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-l MoAb (ER4G) induces apoptosis of rat mesangial cells (GMC) in vitro. Since the classical complement pathway plays an essential role in Thy- I 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-l MoAb, ER4G (IgG2a) and ER14 (IgG I ), were used. It was established that ER4G binds Clq efficiently, while ER14 reacts poorly with Clq. For the experiments of apoptosis, quiescent rat GMC were exposed for I h at 37°C to a fixed concentration of anti-Thy- I MoAb and incubated further for 16 h at 37°C in the presence or absence of C lq. 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 FACS using FITC-annexin V (the annexin V methods) or bicolour FACS analysis using FITC-annexin V and propidium iodide (the annexin V/PI method). With the annexin V method, M-GMC revealed 9.4 + 14% 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 + 57% apoptosis (P < 0.01 relative to control). Incubation of ER4G-GMC together with 100 μg/ml of Clq significantly increased GMC-apoptosis up to 39.4 ± 49% (P < 001 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, C1q 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 FACS 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.