A New Complement-Mediated Cytolytic Mechanism—the C1-Bypass Activation Pathway

Abstract

A new cytolytic pathway is described whereby cells sensitized with cytotoxic antibody can be specifically iysed in the complete absence of intact, classical complement pathway function. Evaluation of this model with sera deficient in the 4th (C4), 2nd (C2), and 6th (C6) components of complement has revealed that this new Iytic mechanism, termed the Cl-bypass activation pathway, is initiated by the antibody-mediated activation of C1. Utilizing components of the previously described alternate complement pathway, this pathway bypasses C4 and C2 to activate the third to ninth components of complement (C3-9) with induction of membrane damage.Serum complement (C) is the principal humoral cytotoxic effector system within the body, and a principal mediator of the biological events of inflammation. Until recently, complement activity was thought to result from the sequential activation of a single series of effector proteins. That is, antigen-antibody complexes were shown to activate in turn the first three components of complement C1, C4, and C2, termed the early components, and activation of these early components led to activation of C3-9, the later components in the complement sequence. A few years ago work from several laboratories established the existence of an alternate pathway of complement activation that bypassed C1, C4, and C2 of the "classical" pathway to enter the complement sequence at the level of C3 (1-4).Previous studies from our laboratory have shown that the alternate pathway is very inefficient at mediating the lysis of both erythrocytes and nucleated cells sensitized with cytotoxic antibody (5). In this report, for the first time, a new cytolytic system is described whereby cells sensitized with cytotoxic antibody can be specifically lysed in the complete absence of intact classical pathway function. Examination of this model has disclosed the existence of a novel cytotoxic complement pathway that is initiated by the attachment of C1 to membrane-bound antibody. The formation of an antigen-antibody-Cl complex (EAC1) leads to activation of components of the bypass or alternate complement pathway, and these in turn activate C3 and the later components of the complement sequence. MATERIALS AND METHODSPreparation of Anti-Forssman Antiserum and Purification of IgG Immunoglobulin. Anti-Forssman antiserum was prepared in a New Zealand white rabbit by repeated intravenous injections of increasing quantities of boiled sheep erythrocyte stromata over a 2.5-week period (10). 2 Weeks after the last intravenous injection of antigen, the rabbit was injected in each footpad with 0.2 ml of an emulsion containing equal volumes of antigen and Freund's complete adjuvant. 2 Weeks later, the animal was bled and the serum was used as a source of IgG hemolytic antibody. The IgG fraction of this antiserum was purified by gel filtration on Sephadex G-200 followed by sucrose density ultracentrifugation as described (6). 0.1 ml of a 1:1280 dilution of this antiserum was sufficient to produce 50% hemolysis of 0.1 ml of ...

The key role of peptidoglycan in the opsonization of Staphylococcus aureus.

“…However, it appears that opsonization can proceed via another heat-labile pathway when C2 is blocked but not wheni Cl is blocked. Other investigators (51,52) have demonstrated that C1 can play a role in the activation of the alternative complemiient pathway (the "Cl-bypass" mechanism). From the results in this study, it appears that the C l-bypass mav be operative in the opsonization of S. atureus H peptidoglycani by C2-deficient serum, and that blockiing Cl thereby interferes with alternative pathway activation.…”

Human mannose-binding protein activates the alternative complement pathway and enhances serum bactericidal activity on a mannose-rich isolate of Salmonella.

“…In addition, neither mannoserich SH5770 nor mannose-free S. adelaide activated significant amounts of Cl s in the presence of MBP, Cl r, and C ls. We considered briefly the possibility that MBP may activate complement via the unconventional Cl-bypass pathway (41,42). However, MBP-mediated complement fixation occurred in the absence of calcium, and therefore Cl -bypass was not considered further.…”

Development of a hemolytic assay for mouse complement components in sera and the variation of their levels with age.

“…Because of these contradictory results, we examined the possibility of participation of alternative pathway factors : 1) heat treatment of the partially purified C4 fraction from mouse EDTA-plasma showed no effect on C4 activity (50°C, 20 min), 2) C4 activity could not be measured in VBS containing 10 mM EGTA and 5 mM MgCl2, 3) zymosan treatment of mouse serum also showed no effect on C4 activity (17°C, 15 min). From these results, our assay method proved to be functional C4 titration and also ruled out the participation of the Cl-bypass pathway which was described by May and Frank (1973) as a mechanism for lysing sheep E heavily sensitized with rabbit antibody using C4 deficient guinea pig serum. Therefore, it will be difficult to consider that the Ss protein is a functional C4 molecule itself.…”