Figure 3

Analysis of IL-6 release, COX-2 expression, and IκB degradation reveals that IFNγ can not be regarded as a general inhibitor of IL-1β action on A549 cells. (A) A549 cells were incubated as unstimulated control, or stimulated with IL-1β (12.5 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 24 h, cell-lysates were assayed for COX-2 expression by Western blot analysis. One representative of three independently performed experiments evaluating COX-2 expression is shown. (B) A549 cells were incubated as unstimulated control, or stimulated with IL-1β (12.5 ng/ml). Where indicated, A549 cells were preincubated for 16 h with either IFNγ at 12.5 ng/ml (without later addition of IL-1β) or with the indicated concentrations of IFNγ in combination with IL-1β (12.5 ng/ml). IL-1β was added directly thereafter to the cultures without further washing. After 24 h, cell-free supernatants were assayed for IL-6 secretion by ELISA. Data are expressed as mean IL-6 concentrations ± SD (n = 3). **p < 0.01 compared with untreated control. (C) A549 cells were incubated as unstimulated control or stimulated with IL-1β (12.5 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 15 min or 30 min, cells were harvested and homogenates were evaluated for IκBα-protein by Western blot analysis. One representative for three independently performed experiments is shown.