement membrane attack complex in laser-induced choroidal neovascularization

Choroidal neovascularization (CNV), or choroidal angiogenesis, is the hallmark of age-related macular degeneration and a leading cause of visual loss after age 55. The pathogenesis of new choroidal vessel formation is poorly understood. Although inflammation has been implicated in the development of CNV, the role of complement in CNV has not been explored experimentally. A reliable way to produce CNV in animals is to rupture Bruch's membrane with laser photocoagulation. A murine model of laser-induced CNV in C57BL/6 mice revealed the deposition of C3 and membrane attack complex (MAC) in the neovascular complex. CNV was inhibited by complement depletion using cobra venom factor and did not develop in C3(-/-) mice. Anti-murine C6 Abs in C57BL/6 mice inhibited MAC formation and also resulted in the inhibition of CNV. Vascular endothelial growth factor, TGF-beta2, and beta-fibroblast growth factor were elevated in C57BL/6 mice after laser-induced CNV; complement depletion resulted in a marked reduction in the level of these angiogenic factors. Thus, activation of complement, specifically the formation of MAC, is essential for the development of laser- induced choroidal angiogenesis in mice. It is possible that a similar mechanism may be involved in the pathophysiology of other angiogenesis essential diseases.     

Molecular biology of complement

Complementary DNA clones corresponding to most of the proteins of a major amplification and effector of immune host defenses, the complement system, have been isolated and characterized. Availability of these molecular probes has substantially increased our information about and understanding of the structure of the complement proteins and regulation of complement gene expression. Information about the proteins has led to the generation of potential pharmacological agents for the selective control of inflammation. Understanding of the regulatory mechanism has provided insights into the mechanisms accounting for the response to several cytokines including interferon-gamma, interleukin-1 and tumor necrosis factor. Finally, complement molecular genetics has been stimulated so that the basis for several complement deficiency disorders has been elucidated.