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 mechanisms. Functional consequences of increased production of reactive oxygen species were investigated in vitro after stimulation with several proinflammatory cytokines. Time dependent increases in DCF-fluorescence as a measure of reactive oxygen load were quantified in single cells after incubation with TNF-α, IL-1 and IFN-γ. The increased DCF-fluorescence was inhibited by cell permeant antioxidative substances Tiron and Tempol. NMMA, an inhibitor of nitric oxide synthase reduced endothelial DCF-fluorescence only marginally, indicating a minor participation of nitric oxide production in this detection system. Cytokine induced endothelial DCF-fluorescence increased in the presence of NADH, whereas coincubation with NADPH or xanthine was without effect. Flavoenzyme inhibitor diphenyliodonium abolished stimulated DCF-fluorescence. Cytokine induced release of MCP-1 and IL-6 by endothelial cells was completely inhibited in the presence of Tiron and Tempol, whereas NMMA was less effective. Collectively these data indicate that cytokine stimulated endothelial cells increase their reactive oxygen species production probably via NADH oxidase and this production may critically be involved in the secretion of MCP-1 and IL-6.