Auto-Immune Responses to Oxidatively Altered Self Proteins in the Retina Lead to the Development of Retinal Degeneration

Abstract

Purpose:: Patients with age-related macular degeneration (AMD) have circulating auto-antibodies to carboxyethylpyrrole (CEP)-modified albumin. CEP albumin adducts are generated in the retinal photoreceptors in response to oxidative stress. The main goal of this work is to determine immunized mice with CEP-modified albumin leads to the generation of an immune response that induces AMD.

Methods:: C57BL/6 were immunized in the footpad with mouse serum albumin adducted with CEP (CEP-MSA) or non-adducted MSA emulsified in complete Freund adjuvant (CFA) followed with a subcutaneous challenge at day 10 with incomplete Freund adjuvant-CEP-MSA. Mice received a second subcutaneous challenge two months later with CFA-MSA. Fundus exams and electroretinograms (ERG) were performed at different time points. Anti-CEP antibodies in the serum were measured by ELISA. Animals were sacrificed at two months for histological examination of enucleated eyes. T cell priming to CEP-MSA was assessed in the lymph nodes and spleens using Interferon gamma (IFN-g), IL-4 and IL-2 ELISPOT assay in response to in vitro CEP-MSA stimulation. The role of adaptive immunity was tested in CFA-CEP-MSA immunized RAG KO mice deficient in T and B cells.

Results:: All C57BL/6 mice (7/7) that were immunized and challenged with CFA-CEP-MSA had increased titers of anti-CEP antibodies, in contrast to controls (0/7). Moreover, a significant number of CEP-MSA specific IFN-g and IL-2 producing T cells in lymph nodes and spleen of immunized mice and no IL-4 producing T cells were detected. Although clinical examination was unremarkable, histological analysis showed significant amount of photoreceptor loss and scattered vacuolization of the RPE in CEP-MSA C57BL/6 immunized mice only, similar to those described in early AMD. No ERG changes were noted. Immunized and challenged RAG KO mice had no pathological changes in the retina, correlating with the lack of T cell priming and the absence of anti-CEP auto-antibodies.

Conclusions:: Immune responses to a CEP adducted self-protein in the retina can lead to the generation of an adaptive immune response that results in retinal damage and possibly AMD. This demonstrates for the first time that an in vivo chemical modification induced by oxidative stress in a specific organ can lead to autoimmunity. Moreover, this represents a novel and unique animal model to study immune mechanisms that could lead to the development of AMD and could be used to develop new therapies.

Keywords: age-related macular degeneration • autoimmune disease • oxidation/oxidative or free radical damage