Nuclear factor-κB-dependent induction of interleukin-8 gene expression by tumor necrosis factor α: Evidence for an antioxidant sensitive activating pathway distinct from nuclear translocation

Tumor necrosis factor α (TNFα) is a pluripotent activator of inflammation by inducing a proinflammatory cytokine cascade. This phenomenon is mediated, in part, through inducible expression of the CXC chemokine, interleukin-8 (IL-8). In this study, we investigate the role of TNFα- inducible reactive oxygen species (ROS) in IL-8 expression by 'monocyte-like' U937 histiocytic lymphoma cells. TNFα is a rapid activator of IL-8 gene expression by U937, producing a 50-fold induction of mRNA within 1 hour of treatment. In gene transfection assays, the effect of TNFα requires the presence of an inducible nuclear factor-κB (NF-κB) (Rel A) binding site in the IL-8 promoter. TNFα treatment induces a rapid translocation of the 65 kD transcriptional activator NF-κB subunit, Rel A, whose binding in the nucleus occurs before changes in intracellular ROS. Pretreatment (or up to 15 minutes posttreatment) relative to TNFα with the antioxidant dimethyl sulfoxide (DMSO) (2% [vol/vol]) blocks 80% of NF-κB-dependent transcription. Surprisingly, however, DMSO has no effect on inducible Rel A binding. Similar selective effects on NF-κB transcription are seen with the unrelated antioxidants, N-acetylcysteine (NAC) and vitamin C. These data indicate that TNFα induces a delayed ROS-dependent signalling pathway that is required for NF-κB transcriptional activation and is separable from that required for its nuclear translocation. Further definition of this pathway will yield new insights into inflammation initiated by TNFα signalling.