Antibody-mediated Death Oo Recoverin Positive Retinal Neurons

Abstract

Abstract: : Purpose: To investigate the mechanism by which anti-recoverin immunoglobulins kill retinal neurons in vivo and in vitro. Methods: Polyclonal and monoclonal antibodies were generated against recombinant mouse recoverin using standard procedures. Primary cell cultures of retinal neurons were made from 0-2 day postnatal rat pups and maintained in culture for 10 - 14 days before immunoglobulins were added whereas immunoglobulins were injected intravitreally into adult rat eyes and at selected times the rats were killed, the eyes enucleated and processed for immunofluorescence microscopy. Antibody uptake by retinal cells in vitro and in vivo was analyzed and citotoxic effect on retinal cells was measured by neuronal cell counts. Fc, Fab, and F(ab)₂ fragments were made and tested for uptake and ability to kill retinal neurons. Results: Most retinal neurons take up IgG in vivo and in vitro but only recoverin positive cells are killed. Immunoglobulin fragments with at least one binding site were effective in killing retinal neurons. A mAb against recoverin reacted with native recoverin but when used for immunocytochemistry labeled nuclei of morphologically distinct, recoverin negative cells in culture. Conclusions: Most retinal neurons can take up immunoglobulins but the presence of recoverin is necessary for cell death in a time-dependant manner. Monoclonal and monovalent antibodies can kill recoverin positive cells. A unique mAb against recoverin may help to identify the molecular basis for antibody mediated cell death which occurs in our experiments and in Cancer Associated Retinopathy.

Keywords: apoptosis/cell death • photoreceptors • animal model