TNF inducible IL-8 expression in the U937 monocytic cell line. (A) TNF rapidly induces IL-8 mRNA abundance. U937 cells were untreated (control) or stimulated with 20 ng/mL TNF for the indicated times (in minutes, at top). Total RNA was extracted and analyzed by Northern analysis by hybridization with a hIL-8 cDNA probe (top) and an 18S cDNA probe (bottom) as an internal control. Shown is a representative Northern blot. Relative to control, IL-8 increases 10-fold (30 minutes), 37-fold (45 minutes), and 56-fold (60 minutes). This experiment was reproduced 2 times with similar results. (B) TNF induces a dose-dependent increase in IL-8 mRNA abundance. Cells unstimulated/stimulated with the indicated doses (in ng/mL) TNF for 1 hour 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. Relative to control, 0.0064 ng/mL TNF increased normalized IL-8 signal by 2-fold, 0.032 ng/mL TNF increased IL-8 signal by 7-fold, 0.16 ng/mL TNF increased IL-8 signal by 21-fold; 0.8 ng/mL increased IL-8 signal by 38-fold. This experiment was reproduced 2 times with similar results. (C) TNF induces a time-dependent increase in IL-8 protein secretion. Duplicate cultures were untreated (control) or stimulated with 20 ng/mL TNF. At indicated times, cell culture supernatants were harvested and immunoreactive IL-8 determined by ELISA. Shown is the mean ± SD of n = 2 independent experiments. TNF increases IL-8 secretion by 2.4-fold at 2 hours and 10-fold by 16 hours. Basal secretion is 500 pg/mL and unchanged any time of this experiment (P< .0001 for unstimulated v stimulated).