Inhibition of Tumor Necrosis Factor-α– and Interleukin-1–Induced Endothelial E-Selectin Expression by Thiol-Modifying Agents

The expression of endothelial-leukocyte adhesion molecules has been postulated to be regulated by redox-sensitive events. Tumor necrosis factor-alpha (TNF-alpha)- and interleukin-1 (IL-1)-induced E-selectin expression was analyzed after pretreating human umbilical vein endothelial cells with different thiol-modifying agents, ie, diamide, phenylarsine oxide, N-ethylmaleimide, and diethyl maleate. E-selectin protein expression was quantified by indirect immunofluorescence. All compounds suppressed the cytokine-induced E-selectin expression in a concentration-dependent manner, whereas the antioxidant N-acetylcysteine showed no effect. The inhibitory effect of diamide (100 micromol/L, 1 hour) was reversible within 6 hours when the cells were allowed to recover before application of cytokines. Reversibility was strongly delayed when cells were deprived of glutathione by buthionine sulfoximine pretreatment. Glutathione depletion alone did not influence cytokine-induced E-selectin expression. Analysis of cellular glutathione status showed a 3-fold increase in oxidized glutathione after diamide treatment. Monochlorobimane labeling also revealed a decrease in total cellular thiols. During recovery, the glutathione status was restored within 1 hour, whereas total thiol content and E-selectin expression needed at least 6 hours to return to baseline. Complete inhibition of E-selectin expression by the vicinal thiol blocker phenylarsine oxide (0.5 micromol/L) was reversed by dithiols like dithiothreitol or dimercaptopropanol, but not by the monothiol 2-mercaptoethanol. These data suggest that proteins with essential thiols, most probably vicinal thiols. are involved in the IL-1- and TNF-alpha-mediated induction of E-selectin. These thiols must be in the reduced state; oxidation or other modification thereof attenuates or abolishes the cells' response to the cytokines.