Secretion of MCP-1 and IL-6 by cytokine stimulated production of reactive oxygen species in endothelial cells

Endothelial cells are known to produce reactive oxygen species by several m echanisms. Functional consequences of increased production of reactive oxyg en species were investigated in vitro after stimulation with several proinf lammatory cytokines. Time dependent increases in DCF-fluorescence as a meas ure of reactive oxygen load were quantified in single cells after incubatio n with TNF-alpha, IL-1 and IFN-gamma. The increased DCF-fluorescence was in hibited by cell permeant antioxidative substances Tiron and Tempol. NMMA, a n inhibitor of nitric oxide synthase reduced endothelial DCF-fluorescence o nly marginally, indicating a minor participation of nitric oxide production in this detection system. Cytokine induced endothelial DCF-fluorescence in creased in the presence of NADH, whereas coincubation with NADPH or xanthin e was without effect. Flavoenzyme inhibitor diphenyliodonium abolished stim ulated DCF-fluorescence. Cytokine induced release of MCP-1 and IL-6 by endo thelial cells was completely inhibited in the presence of Tiron and Tempol, whereas NMMA was less effective. Collectively these data indicate that cyt okine stimulated endothelial cells increase their reactive oxygen species p roduction probably via NADH oxidase and this production may critically be i nvolved in the secretion of MCP-1 and IL-6.