THE ALTERNATIVE COMPLEMENT PATHWAY
The alternative complement pathway constitutes the humoral component of natural defence against infections which can operate without antibody participation. Six proteins, C3, B, D, H, I, and P, by themselves perform the functions of initiation, recognition, and amplification of the pathway, which results in the formation of the activator-bound C3/C5 convertase (30). A variety of activators of this pathway have been described such as certain particulate polysaccharides, for example, bacterial (LPS), yeast (zymosan), or plant (inulin) polysaccharides, fungi, bacteria, viruses, certain mammalian cells, and aggregates of immunoglobulins, for example, the Fab portions of IgA or IgE (31). It is not yet known which structures these activators have in common as far as recognition by the pathway is concerned. Likewise, the mechanisms by which these heterogeneous groups of substances can initiate the activation of this pathway, are not fully understood. It is clear, however, that recognition involves C3b (32). The microenvironment of the particle-bound C3b determines whether C3b prefers the binding of B, which causes activation of the pathway, or of H, which cancels the progression of the reaction (33,34). Since C3b becomes covalently bound to receptive surfaces, interaction between the putative recognition site in C3b and the recognised structure in the immediate environment may be quite weak. This strategy would allow a wide spectrum of different but related substances to be recognised by C3b. The degree of specificity of the pathway is low, but by no means non-specific (35).
C3 has been shown to contain a thioester bond (36) which reacts with almost anything that exposes -OH or -NH2 groups, by spontaneously "ticking over" (37). The spontaneous slow hydrolysis of the thioester converts inactive native C3 to a functionally active C3b-like molecule (38). This form of C3, referred to as C3(H2O), constitutes a subunit of the initial C3 convertase, and its continuous production is the chemical basis of what has been called the "tick-over" phenomenon (39). Other properties of the thioester bond have been recognised such as: the chemical basis of the metastable membrane binding site of C3 (40), and that C3b, the product of the reaction catalysed by the C3 convertase, forms a subunit of the enzyme itself. The second property is the cause of positive feedback (41), which is the driving force of amplification of the pathway, a property lacking in the classical complement pathway.
The studies of mixtures of the six isolated proteins have shown that these six proteins are, indeed, sufficient to generate all known biological activities ascribed to the pathway. One such mixture behaved qualitatively and quantitatively like the alternative pathway in whole human serum (42). In addition to these six proteins, the five precursor proteins of the MAC reconstituted the cytolytic and bactericidal alternative pathway, as shown by the lysis of rabbit erythrocytes (43), and the killing of Raji cells (44) or gram-negative bacteria (45).
THE INITIAL C3 CONVERTASE
Without the participation of enzymes, initiation of the alternative pathway is safeguarded by spontaneous low-rate hydrolysis of the thioester in C3 and the resultant continuous supply of C3(H2O). Upon the Mg2+-dependent binding of factor B to C3(H2O), the activation of the proenzyme complex by factor D is triggered. The resultant cleavage of B causes the release of Ba, a 30kD fragment and the formation of C3(H2O)Bb(Mg), the initial C3 convertase, which is confined to the fluid phase (46,47). Bb, a 60kD fragment, is the second formed as a result of the cleavage of B. The initial C3 convertase is under positive regulation by properdin (P), whose function in the alternative pathway is to bind to cell-bound C3b and to stabilise the C3/C5 convertase (48,49), and is under negative regulation by factors H and I. It has been shown that for modified C3, the acquisition of H-binding capacity is slower than that of B-binding sites (50j. Thus, modified C3 has temporarily a greater chance to form the fluid-phase C3 convertase than to become enzymatically degraded by H and I.
THE TARGET CELL-BOUND C3/C5 CONVERTASE
The function of the initial C3 convertase is to produce metastable C3b and to deposit C3b on the surface of surrounding particles. The thioester in metastable C3b is 101°ree; times more reactive than that in native C3. Since C3b deposition is catalysed by a fluid-phase enzyme, it is expected to be a random process. If metastable C3b molecules fall randomly onto the surface of surrounding cells, then early during the reaction, deposition will have begun on some cells and not others. If these cells are activators of the pathway, there will be a marked initial heterogeneity of C3b distribution among them before a normal distribution is established (51).
