Regulation of ICAM-1/CD54 expression on human endothelial cells by hydrogen peroxide involves inducible NO synthase

Expression of the inducible isoform of nitric oxide synthase (iNOS) is stimulated by cytokines in human epithelial cells. This work indicates that incubation of human umbilical cord endothelial cells with combinations of interleukin‐1β, tumor necrosis factor α, and interferon‐γ stimulated the synthesis of iNOS mRNA, as detected by reverse transcriptase‐polymerase chain reaction. It is important to note that 50, 100, and 200 μM hydrogen peroxide was able to stimulate iNOS directly. Furthermore, 100 μM H2O2 enhanced synthesis of the oxidation products, nitrite (NO) and nitrate (NO) at 12 and 36 h. iNOS protein, detected by Western blot analysis, as well as L‐citrulline levels, were also increased. When endothelial cell monolayers were incubated for 1 h with 100 μM H2O2 and subsequently with cytokines, iNOS mRNA was further augmented. Under the same conditions, we regularly observed an inhibition (25%) of intercellular adhesion molecule‐1 (ICAM‐1/CD54) expression. The latter was reversed when the NOS inhibitor NG ‐monomethyl‐L‐arginine was added, as shown by flow cytometry. These data suggest a specific effect of endogenous hydroperoxides on the biosynthesis and processing of the human endothelial iNOS isoform. We propose that H2O2 induces a temporary NO‐dependent modulation of adhesion molecule expression to limit the tissue destruction that accompanies the vascular recruitment of leukocytes. J. Leukoc. Biol. 67: 327–334; 2000.