Background/aims: Interleukin (IL) 8 is a major neutrophil-activating cytokine synthesized by intestinal epithelial cell lines. The aim of this study was to improve the understanding of the regulation of IL-8 synthesis by investigating the roles of protein kinase C (PKC), protein kinase A (PKA), and protein tyrosine kinase (PTK) in the induction of IL-8.
Methods: HT-29 cells were stimulated with IL-1 beta or tumor necrosis factor alpha (TNF-alpha) together with activators or inhibitors of PKC and PKA or with inhibitors of PTK. The presence of IL-8 protein was detected by enzyme-linked immunosorbent assay and that of IL-8 messenger RNA by Northern blotting and in situ hybridization.
Results: TNF-alpha and IL-1 beta dose-dependently induced IL-8 production in HT-29 cells. Activation of PKC by phorbol myristate acetate also stimulated IL-8 production; however, the effects of IL-1 beta or TNF-alpha did not require PKC, as shown by the PKC inhibitor staurosporin or PKC depletion. Stimulation of PKA by forskolin or inhibition by H89 or H7 had no influence on the synthesis of IL-8. However, induction of IL-8 by IL-1 beta or TNF-alpha was reduced by the PTK inhibitors herbimycin (by 79% or 89%, respectively) and genistein (by > 95%).
Conclusions: The synthesis of IL-8 is stimulated in HT-29 cells by IL-1 beta or TNF-alpha. This stimulation is independent from PKC or PKA but depends on protein tyrosine phosphorylation.