C1q, a subunit of the first component of complement, enhances antibody-mediated apoptosis of cultured rat glomerular mesangial cells

We have shown previously that IgG2a anti-Thy-1 MoAb (ER4G) induces apoptosis of rat mesangial cells (GMC) in vitro. Since the classical complement pathway plays an essential role in Thy-1 nephritis, we analysed whether Clq, a subunit of the first component of complement, enhances the ER4G-mediated apoptosis of rat GMC. Two different subclasses of anti-Thy-1 MoAb, ER4G (IgG2a) and ER14 (IgG1), were used. It was established that ER4G binds Clq efficiently, while ER14 reacts pearly with Clq. For the experiments of apoptosis, quiescent rat GMC were exposed for 1 h at 37 degrees C to a fixed concentration of anti-Thy-1 MoAb and incubated further for 16 h at 37 degrees C in the presence or absence of Clq. GMC exposed to medium (M-GMC) followed by incubation of the cells with medium alone was used as controls. Apoptosis was assessed by morphological studies and quantitative analysis on FAGS using FITC-annexin V (the annexin V methods) or bicolour FAGS analysis using FITC-annexin V and propidium iodide (the annexin V/PI method). With the annexin V method, M-GMC revealed 9.4 +/- 1.4% apoptosis. Clq had only marginal effects on apoptosis of M-GMC. GMC exposed to ER4G (ER4G-GMC) and further incubated with medium in the absence of Clq resulted in 25.7 +/- 5.7% apoptosis (P < 0.01 relative to control). Incubation of ER4G-GMC together with 100 mu g/ml of Clq significantly increased GMC-apoptosis up to 39.4 +/- 4.9% (P<0.01 relative to ER4G-GMC incubated in the absence of Clq). This enhancing effect of Clq on apoptosis of ER4G-GMC was time-and dose-dependent. In contrast, Clq did not significantly alter the apoptosis of either GMC exposed to ER14 (ER14-GMC) or to F(ab')(2)-ER4G (F(ab')(2)-ER4G-GMC), while ER14-GMC or F(ab')(2)-ER4G-GMC incubated with medium resulted in significant apoptosis compared with control. These results were supported by morphological studies and bicolour FAGS analyses in time course experiments using the annexin V/PI method. The effect of Clq is dependent on the presence of intact Clq-containing globular heads and does not occur with collagen-like fragments of Clq. Furthermore, incubation of ER4G-GMC with antimouse K-chain antibodies also increased ER4G-mediated GMC-apoptosis. These results indicate for the first time that Clq enhances antibody-mediated apoptosis of rat GMC in vitro, presumably by its binding to ER4G and probably by additional cross-linking of Thy-1 on the surface of GMC.