TNF stimulation of IL-8 is dependent on intracellular oxidation. (A) Inducible IL-8 protein secretion is sensitive to DMSO. Triplicate cultures of U937 cultures were untreated (control) or stimulated with 20 ng/mL TNF in the absence or presence of 2% (vol/vol) DMSO. At indicated times, cell culture supernatants were harvested and immunoreactive IL-8 determined by ELISA. Shown is the mean ± SD of n = 3 independent experiments (for both time points shown, P < .0001 for nonpretreated vpretreated with 2% (vol/vol) DMSO). (B) DMSO causes a dose-dependent inhibition of TNF-inducible IL-8 mRNA. Cells were unstimulated/stimulated with 20 ng/mL TNF (1 hour) in the absense/presence of DMSO of indicated concentrations (vol/vol). Cells were lysed and the total RNA subject to Northern analysis by hybridization with a hIL-8 cDNA probe (top) and an 18S cDNA probe (bottom) as an internal control. (C) DMSO causes a dose-dependent inhibition of TNF-inducible IL-8 promoter activity. Triplicate cell cultures were transfected with the –162 IL-8/LUC reporter plasmid and an SV40/alkaline phosphatase plasmid as an internal control. Cells were unstimulated/stimulated with 20 ng/mL TNF in the absence or presence of indicated concentrations of DMSO (in % [vol/vol]). Fold-induction of the Luciferase activity of stimulated (calculated from unstimulated) cells is shown (P < .0001 for stimulated v pretreated with 2% (vol/vol) DMSO before stimulation.