The quantitative role of alternative pathway amplification in classical pathway induced terminal complement activation

Abstract

Complement activation with formation of biologically potent mediators like C5a and the terminal C5b-9 complex (TCC) contributes essentially to development of inflammation and tissue damage in a number of autoimmune and inflammatory conditions. A particular role for complement in the ischaemia/reperfusion injury of the heart, skeletal muscle, central nervous system, intestine and kidney has been suggested from animal studies. Previous experiments in C3 and C4 knockout mice suggested an important role of the classical or lectin pathway in initiation of complement activation during intestinal ischaemia/reperfusion injury while later use of factor D knockout mice showed the alternative pathway to be critically involved. We hypothesized that alternative pathway amplification might play a more critical role in classical pathway-induced C5 activation than previously recognized and used pathway-selective inhibitory mAbs to further elucidate the role of the alternative pathway. Here we demonstrate that selective blockade of the alternative pathway by neutralizing factor D in human serum diluted 1 : 2 with mAb 166-32 inhibited more than 80% of C5a and TCC formation induced by solid phase IgM and solid- and fluid-phase human aggregated IgG via the classical pathway. The findings emphasize the influence of alternative pathway amplification on the effect of initial classical pathway activation and the therapeutic potential of inhibiting the alternative pathway in clinical conditions with excessive and uncontrolled complement activation.

Fig. 1

Specificity of inhibition by antifactor D and anti-C2 mAbs in haemolytic assays. Anti-factor D mAb 166–32 (•) completely inhibited alternative pathway haemolytic activity in normal human serum (NHS) while anti-C2 mAb 175–62 (▴) and the control mAb (○) had no effect (a). Anti-factor D showed no inhibition of classical pathway haemolysis in NHS in contrast to anti-C2 (b). Normal alternative pathway haemolysis (□) and no classical pathway haemolysis (▪) were found in C2-depleted (C2D) serum (c), while the opposite pattern was obtained in factor B depleted serum (d). Anti-factor D inhibited alternative pathway haemolysis in C2D serum completely while anti-C2 had no effect (e). The opposite pattern was obtained in factor B depleted serum (f).

Fig. 2

Anti-factor D inhibition of classical pathway activation in NHS. Anti-factor D mAb 166–32 (•) abolished solid phase (a) and fluid phase (b) formation of TCC induced by human IgM on the solid phase (Δ; no antibody added). Similarly, antifactor D (•) profoundly inhibited fluid phase formation of TCC induced by human aggregated IgG (HAIGG) in the fluid phase (Δ; no antibody added) (c). The points and error bars indicate mean values and range from two independent experiments.

Fig. 3

Effect of antifactor D, anti-C2 and control antibody on HAIGG-induced activation in NHS. Anti-factor D mAb 166–32 inhibited more than 90% of fluid phase TCC formation induced by human HAIGG whereas anti-C2 and a combination of anti-C2 and antifactor D completely blocked TCC formation (a). The control antibody had no effect on TCC formation. Similar results were obtained for the activation products C3bc (b), C5a (c) and C3bBbP (d). The data and error bars as for Fig. 2.

Fig. 4

Fluid phase C3bBbP formation after HAIGG-induced activation in NHS, C1q depleted serum (C1qD) and C1qD reconstituted withpurified C1q. Addition of HAIGG to NHS induced activation and marked C3bBbP formation in a dose–response manner. By contrast, addition of HAIGG to C1qD serum induced no C3bBbP formation, showing that HAIGG had no direct effect on the alternative pathway. After reconstituting C1qD serum with purified C1q, C3bBbP was again generated in a dose–response manner with increasing amounts of HAIGG, consistent with a restored classical pathway activity leading to alternative pathway amplification. T_0,baseline value in NHS (left), C1qD (middle) and C1qD + C1q (right). The data and error bars as for Fig. 2.