Lectin pathway activation of C3 is known to involve target recognition by mannan-binding lectin (MBL)or ficolins and generation of classical pathway C3 convertase via cleavage of C4 and C2 by MBL-associated serine protease 2 (MASP-2). We investigated C3 activation in C2-deficient human sera and in sera with other defined defects of complement to assess other mechanisms through which MBL might recruit complement. The capacity of serum to support C3 deposition was examined by ELISA using microtiter plates coated with O antigen-specific oligosaccharides derived from Salmonella typhimurium, S. thompson, and S. enteritidis corresponding to serogroups B, C, and D (BO, CO, and DO). MBL bound to CO, but not to BO and DO, and efficiently supported C3 deposition in the absence of C2, C4, or MASP-2. The existence of an MBL-dependent C2 bypass mechanism for alternative pathway-mediated C3 activation was clearly demonstrated using CO, solid-phase mannan, and E. coli LPS. MASP-1 might contribute, but was not required for C3 deposition in the model used. Independent of MBL, specific antibodies to CO supported C3 deposition through classical and alternative pathways. MBL-dependent C2 bypass activation could be particularly important in various inherited and acquired complement deficiency states.

Lectin pathway activation of C3 is known to involve target recognition by mannan-binding lectin (MBL)or ficolins and generation of classical pathway C3 convertase via cleavage of C4 and C2 by MBL-associated serine protease 2 (MASP-2). We investigated C3 activation in C2-deficient human sera and in sera with other defined defects of complement to assess other mechanisms through which MBL might recruit complement. The capacity of serum to support C3 deposition was examined by ELISA using microtiter plates coated with O antigen-specific oligosaccharides derived from Salmonella typhimurium, S. thompson, and S. enteritidis corresponding to serogroups B, C, and D (BO, CO, and DO). MBL bound to CO, but not to BO and DO, and efficiently supported C3 deposition in the absence of C2, C4, or MASP-2. The existence of an MBL-dependent C2 bypass mechanism for alternative pathway-mediated C3 activation was clearly demonstrated using CO, solid-phase mannan, and E. coli LPS. MASP-1 might contribute, but was not required for C3 deposition in the model used. Independent of MBL, specific antibodies to CO supported C3 deposition through classical and alternative pathways. MBL-dependent C2 bypass activation could be particularly important in various inherited and acquired complement deficiency states.

Soderstrom, C. Normal human serum depleted of Clq, factor D and properdin: its use in studies of complement activation. APMIS 99; 1120-1128, 1991 Normal human sera were depleted of Clq, factor D (D) and properdin (P) by a simple and reproducible procedure providing reagents for analysis of complement-dependent functions. Classical pathway activity was restored with purified Clq, and alternative pathway activity with purified D and P. Since both activation pathways were abolished, antibodies and other components could be removed without loss of complement activity during immunoabsorption procedures. Synergism between the two pathways during haemolysis of rabbit erythrocytes was clearly demonstrated, and was also found on analysis of C3 cleavage in serum incubated with other alternative pathway activators such as zymosan and inulin. Experiments with a Neisseria meningitidis serogroup W-135 strain isolated from a patient with inherited P deficiency showed that both pathways were capable of supporting antibody-dependent killing of the bacteria in serum. The alternative pathway was possibly more efficient than the classical pathway in the assay system. In C Iq,D,P-depleted serum with high concentrations of anticapsular IgG antibodies, the addition of D alone resulted in efficient alternative pathway-mediated killing. The alternative pathway was equally efficient in a C1 q,D,P-depleted serum with low concentrations of anticapsular antibody, but in this case the reaction required both D and P.

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Serum bactericidal activity and chemiluminescence (CL) responses of polymorphonuclear leukocytes (PMNL) to pathogenic Neisseria meningitidis serogroups B and W-135 and to nonpathogenic serogroup 29E were examined with pooled normal human serum depleted of the complement proteins C1q, factor D, properdin and C5. Purified C1q, factor D, properdin and C5 were added alone or in combination. For investigation of serogroup W-135 meningococci, a C1q, factor D and properdin-depleted postvaccination serum with high concentrations of anticapsular antibodies was also used. Serogroup B and W-135 cultured to log phase were resistant to the bactericidal activity of pooled normal human serum but were efficiently killed through the classical pathway alone when the bacteria were cultured to stationary phase. Nonpathogenic serogroup 29E meningococci in log or stationary growth phases were efficiently killed in serum, predominantly through the classical pathway. Serogroup W-135 meningococci grown to log phase were resistant to classical pathway-mediated bactericidal activity in postvaccination serum but were killed on addition of alternative pathway proteins. Stationary phase serogroup W-135 meningococci were killed through both pathways in the postvaccination serum. In the pooled normal human serum CL responses of PMNL were consistently more pronounced with fully reconstituted C1q, factor D, properdin, C5-depleted serum than with serum reconstituted with C1q, factor D and properdin suggesting contribution of actions related to terminal components. In the absence of C1q, serogroup W-135 meningococci in postvaccination serum induced a significant but delayed alternative pathway-mediated CL response. CL responses induced by serum-opsonized meningococci, in contrast to serum bactericidal activity, were not influenced by culture conditions. The findings suggest that phagocytic mechanisms may be of critical importance for resistance to pathogenic meningococci in nonvaccinated adults.