Inhibition of tumor necrosis factor-alpha- and interleukin-1-induced endothelial E-selectin expression by thiol-modifying agents

The expression of endothelial-leukocyte adhesion molecules has been postula ted to be regulated by redox-sensitive events. Tumor necrosis factor-alpha (TNF-alpha)- and interleukin-1 (IL-1)-induced E-selectin expression was ana lyzed after pretreating human umbilical vein endothelial cells with differe nt thiol-modifying agents, ie, diamide, phenylarsine oxide, N-ethylmaleimid e, and diethyl maleate. E-selectin protein expression was quantified by ind irect immunofluorescence. All compounds suppressed the cytokine-induced E-s electin expression in a concentration-dependent manner, whereas the antioxi dant N-acetylcysteine showed no effect. The inhibitory effect of diamide (1 00 mu mol/L, 1 hour) was reversible within 6 hours when the cells were allo wed to recover before application of cytokines. Reversibility was strongly delayed when cells were deprived of glutathione by buthionine sulfoximine p retreatment. 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. Monochlorobi mane labeling also revealed a decrease in total cellular thiols. During rec overy, the glutathione status was restored within 1 hour, whereas total thi ol content and E-selectin expression needed at least 6 hours to return to b aseline. Complete inhibition of E-selectin expression by the vicinal thiol blocker phenylarsine oxide (0.5 mu mol/L) was reversed by dithiols like dit hiothreitol or dimercaptopropanol, but not by the monothiol 2-mercaptoethan ol. These data suggest that proteins with essential thiols, most probably v icinal thiols. are involved in the IL-1- and TNF-alpha-mediated induction o f E-selectin. These thiols must be in the reduced state; oxidation or other modification thereof attenuates or abolishes the cells' response to the cy tokines.