Expression of Fas-Fas Ligand Antigens and Apoptotic Marker APO2.7 by the Human Conjunctival Epithelium. Positive Correlation with Class II HLA DR Expression in Inflammatory Ocular Surface Disorders

Abstract

Fas antigen (CD95) is a membrane receptor that plays a major role in induction of apoptosis. In surface conjunctival epithelial cells the expressions of Fas, Fas ligand, the apoptotic marker APO2.7 and of HLA DR class II antigen, a membrane marker known to be expressed in inflammatory conditions were investigated. Impression cytology specimens were collected in 65 patients: 20 normal ones, 15 contact lens wearers, 20 receiving chronic topical antiglaucoma treatment and 10 with nonspecific chronic conjunctivitis. Cells were processed for flow cytometry, using monoclonal antibodies to Fas, Fas ligand, APO2.7, HLA DR antigens and a negative isotypic control. Percentages of positive cells were recorded and levels of fluorescence quantified using fluorescent beads at standardized fluorescence intensities. In addition, a human conjunctival cell line was incubated with anti-Fas stimulating antibodies in order to test Fas-induced apoptosis in vitro. Fas was found in all specimens in most of the conjunctival cells, but quantitation of levels of fluorescence showed a significantly higher expression in pathologic eyes than in normal ones. Fas ligand and APO2.7 were variably expressed by conjunctival cells, but in a significantly higher percentage of cells in pathological eyes than in normal ones. In these eyes a strong expression of HLA DR was also observed, whereas normal eyes showed lowest levels. Highly significant correlations were found between Fas, Fas ligand, APO2.7 and HLA DR levels. Anti-Fas antibodies in vitro induced strong apoptosis in epithelial cells as confirmed by APO2.7 expression and DAPI staining. This study confirms that conjunctival epithelial cells normally express Fas antigen, and more inconstantly its ligand, as do corneal ones or keratinocytes. Fluorescence quantitation by flow cytometry showed much higher expression in inflammatory eyes than in normal ones, and demonstrated a strong correlation between apoptotic and inflammatory pathways in the ocular surface.