Complement component C3 production in IL-1β-stimulated human intestinal epithelial cells is blocked by NF-κB inhibitors and by transfection with Ser 32/36 mutant IκBα

Background. Recent studies suggest that interleukin-1β (IL-1β) stimulates the production of the acute phase protein complement component C3 in human intestinal epithelial cells. The transcription factor NF-κB activates different genes involved in the response to cytokines. It is not known if IL-1β-induced C3 production in the enterocyte is regulated by NF- κB. Materials and methods. Cultured Caco-2 cells, a human intestinal epithelial cell line, were treated with one of the NF-κB inhibitors, tosyl- lys-chloromethylketone (TLCK), genistein, or pyrrolidine dithiocarbamate (PDTC), or with N-acetyl-leu-leu-norleucinal (LLnL), a proteasome inhibitor known to block the degradation of IκB, the cytosolic inhibitor of NF-κB. Following this treatment, the Caco-2 cells were stimulated with IL-1β, and C3 levels in the culture medium were measured after 24 h by ELISA. C3 mRNA levels were determined after 4 h by Northern blot analysis. In other experiments, Caco-2 cells were transfected with a mutant IκBα in which serines 32 and 36 were substituted by alanine. This mutation prevents IkBα phosphorylation and subsequent NF-κB nuclear translocation. After transfection, the cells were stimulated with IL-1β, and C3 levels in the culture medium were measured after 24 h. Cytosolic IκBα was determined by Western blot analysis. Results. TLCK, genistein, and LLnL each inhibited IL- 1β-induced C3 production in a dose-dependent fashion. These responses were associated with decreased C3 mRNA levels. In contrast, PDTC did not influence C3 production or C3 mRNA in the Caco-2 cells. Transfection of the Caco-2 cells with the Ser 32/36 mutant IkBα resulted in maintained IκBα levels and decreased IL-β-induced C3 production. Conclusions. IL-1β-stimulated C3 production in the enterocyte may be regulated by NF-κB.