NF-kappa B activation is required for C5a-induced interleukin-8 gene expression in mononuclear cells

C5a, a potent peptide chemoattractant, stimulates interleukin-8 (IL-8) secr etion from peripheral blood mononuclear cells (PBMC), Experiments were cond ucted to understand the mechanisms for C5a-induced IL-8 production, which w as 14-fold greater than that in unstimulated cells by 2 hours, IL-8 secreti on was accompanied by accumulation of IL-8 mRNA in the cytosol and by nucle ar expression of a kappa B DNA binding activity within 30 minutes, AP-1 but not NF-IL-6 DNA binding activity was also detected in C5a-stimulated PBMC; however, its delayed expression (maximal at 4 hours) suggested a less impo rtant role in the rapid production of IL-8. The correlation between C5a-ind uced kappa B binding activity and IL-8 gene expression was examined in the RAW264.7 macrophage cells using reporter genes directed by the KB sequence from I kappa B alpha and IL-8 promoter regions. C5a-induced reporter gene e xpression was abolished by introducing mutations into the kappa B sites and by coexpression of a dominant negative I kappa B alpha construct resistant to agonist-induced phosphorylation. Pertussis toxin, which ADP-ribosylates the G(i) proteins known to couple to the C5a receptor, produced minimal in hibition of C5a-induced IL-8 expression and had little effect on C5a-induce d calcium mobilization in RAW264.7 cells. These results suggest that NF-kap pa B activation is required for C5a-induced IL-8 gene expression and that t his response is mediated primarily through a pertussis toxin-insensitive pa thway. (C) 1999 by The American Society of Hematology.