Background Information

Complement is an important part of the body's constitutive immune defenses, providing an effector system for specific immunity as well as the innate responses. In addition to its well-characterized functions as opsonin, chemoattractant, and bactericidal agent, complement plays many housekeeping roles such as regulating the clearance of immune complexes, removing debris from damaged tissues, and maintaining antigen in germinal centers for stimulation of immunological memory cells. In addition to the 20 or so circulating components and control proteins, there are membrane-associated complement receptors and regulatory proteins on many different types of cells, and the cross-talk between activated components and cellular constituents is being seen to be more and more complex and fascinating as research in this area progresses. Like other parts of the immune response, complement, being only a single system, cannot stand alone, and its interaction and overlap with other mechanisms are still exciting areas of research.

Reasons for measuring complement activity include assessment of potential complement deficiency in an individual's immune-function workup, determination of changes in comple ment function resulting from disease or treatment, estimation of the extent of involvement of complement in the pathogenesis of a disease state or experimental model, and evaluation of the ability of a substance to activate complement in vivo or in vitro (including use of complement fixation tests to detect antibodies or immune complexes).

Evaluation of complement deficiency

To effectively analyze an individual's complement system, one must consider the history along with the assay results. It is much more time- and cost-effective to screen serum using CH50 and AH50 (unit 13.2) assays to locate a defect than to measure all of the components individually (see Table 13.1.7). Deficiencies of the late components (C3, C5-C9) or the control proteins factors H, I, and properdin tend to be associated with recurrent infections, often with Neisseria. Early classical pathway defects (C1, C2, or C4) are associated with collagen vascular diseases, including systemic lupus erythemato-sus, although recurrent infections with H. in-fluenzae and pneumococci are also reported in this group. Since complement deficiencies often cause no symptoms, CH50 or AH50 screens may give the only clues to their presence.

Table 13.1.7 Locating Complement Deficiency in Patient Serum

Assay and results

Suggested action (diagnosis)

CH50 normal AH50 normal

CH50 0 or very low AH50 normal

CH50 Normal AH50 0 or very low

CH50 0 or very low AH50 0 or very low

No action (no defect)

Assay C1q, C1r, C1s, C2 and C4 levels and/or functions (defect in classical pathway)

Assay properdin, factor B, factor D, factor H, and factor I (defect in alternative pathway or control proteins) Assay C3, C5, C6, C7, C8, C9, and factors H and I (defect in terminal components or control proteins shared by both pathways)

Evaluation of complement activation by test substance

Two methods are generally used to test a substance to determine whether it activates complement and, if so, which pathway is utilized: ELISA specific for the individual component fragments generated during complement activation, and functional (hemolytic) measurements of individual components that decrease in proportion to the amount of activation (Harbeck and Giclas, 1991; Harrison and Lachmann, 1986; Rapp and Borsos, 1970; Whaley, 1985). The usual classical pathway activators are immune complexes made up of IgG or IgM antibodies combined with antigen, but other substances can also interact with C1q and thereby activate the classical pathway in an immunoglobulin-independent fashion (Sim, 1993). This mechanism may be relevant when the activator is a virus, subcellular membranes associated with effete or lysed cells, bacterial products such as lipid A, chemical compounds such as some drugs, complexes containing C-reactive protein, or similar agent. Control of the classical pathway is effected by C1-esterase inhibitor (C1-INH), which binds to the activated forms of C1r and C1s and removes them from circulation.

The factors that control production of complement components are similar to those that control production of other plasma proteins. Most components are produced by cells in the liver, but many can be made by other cells, including fibroblasts, macrophages, and mono-cytes. C1 subcomponents are produced by columnar epithelial cells of the small intestine, lung, and bladder. The concentration of any component represents the balance between its synthesis and degradation rates and is also affected by changes in distribution between in-travascular and extravascular compartments.

Most complement deficiencies result from abnormal function of the gene for the component in question, so that no protein is produced or secreted and the diagnosis of deficiency is relatively easy. There are also cases where dysfunctional proteins are made. This is true of C1-INH: 15% of individuals with the hereditary form of C1-INH deficiency have normal or elevated levels of the protein, but insufficient function to protect them from angioedema.

C8 deficiency is also difficult to detect by measuring the protein level because three different genes are involved in producing the C8 molecule; although one gene is defective, the product of the other genes is present and will be recognized by the antisera used for the test. Thus, a low C8 level should always be checked by testing C8 function as well (Opferkuch et al., 1978; Harbeck and Giclas, 1991).

C1 is a 1,000,000-dalton macromolecular complex composed of three subunit molecules, C1q, C1r, and C1s, that must be measured individually. The rest of the complement components and control proteins are single molecules in their native forms, but activation results in cleavage of many of the components and generates split products that cross-react with the antisera. This makes quantitation difficult, so looking at split products or measuring function may be necessary to determine how much of a component is in the native state. C1-INH is a multispecific protease inhibitor that blocks the enzymatic activities of C1r and C1s as well as the coagulation-derived enzymes kallikrein, plasmin, and activated Hageman factor (XIIa).


Classical Pathway Evaluation

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