Expression and release of tumor necrosis factor-alpha by explants of mouse cornea

Purpose. To elucidate a possible target of immunosuppressive agents widely used in the treatment of corneal disorders, the authors determined whether corneal cells are capable of expressing and releasing tumor necrosis factor-alpha (TNF alpha) on lipopolysaccharide (LPS) stimulation, and they investigated whether TNF alpha production can be modulated by pharmacologic agents. Methods, Trephined central corneas from C57BL/6 mice were kept in culture for 3 days. Release of TNF alpha after a 24-hour stimulation with LPS (1 mu g/ml) into the culture medium was determined both by bioassay and by enzyme-linked immunosorbent assay. Expression of TNF alpha mRNA after 6-hour stimulation was examined by polymerase chain reaction. Immunofluorescent staining on cryostat sections of cultured corneas was performed to localize TNF alpha in the tissue. Corneal explants were pretreated with immunosuppressive agents (prednisolone, budesonide, cyclosporin A) for 48 hours, followed by 6- or 24-hour stimulation with LPS in the continuous presence of the agents. Results, Lipopolysaccharide stimulated TNF alpha release into the culture medium, The addition of budesonide (10(-7) M) or prednisolone (10(-6) M) significantly inhibited LPS-induced TNF alpha release, whereas cyclosporin A (10(-7)-10(-5) M) had no marked effect. Levels of TNF alpha mRNA in corneal explants increased fivefold after stimulation with LPS. Immunohistochemical staining revealed that TNF alpha was expressed in the epithelial cells. Budesonide markedly decreased mRNA expression and abolished immunostaining of TNF alpha stimulated by LPS, Conclusions. TNF alpha is produced and released by the epithelial cells of mouse central cornea in response to LPS. Contrary to cyclosporin A, corticosteroids such as prednisolone and budesonide potently inhibit TNF alpha production.