PURPOSE: Proinflammatory cytokines are integral components of the allogeneic response to a corneal transplant and contribute to the pathogenesis of graft failure that results from irreversible damage to donor corneal endothelium. As yet, the mechanism and effectors of tissue damage during graft rejection remain unidentified. In the current study, the synergistic apoptotic effect of sustained proinflammatory cytokine insult was investigated in excised cornea and in transformed and primary corneal endothelial cells. METHODS: Apoptosis was assessed by tissue- and flow cytometry-based TUNEL staining. Downstream signaling events of cytokine stimulation and subsequent activation status of endothelium were studied by RT-PCR and Western blot analysis. Cellular production of NO was examined by the Griess reaction. RESULTS: Prolonged exposure (48 hours) of corneal endothelium to IL-1, IFNgamma, and TNF (100 ng/mL each) resulted in induction of apoptosis. Synergy in induction of apoptosis was found after exposure to cytokine combinations. Cytokine-mediated cytotoxicity was correlated with high and sustained (up to 36 hours) endothelial activation (specifically through NF-kappaB, p38, and STAT-1), upregulation of inducible nitric oxide synthase (iNOS), and elevated de novo production of NO. Pharmacologic inhibition of iNOS elicited complete cytoprotection from inflammatory cytokine insult. CONCLUSIONS: The specific release of proinflammatory cytokines from alloreactive infiltrating cells, in combination with the inflamed environment of a corneal allograft, results in apoptosis in the corneal endothelium. This effect is mediated by the de novo generation of NO and sustained activation of NF-kappaB, p38, and STAT-1. Inflammatory cytokine-induced apoptosis presents a new target for the development of interventions to prevent or attenuate endothelial injury in graft rejection.
PURPOSE: Proinflammatory cytokines are integral components of the allogeneic response to a corneal transplant and contribute to the pathogenesis of graft failure that results from irreversible damage to donor corneal endothelium. As yet, the mechanism and effectors of tissue damage during graft rejection remain unidentified. In the current study, the synergistic apoptotic effect of sustained proinflammatory cytokine insult was investigated in excised cornea and in transformed and primary corneal endothelial cells. METHODS: Apoptosis was assessed by tissue- and flow cytometry-based TUNEL staining. Downstream signaling events of cytokine stimulation and subsequent activation status of endothelium were studied by RT-PCR and Western blot analysis. Cellular production of NO was examined by the Griess reaction. RESULTS: Prolonged exposure (48 hours) of corneal endothelium to IL-1, IFNgamma, and TNF (100 ng/mL each) resulted in induction of apoptosis. Synergy in induction of apoptosis was found after exposure to cytokine combinations. Cytokine-mediated cytotoxicity was correlated with high and sustained (up to 36 hours) endothelial activation (specifically through NF-kappaB, p38, and STAT-1), upregulation of inducible nitric oxide synthase (iNOS), and elevated de novo production of NO. Pharmacologic inhibition of iNOS elicited complete cytoprotection from inflammatory cytokine insult. CONCLUSIONS: The specific release of proinflammatory cytokines from alloreactive infiltrating cells, in combination with the inflamed environment of a corneal allograft, results in apoptosis in the corneal endothelium. This effect is mediated by the de novo generation of NO and sustained activation of NF-kappaB, p38, and STAT-1. Inflammatory cytokine-induced apoptosis presents a new target for the development of interventions to prevent or attenuate endothelial injury in graft rejection.