Identification of TNF-inducible IL-8 cis elements in U937 cells. (A) 5′-deletions of the human IL-8 promoter/Luciferase reporter (hIL-8/LUC) were transfected into U937 cells. Sixteen hours later, cells were stimulated with 20 ng/mL TNF for 6 hours before luciferase assay. Shown is the normalized Luciferase activity from a representative transfection plotted on a semilogarithmic graph. Above the TNF-stimulated bar is the fold activation of the Luciferase activity by TNF (fold: activity of stimulated divided by activity of unstimulated). (B) Site mutations of NF-κB, NF-IL6, and AP-1 sites in the context of the –162hIL-8/LUC were analyzed for their inducibility by TNF. Shown is the normalized Luciferase activity in a representative transfection (mean ± SD,P < .0001 for all comparisons of unstimulated vstimulated, except ▵NF-κB). (C) Multimers of NF-κB, NF-IL6, and AP-1 sites ligated upstream of an inert hIL-8 TATA box were analyzed for their inducibility by TNF. As a positive control, the AP-1 multimer was treated for 6 hours with 1 μmol/L phorbol myristyl acetate (PMA). Shown is the normalized Luciferase activity from a representative transfection. TNF stimulated the NF-κB multimer, and PMA stimulated the AP-1 multimer (P < .0001 for comparisons of unstimulated v stimulated).