In vitro model for antibody mediated death of mammalian photoreceptor cells

Abstract

Abstract: : Purpose: To establish an in vitro model to study the mechanisms by which rat photoreceptor cells are programmed to die by exposure to antibodies against Recoverin Methods: Polyclonal and monoclonal antibodies were generated against purified recombinant mouse Recoverin using standard procedures. Primary retinal cell cultures of 0 –1 day old rat pups were generated and grown for 10 days prior to exposure to IgG purified from mouse monoclonal or rabbit polyclonal antibodies against Recoverin. At selected times, cells were fixed and stained with antibodies against Rhodopsin, Arrestin or Recoverin and the cells visualized with secondary reagents labeled with "Alexa" fluorochromes. We analyzed these cells with a Nikon Eclipse 800 flourescence microscope and recorded images with a Micromax digital camera (Princeton Instruments). Small hairpin RNAs specific to rat recoverin were used to knock down the expression of endogenous recoverin in the cultured neurons Results: Most of retinal neurons took up the IgGs in vitro but only Recoverin–positive cells were killed when cells were incubated with polyclonal but not monoclonal antibodies. Normal mouse and rabbit IgGs were taken up by the cells but were not cytotoxic. To test whether the inability of monoclonal antibodies to form an IgG–Recoverin lattice was the reason for lack of cell death with the Recoverin mAb, goat anti–mouse IgG was subsequently added to the antibody treated cultures. None of the cells were killed by this procedure. Partial knock down of recoverin expression in the cultured cells may be sufficient to protect Recoverin–positive cells against antibody mediated cell death. We are currently testing this hypothesis Conclusions: Unlike polyclonal anti–Recoverin antibodies which kill photoreceptor cells, a monoclonal antibody against Recoverin (Mab10) did not kill Recoverin–positive cells even when present at a concentration of 200 ug/ml for up to 144 hours. The inability of the subsequent addition of anti–mouse IgG to kill the retinal neurons suggests that formation of an "antigen–antibody lattice" is not sufficient to kill the cells. Alternatively, the specific Recoverin epitope recognized by Mab10 may not be involved in antibody mediated cell death. Significant knockdown of recoverin expression may provide protection to photoreceptor cells and represent a therapeutic strategy to preserve the vision of some patients with CAR

Keywords: apoptosis/cell death • photoreceptors • gene transfer/gene therapy