Anti-recoverin antibodies cause the apoptotic death of mammalian photoreceptor cells in vitro

We developed an in vitro model to study the effect of anti-recoverin antibodies on retinal cells and the mechanism(s) by which they kill photoreceptors in cancer-associated retinopathy (CAR). Rat retinal cells were grown in a defined medium, and cell types mere identified by using antibodies against rhodopsin, recoverin, syntaxin, and thy-1, Purified immunoglobulin (IgG) against recoverin was added to the cultures at different concentrations for 24, 48, or 72 hr, and the survival of the cells was determined by fluorescence microscopy. Preimmune IgG and normal medium were used as controls. The cell death detection enzyme-linked immunosorbent assay and the terminal deoxyuridine triphosphate nick-end labeling assay were used to demonstrate cells undergoing apoptosis, Double labeling was used to visualize cell types and apoptotic death. Rods, amacrine cells, and ganglion cells mere identified in the cultures. Rod cells, but not ganglion cells and amacrine cells, markedly decreased in the presence of 200 mu g/ml of anti-recoverin IgG for 24, 48, and 72 hr, Anti-recoverin antibodies caused apoptosis in rod cells but not in amacrine cells. Almost all cells were shown to take up IgG from the medium. In conclusion, our retinal cell cultures provide a system for investigating antibody-mediated photoreceptor cell death and demonstrate that anti-recoverin antibodies cause the apoptotic death of rod cells, with no effect on amacrine cells. The results suggest that anti-recoverin antibodies play a key role in the apoptotic death of photoreceptors in CAR. (C) 1999 wiley-Liss,Inc.