Suppression of nuclear factor kappa B and CD18-mediated leukocyte adhesion to the corneal endothelium by dexamethasone

Purpose. To demonstrate that leukocyte adhesion to cultured corneal endothelial cells is mediated by the CD18 antigen, and to determine whether dexamethasone directly suppresses adhesion by inhibiting activation of nuclear factor kappa B (NFκB). Methods. Cultured bovine corneal endothelium was stimulated for 6 hours by 40 u/ml tumor necrosis factor alpha (TNFα). Dexamethasone was added 1 hour before TNFα stimulation in the dexamethasone group. After stimulation, neutrophils separated from a healthy human volunteer were added with or without anti-CD18 antibody. The culture plate was settled for 15 minutes at 37°C, and then neutrophils were activated by N-formylmethionyl-leucyl-phenylalanine for 5 minutes. Nonadherent neutrophils were removed by sealing and inverting the culture well. The intracellular localization of NFkB after TNFα simulation was determined by confocal immunocytochemistry using an anti-p65 antibody. Results. Neutrophil adhesion to cultured corneal endothelial cells increased significantly on exposure to TNFa (451.4 ±45.4 cells/mm², n = 16) compared to control (156.7 ± 27.3 cells/mm² n = 16, P <0.01). This increased adhesion was suppressed by the addition of anti-CD18 antibody (157.6 ± 25.1 cells/mm², n = 8, P < 0.01) and by pretreatment with 10^-7 M dexamethaSone (207.9 ± 31.5 cells/mm², n = 10, P < 0.01). Immunocytochemistry 60 minutes after stimulation revealed that NFκB was located in the cytoplasm in unstimulated cells; however, the addition of TNFα caused NFκB to translocate into the nucleus. Pretreatment with dexamethasone tapered NFκB translocation into the nucleus. Conclusions. Leukocyte adhesion to the corneal endothelium was shown to be mediated by CD18 expressed on activated leukocytes. Pretreatment of the endothelium with dexamethasone inhibited leukocyte adhesion; this may be due in part to the suppression of NFκB entry into the nucleus.