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 apo ptosis of rat mesangial cells (GMC) in vitro. Since the classical comp lement 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 a nti-Thy-1 MoAb, ER4G (IgG2a) and ER14 (IgG1), were used. It was establ ished that ER4G binds Clq efficiently, while ER14 reacts pearly with C lq. For the experiments of apoptosis, quiescent rat GMC were exposed f or 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 c ells with medium alone was used as controls. Apoptosis was assessed by morphological studies and quantitative analysis on FAGS using FITC-an nexin V (the annexin V methods) or bicolour FAGS analysis using FITC-a nnexin V and propidium iodide (the annexin V/PI method). With the anne xin V method, M-GMC revealed 9.4 +/- 1.4% apoptosis. Clq had only marg inal 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-apopt osis 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-GM C 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(a b')(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 bi colour 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 fragme nts 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 apopto sis of rat GMC in vitro, presumably by its binding to ER4G and probabl y by additional cross-linking of Thy-1 on the surface of GMC.