The experiments reported herein indicate that the AP is fully capable of initiating C3 activation induced by adCII-IC in vitro. When both the CP and AP are intact, C3 activation appears to be initiated by the AP at twice the level observed with the CP. Initiation of the complement system by the AP, but not the CP, requires the presence of N-glycan on the IgG molecule. Generation of G0-IgG leads to a low level of C3 activation using the LP. However, both the CP and AP are also activated by G0-IgG generating considerably more C3 activation than seen with the LP.
Amplification of C3b deposition by the AP is required to produce synovitis in CAIA, and in other models of adherent IC disease, after initiation of the complement system potentially by all three pathways. However, whether the AP is capable of primarily initiating complement activation as opposed to amplification, has remained unclear. The AP is thought to exhibit low-grade continuous activation by spontaneous hydrolysis, termed the “tickover” mechanism (3
). The C3b generated by this mechanism binds via covalent interactions to amino or hydroxyl groups on nearby surfaces as well as to soluble or adherent IgG. Amplification by the AP results in further C3 cleavage induced by factor B in the presence of factor D.
The role of antibody in activation of the AP has been reviewed (24
) with the best studied example being the solubilization of IC (25
). These experiments indicated that the AP could both primarily initiate and amplify C3b deposition in immune precipitates, leading to solubilization. However, initiation of C3b deposition by the CP greatly accelerated the rate of solubilization (26
). IC-induced activation by the CP has been assumed to be of primary importance in human diseases, until recent evidence has shown the importance of the AP (2
The AP exists in a dynamic state of equilibrium with activation by factor B enhanced by properdin and inhibited by factors H and I (4
). Disruption of a balance between activation and inhibition of the AP may lead to disease as exemplified by the association of factor H deficiency with age-related macular degeneration and atypical hemolytic uremic syndrome (27
). The mechanism of IgG induction of the AP, and the necessity for N
-glycans in this process, remains unknown. Possible mechanisms for N
-glycans in IgG influencing the initiation of complement by the AP may occur at four different points: binding of C3b to IgG, binding of properdin to C3b, binding of factor B to C3b, or binding of factor H to C3b.
Multiple binding sites for C3b exist on the heavy chains of the Fc portion of IgG, in both the Ch1 and Ch2 domains (29
). Binding of C3b to IgG occurs via both ester and amide linkages, with a serial dimer of two C3b molecule favored. Although suggested by the results of early studies (30
), C3b appears not to bind directly to the N
-glycans linked to Asn 297 in the Ch2 domain of the Fc portion. In contrast, C3b binds avidly to certain terminal sugars in polysaccharides on the surface of bacteria (31
). C3b-C3b dimers bound to IgG are protected from inactivation by factor I possibly through strong binding of properdin to the dimers and steric hindrance of one of the factor H binding sites (32
). The effect of N
-glycans in IgG on C3b binding is not known although the possibility exists that N
-glycans are necessary to maintain proper conformation of the IgG. The results of one study suggest that N
-glycans can influence the CP but not the AP (18
). However, variations in experimental conditions may show differences in dependency of the AP on N
-glycans. In contrast, removal of N
-glycans from IgG markedly inhibits Fc binding to FcR (14
). The mechanisms of oligosaccharide interactions with Ch2 residues and of the effects on Fc functions have been discussed (33
Studies on the X-ray structure of C3b reveal marked conformational changes after enzymatic cleavage of C3 to C3b with exposure of the internal thioester bond and of binding sites for properdin, factor B, and factor H (34
). Properdin is a positive regulator of complement activation that stabilizes the AP convertases (C3bBb). Binding of properdin to C3b on a RBC surface occurs before the binding of factor B to C3b and greatly enhances this interaction (35
). Furthermore, properdin inhibits factor I binding to and action on cell-bound C3b but does not compete with binding of factor B or factor H (35
). Properdin binds to C3b on a single site located within residues 1402–1435 on the C-terminus of the α-chain (37
). Furthermore, properdin binds avidly to sulfated glycoconjugates, theoretically increasing the affinity of properdin for C3b if appropriate sulfated glycans are located nearby (38
). These glycans could be on C3b itself, or could originate from the IgG or surface to which the C3b was bound. Hypothetically, the requirement for N
-glycans in AP initiation by IgG observed in our studies could be due to enhancement of properdin binding to C3b. Lastly, the results of recent studies indicate that properdin may initiate complement activation by primarily binding to microbial surfaces through C3b, iC3b, or other ligands, then binding more C3b or C3bBb through its unoccupied site with further in situ assembly of AP convertases (39
). It is not known if IgG could offer such a site for primary binding of properdin, or what might be the role of N
-glycans on IgG in this proposed initiation mechanism for the AP.
Factor B may bind to C3b in the region between residues 727 and 768, although other sites on C3b may influence this binding (34
). The possible influence of N
-glycans on Asn 297 of IgG on factor B binding are unknown although these polysaccharides linked to IgG may enhance the binding of factor B to C3b through secondary interactions.
Factor H regulates the complement system by acting as a cofactor for factor I-mediated cleavage of C3b and by accelerating the decay of the AP C3 and 5 convertases. Factor H is present at a high concentration in plasma, ~ 500 μg/ml, and binds to multiple polyanions on cell surfaces to protect them from attack by the AP (42
). Although factor H possesses three binding sites for C3b, the C-terminal domains 19–20 offer the most critical binding site (43
). The presence of polyanions on cells greatly enhances the binding of factor H to C3b through domains 19–20 (43
). Three sites exist on C3b for binding of factor H, with two sites partially overlapping with factor B, factor H domains 19–20, and CR1 where all 3 molecules may bind to a site on C3d (34
). Although factor H binds to glycosaminoglycans (45
), whether factor H binding to C3b could be sterically inhibited by N
-glycans on IgG is unknown.
The results of our in vitro studies clearly show that G0-IgG in adherent anti-CII mAb can activate C3 through both the CP and AP. RA is associated with an increased prevalence of G0-IgG molecules (11
). G0-IgG was concluded to be pathogenic in an experimental model of inflammatory arthritis as passive transfer of agalactosyl isoforms of polyclonal anti-CII Ab to mice primed to CII induced more disease than did transfer of the untreated Ab (47
). IgG RF from patients with RA demonstrated self-association with formation of cyclic dimers (48
). It has been hypothesized that increased amounts of G0-IgG in IgG RF may predispose to more self-association with the potential to increase pathogenicity (50
The AP plays an important pathophysiologic role in multiple immunologic diseases, some involving adherent immune complexes (3
). Although anti-CII antibodies may not play a primary role in the pathogenesis of RA, studies on adCII-IC may provide important information on pathogenic mechanisms of adherent IC in general. The overall results of our studies indicate that in addition to the key role played by the amplification loop of the AP in experimental models of arthritis, the AP may primarily initiate complement activation. Our observations that this process is dependent on N
-glycans in IgG provide a foundation for further studies on the involved mechanism. Additional studies are in progress evaluating all three pathways of complement in the initiation of activation in vivo.