Abstract
Abstract: :
Purpose: While corneal transplantation is the most common and successful form of solid organ transplantation, immune rejection of grafts represents the main threat to transplant survival. The allospecific (DTH-related) mechanisms responsible for corneal graft failure at the local level remain unclear. We hypothesize that graft cell apoptosis is a major pathway of tissue damage and ultimately graft failure. Our purpose is to delineate the factors contributing to apoptosis in corneal cells, both in vitro and in animal models of corneal inflammation and transplantation. Methods: Cell lines of mouse corneal endothelial cells (CEC) and mouse keratocytes (MK/T-1 cells) were grown in culture and stimulated with agonistic Fas MAb, IL-1ß, TNF-α, or IFN-γ alone or in combination. Expression of both caspases was measured by Western blotting and apoptosis was measured by TUNEL assay. A mouse model of corneal inflammation was prepared by placement of intrastromal sutures. Enucleated eyes were examined for presence of active caspases in the cornea by immunohistochemistry or for apoptosis by TUNEL assay. Finally, orthotopic corneal transplantation was performed (C57BL/6 to BALB/c) in both inflamed (high-risk) and uninflamed (normal-risk) host beds. Following transplantation, enucleated eyes were examined for presence of active caspases and apoptosis by IHC and TUNEL assay. Results: Cytokine-stimulated CEC and MK/T-1 cells expressed active caspases and underwent apoptosis in vitro in response to Fas MAb, but only in combination with other cytokines. CEC required Fas MAb plus IFN-γ, whereas MK/T-1 cells required Fas MAb plus either IL-1ß or TNF-α to induce caspase activation. Corneas from inflamed mouse eyes showed significant expression of active caspases and TUNEL staining compared to uninflamed corneas. Grafted corneas from high-risk recipients also showed significant caspase activation and apoptosis compared to grafted corneas from normal-risk recipients and ungrafted corneas. Conclusions: We believe these data point to a role for apoptosis in the inflamed cornea and in allospecific corneal graft destruction. The in vitro data indicate that Fas receptor stimulation is required for apoptosis in corneal endothelial cells and keratocytes, but that synergistic cytokine receptor stimulation is also required. The in vivo data indicate that factors present in the cornea during inflammation and ‘high-risk' corneal transplantation lead to apoptosis of resident corneal cells. We speculate that this ‘bystander' apoptosis in the cornea represents a mechanism of corneal graft destruction and ultimately, graft failure.
Keywords: apoptosis/cell death • cornea: basic science • inflammation