The C5 convertase is not required for activation of the terminal complement pathway in murine experimental cerebral malaria

Abstract

Cerebral malaria (CM) is the most severe manifestation of clinical malaria syndromes and has a high fatality rate especially in the developing world. Recent studies demonstrated that C5(-/-) mice are resistant to experimental CM (ECM) and that protection was due to the inability to form the membrane attack complex. Unexpectedly, we observed that C4(-/-) and factor B(-/-) mice were fully susceptible to disease, indicating that activation of the classical or alternative pathways is not required for ECM. C3(-/-) mice were also susceptible to ECM, indicating that the canonical C5 convertases are not required for ECM development and progression. Abrogation of ECM by treatment with anti-C9 antibody and detection of C5a in serum of C3(-/-) mice confirmed that C5 activation occurs in ECM independent of C5 convertases. Our data indicate that activation of C5 in ECM likely occurs via coagulation enzymes of the extrinsic protease pathway.

FIGURE 1.

C4^−/− and factor B^−/− mice are fully susceptible to the development of ECM, whereas C3^−/− mice are partially resistant. Wild type, C4^−/−, factor B^−/− (FB^−/−), and C3^−/− mice were injected intraperitoneally with 5 × 10⁵ PbA-iRBC, and clinical scores and survival were monitored twice daily for 10 days as described under “Experimental Procedures.” A, C4^−/− mice (n = 16) were fully susceptible to disease-induced mortality as compared with wild type mice (n = 17). B, factor B^−/− mice (n = 16) were fully susceptible to disease-induced mortality as compared with wild type mice (n = 19). C, C3^−/− mice (n = 19) are not significantly protected from disease-induced mortality (p = 0.21, log rank test) as compared with wild type mice (n = 18). D, clinical scores for wild type and C3^−/− mice are significantly different from day 7.5 through day 10 (p < 0.05, Wilcoxon rank sum test). E, parasitemia on day 6 after infection was not significantly different between wild type and C3^−/− mice (p > 0.05, Student's t test). F, INF-γ, IL-6, and IL-1 levels were determined by ELISA (n = 3–5 mice/group). G, brain tissue was isolated from PBS-perfused wild type and C3^−/− mice at day 6 (n = 4/group) and subjected to flow cytometric analysis as described previously (43). The number of CD8⁺ T cells in the brains of wild type and C3^−/− mice was not significantly different (p > 0.05, Student's t test).

FIGURE 2.

Terminal pathway activation occurs in C3^−/− mice in ECM. Wild type mice were injected with 5 × 10⁵ PbA-iRBC as described under “Experimental Procedures” A, anti-C9 antibody (Ab)-treated mice were significantly protected from ECM (p = 0.0001, log rank test) and had reduced clinical scores (B) as compared with mice treated with isotype control antibody (p < 0.05 on days 6–9, Wilcoxon rank sum test). C, wild type (n = 8) and C3^−/− mice (n = 5) were injected with PbA-iRBC as in A. On day 6 after infection, mice were bled, and serum C5a levels were determined by ELISA. C5a levels in C3^−/− mice were elevated, but not significantly, as compared with wild type mice. D, treatment of wild type mice with CR2-Crry (100 μg, n = 3) on days 5 and 6 did not significantly alter the course of ECM as compared with PBS-treated mice (n = 3). E, sCrry/GFAP mice (n = 10) were fully susceptible to disease-induced mortality as compared with wild type mice (n = 9).