Enhanced characterization of serum autoantibody reactivity following HSP 60 immunization in a rat model of experimental autoimmune glaucoma

Purpose: Antibodies against heat shock proteins have been identified in sera of human glaucoma patients in several studies and immunization with heat shock protein 60 (HSP 60) causes retinal ganglion cell (RGC) loss in an animal model of experimental autoimmune glaucoma. The aim of this study was to observe the time course of increased anti-retina antibody appearance in the serum and characterize the identification of prominent autoantibodies that accompany HSP 60 immunization in a rat model of experimental autoimmune glaucoma. Methods: Eight weeks after immunization with HSP 60 retinal flatmounts were prepared and RGCs were counted in eight predefined areas and compared to controls. Serum collected before, as well as four and eight weeks after, immunization was used to detect antibody patterns against bovine retinal antigens using Western blotting techniques. These patterns were analyzed by multivariate statistical methods. Autoantibodies that were prominently increased were further identified through mass spectrometry. Intraocular pressure was measured throughout the study. Results: After eight weeks, animals immunized with HSP 60 showed significant RGC loss of retinal flatmounts (P0.02), which was intraocular pressure independent. Early changes in antibody profiles, many of them significant upregulations, were detected. Antigens with significantly upregulated antibody reactivity after four weeks were identified as histone H2B type 1, S-arrestin, glial fibrillary acidic protein, vimentin, and heat shock protein 60. These upregulated autoantibodies returned to normal levels four weeks following their initial upregulation. Antibodies against retinaldehyde binding protein 1 on the other hand became upregulated after eight weeks. Conclusion: This is the first study to identify the appearance and disappearance of retinal autoantibodies in the serum of rats at several time-points following their initial upregulation in response to HSP 60 immunization in a model of experimental autoimmune glaucoma.