Exaggerated IL-8 cytokine response with P. aeruginosa infection in CF is associated with defective ASMase activity. (A) Exaggerated IL-8 response in CF bronchial epithelial cells with PA infection. IB3-1 and S9 cells were infected with PAO1 (25 cfu/cell) for 0, 3, and 5 h (n = 5, *P < 0.05). (B) RT-PCR result 48 hours after RNAi transfection. S9 cells were transfected with either 50 nM or 100 nM of ASMase RNAi or SCR RNAi for 48 hours. (C) Effect of ASMase RNAi on IL-8 expression. S9 cells were transfected with either 50 nM of ASMase RNAi or SCR RNAi 48 hours before PA infection. The cells were either uninfected (control group) or infected with PAO1 (25 cfu/cell) for 5 hours (n = 4, **P = 0.001). (D) Effect of ASMase inhibitor on IL-8 expression. Desipramine 32 μM) was added to S9 cells 1 hour before PA infection. The cells were either uninfected (control group) or infected with PAO1 (25 cfu/cell) for 5 hours (n = 5, **P < 0.001). (E) Effect of ASMase antibody on IL-8 expression. S9 cells were treated with a polyclonal rabbit anti-human ASMase antibody or a control normal rabbit IgG 10 μg/ml 1 hour before PA infection. The cells were either uninfected (control group) or infected with PAO1 (25 cfu/cell) for 5 hours (n = 4, *P < 0.01). (F) Effect of exogenous sphingomyelinase (SMase) on IL-8 expression. Exogenous SMase from Staphylococcus aureus (0.02 U/ml) was added to IB3-1 cells 1 hour before PA infection. The cells were either uninfected (control group) or infected with PAO1 (25 cfu/cell) for 5 hours (n = 4, *P < 0.01).