Excessive secretion of interleukin-8 (IL-8), a pro-inflammatory chemokine secreted by activated endothelium during acute inflammation, causes neutrophil-induced vascular injury during processes such as bacterial sepsis, multiple trauma, or reperfusion of ischemic tissue. Nitric oxide (NO), a key signaling molecule, exhibits anti-inflammatory properties where endothelial activation and extreme IL-8 secretion are present. Research performed by the sponsor shows that NO may exert different regulatory effects on IL-8 expression according to the state of endothelial cell activation. This application's broad objective is to determine NO's role in regulating vascular endothelial responses to acute inflammation examining three specific aims: 1) to characterize the regulation of IL-8 gene expression by the transcription factors AP-1 and NF-kappaB; 2) to determine the effect of NO on the signaling pathway involved in the activation of AP-1 by inflammatory stimuli such as tumor necrosis factor-alpha (TNF-alpha); 3) to determine the effect of NO on the regulatory proteins involved in the activation of NF- kappaB by inflammatory stimuli such as TNF-alpha and interleukin- 1. Molecular techniques such as polymerase chain reaction, transient transfection, dual luciferase reporter assays, site directed mutagenesis, gel mobility shift assays and kinase assays will be used to accomplish these aims. Existing knowledge concerning NO's role in regulating inflammation is incomplete, yet NO is currently under study in human clinical trials as an adjunctive therapy for acute inflammatory vascular injury of the lung. This research will provide information essential to more completely understand the role of NO in the inflammatory process.