The proenzyme, C3bB(Mg), is a reversible trimolecular complex which is activated by D. C3b serves as a substrate modifier enabling D to cleave B, and as an acceptor for Bb. The activated C3 convertase is covalently linked to the surface of target cells through C3b. Its function is to increase the number of target cell-bound C3b molecules in its immediate environment (Sl). Electron microscopic studies of the C3 convertase have shown that Bb consists of two domains, and that Bb is linked to C3b through only one domain, indicating that this is the binding domain and suggesting that the other may be the C-terminal catalytic domain (52). In the formation of the enzyme, Ni2+ can replace Mg2+, yielding a sevenfold more stable complex: C3bBb(Ni) (53). The metal is released on decay-dissociation of the enzyme.
The C3 convertase can function as a C5 convertase provided that an additional C3b molecule is available in close proximity (54,55). The role of this second C3b molecule is to bind C5 and to modify it for cleavage by Bb (56,57). At this stage it should be noted that factor I cannot inactivate C3b in the bimolecular C3 convertase, C3bBb, although it is able to inactivate the additional C3b which converts the C3bBb complex to the C5 convertase, C3bBbC3b, also, that factor H augments the rate of inactivation of C3b by dissociating Bb from the complex C3bBb. The C3/C5 convertase is physically stabilised by the cyclic protein, properdin (58).
DISCRIMINATION AND AMPLIFICATION
The C3b-dependent positive feedback is a unique feature of the alternative pathway. C3b, the product of the reaction catalysed by the C3 convertase, forms a subunit of the C3 convertase itself (59). Each newly produced C3b molecule has the potential to form the enzyme together with B, D, and Mg2+, and thus to produce more C3b and more enzyme. This process occurs rapidly in solution or on the surface of cells (60). In the latter case cells become covered with C3b. The progression of amplification is controlled by H and I, both in the fluid-phase and on non-activating particles. C3b, instead of binding B, binds H and is subsequently cleaved to C3bi (inactivated), therefore H restricts the formation of the C3 convertase and accelerates decay-dissociation of C3bBb (61,62). The action of factor I requires that C3b is in complex with H so that the actual substrate for I is C3bH (63). Factors H and I together constitute a very efficient scavenger system for C3b and also for C3(H2O) (63).
Host cell-associated regulatory proteins serve as formidable protectors against autologous complement attack. Both DAF and MCP are regulators with a wide tissue distribution (64,65). They are functionally complementary in that DAF prevents assembly of the C3 convertase and dissociates the formed enzyme, whereas MCP has cofactor activity for I mediated C3b degradation. CR1, which has a more limited tissue distribution, also has cofactor activity (66). Together, these membrane proteins provide a large measure of protection against C3b deposition on self-tissue.
The recognition of an activator of the alternative pathway is expressed on the molecular level by a reduction in the binding affinity of surface-bound C3b for factor H. It is probable that subtle conformational changes are imposed on bound C3b by its microenvironment and that these changes are responsible for the B versus H binding preference of C3b (67).
A study has been conducted to determine whether the recognition mechanisms established for the human pathway also held true for the alternative pathway of another species. The other species was the rabbit, and results indicated that the molecular mechanism of recognition in both species is analogous and species-specific (68,69).
THE ROLE OF ANTIBODY
Accumulating evidence has shown that antibody, independent of its role in activating the classical complement pathway, is able to function in the alternative complement pathway (70). Metastable C3b is capable of binding directly to immunoglobulin G (IgG). The Fd portion of the heavy chain seems to be the preferred binding site, and both ester and amide bonds have been demonstrated (71). The role of immunoglobulin in alternative pathway function is important because C3b covalently bound to IgG displays relative resistance to inactivation by H and I when compared to free C3b. The resistance appears to be entirely due to the reduced affinity of for H, and this confers on the complex an enhanced capacity to activate C3 in serum (72). This complex of C3b with bactericidal IgG was found to be much more effective than IgG alone in the killing of Escherichia coli by serum (73).
It seems that a number of immunoglobulins are able to activate the alternative complement pathway and play an important role in host defence in the infective process. Aggregated IgG and aggregated IgM both activate the pathway, as well as some aggregated IgA myeloma proteins and some IgE myelomas; although the immunoglobulin concentration required for such activation is actually relatively high.
Both the classical and the alternative complement pathways eventuate in the cleavage and activation of the C5 complement component, this process leads to the assembly of the membrane attack complex.
