Peroxynitrite Mediates IL-8 Gene Expression and Production in Lipopolysaccharide-Stimulated Human Whole Blood

Abstract

Recent evidence indicates that free oxygen radicals, in particular hydroxyl radicals, may act as intracellular second messengers for the induction of IL-8, a potent chemoattractant and activator of neutrophil granulocytes. Here we report that peroxynitrite (ONOO−), formed by a reaction of nitric oxide (NO) with superoxide, mediates IL-8 gene expression and IL-8 production in LPS-stimulated human whole blood. The NO synthase inhibitors aminoguanidine and NG-nitro-l-arginine methyl ester (L-NAME) blocked IL-8 release by ∼90% in response to LPS (1 μg/ml), but did not affect the production of IL-1β or TNF-α. Both aminoguanidine and L-NAME blocked the induction of IL-8 mRNA by LPS. Authentic ONOO− (2.5–80 μM) augmented IL-8 mRNA expression and stimulated IL-8 release in a concentration-dependent manner, whereas the NO-releasing compounds, S-nitroso-N-acetyl-dl-penicillamine and sodium nitroprusside failed to induce cytokine production. Combination of the NO-generating chemicals with a superoxide-generating system (xanthine/xanthine oxidase) markedly increased IL-8 release. Enhanced ONOO− formation was detected in granulocytes, monocytes, lymphocytes, and plasma after challenge with LPS. Furthermore, pyrrolidine dithiocarbamate, an inhibitor of activation of nuclear factor-κB, markedly attenuated the induction of IL-8 mRNA expression and IL-8 release by either LPS or ONOO−. Our study identifies ONOO− as a novel signaling mechanism for IL-8 gene expression and suggests that inhibition of ONOO− formation or scavenging ONOO− may represent a novel therapeutic approach to inhibit IL-8 production that could lead to reduction of neutrophil accumulation and activation.