Macular corneal dystrophy: immunochemical characterization using monoclonal antibodies

Abstract

Macular corneal dystrophy is an inherited corneal disease characterized by corneal opacities resulting from intra- and extracellular deposits within the corneal stroma. Several monoclonal antibodies developed against antigens of corneal fibroblasts were screened for their reactivity with these abnormal deposits in corneas with macular dystrophy using an indirect peroxidase-conjugated immunostaining technique. One of these monoclonal antibodies (designated 8F1-3) reacted very strongly with these abnormal deposits. Although the antigen recognized by this monoclonal antibody was present in the normal corneal stromal and endothelial cells, its concentration in the cells in the corneas with macular dystrophy appeared to be considerably higher, based on the intensity of the immunostaining reaction. Corneal fibroblasts grown in tissue culture were employed for further characterization of the antigen. After fixing with paraformaldehyde and permeabilizing with Triton X-100, immunofluorescent staining of the corneal fibroblasts using these monoclonal antibodies revealed a filamentous pattern of staining which resembled that seen for vimentin filaments. On treatment of corneal fibroblasts with colchicine, the filaments recognized by this antibody were withdrawn from their cytoplasmic array to form a perinuclear cap as also observed for vimentin-containing intermediate filaments. Immunoelectron microscopic studies using colloidal gold-conjugated antimouse IgG indicated that this monoclonal antibody recognized an antigen associated with intermediate-type filament. However, antivimentin antibody did not react with the abnormal deposits in the corneas with macular dystrophy, indicating that the antigen identified in the present study, although associated with intermediate filaments, was not vimentin. Analyses of cytoskeletal antigens by the immunoblotting technique further revealed that this monoclonal antibody recognized two polypeptides with Mr48,000 and 45,000, while antivimentin antibody reacted with 58,000 Mr polypeptide (vimentin).