IFNγ is unable to modulate IL-8 mRNA and protein expression under the influence of IL-1β. A549 cells were incubated as unstimulated control, or stimulated with IL-1β (1 ng/ml). Where indicated, A549 cells were preincubated for 16 h with IFNγ at 20 ng/ml. IL-1β was added directly thereafter to the cultures without further washing. After 10 h, IL-1β-induced IL-8 mRNA accumulation was evaluated by realtime PCR analysis (A) and RPA (B), respectively. IL-8 mRNA expression was normalized to that of GAPDH. (A) Data are expressed as fold-induction compared to unstimulated control ± S.D. (n = 3). IL-8 protein levels in cell-free culture supernatants of those same cultures were determined by ELISA analysis (see results section). (B) One representative of three independently performed RPA analyses is shown. (C) RNA populations were analyzed by standard PCR using primers pairs that specifically detect the two IL-8 splice variants. The minor/rare splice form of IL-8 is denoted as 'IL-8 variant'. (D) A549 cells were incubated as unstimulated control, or stimulated with IL-1β (1 ng/ml). Where indicated, A549 cells were preincubated for 16 h with IFNγ at 20 ng/ml. Furthermore, 1 h before stimulation with IL-1β, BfA (10 μg/ml) was added to all cultures in order to block the cellular secretory machinery. After 8 h of incubation with IL-1β, cells were harvested and cellular IL-8 protein expression was assessed by Western blot analysis. One representative of three independently performed experiments is shown.