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J.M. Doherty, J.W. Streilein; Role of Fas Ligand in the Activation of Macrophages by Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):699.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Fas ligand participates in both immunosuppressive and inflammatory processes within the eye. The goal of this study is to examine Fas ligand expression on corneal endothelial cells (CE) following exposure to activated macrophages. Methods: Immortalized BALB/c CE were grown to confluence in complete medium. Cells were incubated for 24 hrs in serum-free RPMI or in RPMI containing resting or LPS (50 ng)/IFN-g (250 U)/ml-activated macrophages (RAW 264.7) at a ratio of 1 CE: 3 RAW cells. Supernatants collected from the treated macrophage cultures were added to fresh CE. Fas ligand expression by CE was measured by indirect immunocytochemistry and Western Blot analysis. CE viability and NO production were assessed using the MTT assay and Griess reagent, respectively. Results: Cultured CE expressed low levels of Fas ligand. An increase in Fas ligand positive cells (i.e. 60-65% of total number of CE) was observed following exposure of CE to activated macrophages and to a lesser extent their culture supernatants. Interestingly, the most intense Fas ligand staining was observed on CE located immediately adjacent to the macrophages. The addition of resting macrophages and not their culture supernatants to the CE resulted in a 20-25% increase in Fas ligand expression. Concomitantly, a 4.5-fold increase in NO production and a 20% decrease in MTT activity (i.e. loss of CE viability) were observed when activated macrophages were added to CE. Resting macrophages elicited a 1.75-fold increase in NO production and a 129% increase in MTT activity (i.e. cellular activation). Conclusions: Corneal endothelial cells upregulated their expression of Fas ligand in the presence of inflammatory macrophages and their secreted products (i.e. TNF-a). We suggest that the Fas ligand on CE functions by triggering macrophages to produce NO, which in turn can damage corneal endothelial cells in vitro.
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