Absence of complement component 3 does not prevent classical pathway-mediated hemolysis

Abstract

Complement component 3 (C3) is emerging as a potential therapeutic target. We studied complement-mediated hemolysis using normal and C3-depleted human sera, wild-type (WT) and C3-deficient rat sera, and WT and C3 knockout rat models. In all of the in vitro and in vivo experiments, we found that the loss of C3 did not prevent classical pathway-mediated hemolysis, but it did almost abolish alternative pathway-mediated hemolysis. Experiments using preassembled classical pathway C3 convertases confirmed that C4b2a directly activated complement component 5 (C5), leading to membrane attack complex formation and hemolysis. Our results suggest that targeting C3 should effectively inhibit hemolysis and tissue damage mediated by the alternative pathway of complement activation, but this approach might have limited efficacy in treating classical pathway-mediated pathological conditions.

Graphical abstract

Figure 1.

Comparison of the alternative pathway–mediated hemolysis between the C3-sufficient and C3-Dpl human or C3-deficient rat sera. For the measurements of alternative pathway–mediated hemolysis, E^rabb cells were incubated with different concentrations of NHS ranging from 5% to 40%, C3-Dpl human sera (A) or WT C3-deficient (C3KO) rat sera (B) in GVB-Mg⁺⁺/EGTA buffer, and hemolysis was quantitated by measuring levels of released hemoglobin in the supernatants. (C) In some experiments, E^rabb cells were incubated with 100% human or rat sera, and hemolysis was assessed by following the same protocol. These data show almost complete abolition of hemolysis in the C3-Dpl human sera and C3-deficient rat sera. *P < .05.

Figure 2.

Comparison of the classical pathway–mediated hemolysis between the C3-sufficient and C3-Dpl human or C3-deficient rat sera. For the measurements of classical pathway–mediated hemolysis, antibody-sensitized E^shA cells were incubated with different concentrations of NHS, C3-Dpl human sera (A,C), or WT C3-deficient (C3KO) rat sera (B,D) in GVB⁺⁺ buffer, and hemolysis was quantitated by measuring levels of released hemoglobin in the supernatants. These data show significantly reduced classical pathway–mediated hemolysis in the absence of C3 when lower concentrations of sera were used (A-B), but comparable and almost complete hemolysis when higher concentrations of sera were incubated (C-D). *P < .05

Figure 3.

Analyses of the classical pathway–mediated hemolysis in a syngeneic experimental system using preassembled C4b2a. (A) For measuring classical pathway–mediated hemolysis in a syngeneic experimental system, antibody-sensitized rat RBCs were prepared using an anti-rat antiserum, then incubated with different concentrations of WT or C3-deficient (C3KO) rat sera in GVB⁺⁺ buffer, and hemolysis was quantitated by measuring levels of released hemoglobin in the supernatants. These data show significantly reduced classical pathway–mediated hemolysis in the absence of C3 when lower concentrations of sera were used but comparable and almost complete hemolysis when higher concentrations of sera (50%-100%) were incubated. (B) In other experiments, the classical pathway C3 convertases were assembled on E^shA cells after incubation with C3-Dpl sera in the presence of the potent C5 inhibitor SSL7 to suppress hemolysis. Then the E^shA cells with preassembled C4b2a were incubated with sera from different WT and C3 KO rats (n = 6 in each group). Hemolysis was measured to assess the activities of C4b2a in directly activating C5. These results show that the preassembled C4b2a caused almost completely hemolysis in the presence and absence of C3, demonstrating that C4b2a can directly activate C5 in the absence of C3. *P < .05.

Figure 4.

Comparison of complement-mediated hemolysis in WT and C3 KO rats. Identical numbers of labeled E^rabb or E^shA cells were IV infused into WT or C3 KO rats, and the survival of these labeled RBCs at different time points was monitored by flow cytometry. These data show rapid and comparable kinetics of the clearance of the infused E^shA cells (C) but significantly prolonged survival of the infused E^rabb cells in the C3 KO rats (n = 6 in each group) (A). The in vivo experiments were repeated, and this time the rats were euthanized 5 minutes after infusion to collect blood by cardiac puncture for the comparison of levels of serum hemoglobin. These data show that compared with the naïve controls (without any RBC infusion), both the WT and C3 KO rats that received E^shA cell infusion had significantly increased levels of serum hemoglobin with even higher levels in the C3 KO rats than in the WT rats. (D) However, levels of serum hemoglobin in the E^rabb cell infused C3 KO but not WT rats were significantly reduced and comparable to those in the untreated rats (control) (B) (n = 4 in each group). *P < .05.