SAA-induced IL-8 secretion requires transcription and de novo protein synthesis.

(A) Time course of SAA-induced IL-8 production. Neutrophils were stimulated with 2 μM SAA, and the secreted and cell-associated IL-8 concentrations were determined by ELISA after 1, 2, 3, and 4 hours. All data are presented as mean ± SEM of 3 independent experiments, each performed in duplicate. (B) Neutrophils, stimulated with 2 μM SAA for 1, 2.5, and 4 hours or incubated in the absence of SAA for 4 hours, were harvested for RNA preparation. RT-PCR was performed with specific primers for human IL-8 and G3PDH. The PCR products were resolved on a 1% agarose gel by electrophoresis and stained with ethidium bromide. Data shown are from one representative experiment of a total of 3, all with similar results. (C) Neutrophils were preincubated for 1 hour with or without actinomycin D (ActD; 10 μg/mL) or cycloheximide (CHX; 100 μM). The cells were then stimulated with 1 μM SAA for 4 hours. Secreted IL-8 was measured by ELISA as described. Maximal production of IL-8 (∼800 pg/10⁶ cells/mL) was observed in the absence of either inhibitor and was set as 100%, against which the effects of ActD and CHX were measured. Data shown are means ± SEM derived from 3 independent experiments, each performed in duplicate.