The role of C5 in the development of PbA-induced experimental CM in C3H/HeN mice. To determine the phenotype and role of C5 in PbA infection in the C5-sufficient mice strain C3H/HeN, C3H/HeN mice, as well as control strains A/J (CM resistant) and C57BL/6 (CM susceptible), were challenged with PbA. C3H/HeN mice were susceptible to the development of CM (100% developed CM) and displayed similar mortality rates as C57BL/6 (C5-sufficient) mice (C57BL/6 vs. C3H/HeN: P = 0.2237 and χ² = 1.481 using a log-rank test), but significantly higher rates than A/J (C5-deficient) mice (A/J vs. C3H/HeN: P = 0.0033 and χ² = 8.656; and C57BL/6 vs. A/J: P = 0.0017 and χ² = 9.879 using a log-rank test). The data are representative of two independent experiments (n = 10 mice/group/experiment).

Congenic mice confirm a role for C5 in susceptibility to CM. To further explore the hypothesis that C5a contributes to the pathogenesis of CM, we examined the phenotype in recombinant congenic mice derived from systematic inbreeding of A/J and C57BL/6 mice that differ in their C5 status. Transfer of the C5-sufficient allele onto an A/J background in AcB55 mice accelerated the development of CM and fatal outcomes, whereas BcA76 mice that carry the A/J C5 deficiency allele had reduced incidence of CM and improved survival compared with parental C57BL/6 mice. The data are representative of two independent experiments (n = 10 mice/group/experiment). C5-deficient congenic mice survive significantly longer than C5-sufficient congenic mice (P < 0.05 and χ² = 4.038 using a log-rank test).

C5a potentiates PfGPI-induced inflammatory cytokines, and amplified responses are inhibited by C5aR blockade. Human PBMCs were treated in three groups as follows: (a) no antibody treatment (control); (b) 5 μg/ml of mouse anti-C5aR blocking antibody; or (c) mouse anti–human IgG2a κ isotype control. Each group was subsequently treated with 50 nM C5a ± 300 ng/ml HPLC-purified PfGPI for 6–24 h, and the supernatants were assayed for the production of TNF and IL-6. Statistical analysis was performed by two-way analysis of variance and is representative of the interaction effect (C5a*PfGPI). A Student's t test was used to assess whether blockade of C5aR inhibited cytokine induction. (A) C5a potentiated PfGPI-induced TNF production at 6 h (**, P = 0.0064), and this effect was inhibited by blocking C5aR (***, P = 0.0008). (B) C5a potentiated PfGPI-induced IL-6 production at 24 h (***, P = 0.0001), and this effect was inhibited by C5aR blockade (**, P = 0.0013). Data are representative of two independent experiments and are presented as means ± SEM.

Differential susceptibility to PbA-induced experimental CM. The phenotypes of inbred mice strains infected with PbA range from highly susceptible (early onset of cerebral symptoms and high parasitemia) to highly resistant (very little mortality caused by CM). (A) 129sv/J mice were highly susceptible and died significantly earlier than C57BL/6 mice (C57BL/6 vs. 129sv/J: P = 0.001 and χ² = 9.879 using a Mantel-Cox log-rank test). A/J mice survived significantly longer than C57BL/6 mice (A/J vs. C57BL/6: P = 0.001 and χ² = 9.879 using a Mantel-Cox log-rank test). AKR/J mice were highly resistant and had significantly improved survival rates compared with all other strains (A/J vs. AKR/J: P = 0.00001 and χ² = 13.568 using a Mantel-Cox log-rank test). The figure is representative of two independent experiments (n = 10 mice/group/experiment). 100% of 129sv/J and C57BL/6 mice developed CM (as described in Materials and methods), compared with 30% of A/J and 0% of AKR/J mice. (B) 129sv/J mice had significantly higher mean parasitemia than other CM-susceptible or -resistant mice on day 6 after infection (P = 0.006 using a Kruskal-Wallis test). Representative data from one of two independent experiments (n = 5 mice/group/experiment) are shown. Data are presented as means ± SD. (C) Histopathological examination of brains from susceptible (129sv/J, B10.D2/nSnJ, and C57BL/6) and resistant (AKR/J, B10.D2/oSnJ, and BALB/c) mice, isolated on day 6 after inoculation and formalin fixed. 5-μM sections were prepared and stained with Giemsa. CM-susceptible mice displayed characteristic histological evidence of CM, including the accumulation of mononuclear cells and parasitized erythrocytes in the microvasculature. Bar, 50 μM.

Survival and C5a levels in BALB/c, C57BL/6, and A/J mice. Sequence analysis showed that the BALB/c mice used in these studies were C5 sufficient and therefore, by our hypothesis, should be susceptible to PbA-induced CM. (A) However, BALB/c mice are highly resistant to CM despite C5 sufficiency (P < 0.0001 and χ² = 16.531 using a log-rank test). The figure is representative of two independent experiments (n = 10 mice/group/experiment). 100% of C57BL/6 mice developed CM compared with 0% BALB/c mice. (B) Serum C5a levels of C57BL/6, BALB/c, and A/J mice are represented by mean OD at 405 nm (error bars show SD). C57BL/6 mice had higher circulating C5a levels, particularly on days 1 and 5 compared with A/J (C5-deficient) mice, whereas BALB/c mice only show a C5a peak at day 5 and not early in the course of PbA infection (**, P < 0.0001; *, P < 0.05; and #, P < 0.01 using a Mann-Whitney U test). The graph represents pooled data from two independent experiments (n = 5 mice/group/experiment).

Role of C5 in pathogenesis of CM in closely related B10.D2 mouse strains. To examine whether the survival difference was solely attributable to C5, the congenic B10.D2/nSnJ (C5-sufficient) and B10.D2/oSnJ (C5-deficient) mice were infected with PbA. (A) B10.D2/oSnJ mice are resistant to the development of CM and survive significantly longer than C5-sufficient mice (75 vs. 0%; P < 0.0001 and χ² = 12.274 using a log-rank test). The figure is representative of two independent experiments (n = 8 mice/group/experiment). 100% of B10.D2/nSnJ mice developed CM compared with 25% of B10.D2/oSnJ mice. (B) Levels of serum C5a, shown as mean OD at 405 nm, were significantly increased in C5-sufficient (B10.D2/nSnJ) mice compared with C5-deficient (B10.D2/oSnJ) mice at days 1, 3, 5, and 7 after infection (*, P < 0.0001 using a Mann-Whitney U test). The data are representative of two independent experiments (n = 5 mice/group/experiment). Data are presented as means ± SD.

Blockade of C5a–C5aR protects susceptible mice from the development of CM. To identify whether CM outcome in C5-sufficient mice could be modulated, C5aR was blocked using antibody treatment of PbA-infected mice. B10.D2/nSnJ (C5-sufficient) mice that received anti-C5aR serum (n = 8) were significantly protected from developing CM compared with controls receiving nonimmune serum (n = 7; P = 0.0022 and χ² = 9.414 using a log-rank test). 100% of B10.D2/nSnJ mice treated with nonimmune serum developed CM compared with 0% of B10.D2/nSnJ mice treated with anti-C5aR serum.