Role of Anti-Carboxyethylpyrrole (CEP) Specific Th2 T Cells and IgG1 Autoantibody Production in the Recruitment of Inflammatory Cells and Retinal Lesions in the Development of Autoimmune Retinal Degeneration

Abstract

Purpose: : Carboxyethylpyrrole (CEP) albumin adducts are generated in the retinal photoreceptors in response to oxidative stress and patients with age-related macular degeneration (AMD) have circulating CEP autoantibodies. We have previously reported a novel murine model of immune mediated retinal degeneration induced by immunization with CEP-modified albumin (CEP-MSA).The goal of this work is to test how this autoimmune response mediates disease.

Methods: : Balb/c mice were immunized in the footpad with CEP-MSA or non-adducted MSA emulsified in complete Freund’s adjuvant (CFA) followed with a subcutaneous challenge at day 10 with incomplete Freund’s adjuvant-CEP-MSA in the skin of the neck, followed by a second subcutaneous challenge one month later with CFA-MSA. Serum was collected at different time points and isotype switch was identified by ELISA. Specificity of anti-CEP autoantibody to retinal tissue was assessed by performing immunohistochemical staining using sera from immunized mice. Identification of inflammatory cell recruitment to retinal lesions (neutrophils, NIMPR14; macrophages, CD11b and B cells, B220) was done by immunohistochemistry. Functional phenotype of anti-CEP specific T cells was done in in vitro stimulated spleenocytes with CEP-MSA using cytokine ELISA.

Results: : Anti-CEP-MSA autoantibody switch to an IgG1 phenotype by day 30 after immunization and persisted for 1 year. In vitro stimulated CEP-MSA specific T cells had a Th2 phenotype as determined by the production of mainly IL-4. This Th2 phenotype correlated with the switch to IgG1 anti-CEP antibodies. Furthermore, immunohistochemical analysis demonstrated that anti-CEP IgG1 sera antibodies localized to scattered areas of the retinal pigment epithelium where retinal degeneration lesions were present. Moreover, the presence of neutrophils, macrophages and B cells correlated with retinal lesions and the deposit of anti-CEP IgG1 autoantiodies.

Keywords: age-related macular degeneration • immunomodulation/immunoregulation • autoimmune disease