Complement component C3 production in IL-1 beta-stimulated human intestinal epithelial cells is blocked by NF-kappa B inhibitors and by transfection with Ser 32/36 mutant I kappa B alpha

Background, Recent studies suggest that interleukin-1 beta (IL-1 beta) stim ulates the production of the acute phase protein complement component C3 in human intestinal epithelial cells. The transcription factor NF-kappa B act ivates different genes involved in the response to cytokines. It is not kno wn if IL-1-beta-induced C3 production in the enterocyte is regulated by NF- kappa B. Materials and methods. Cultured Caco-2 cells, a human intestinal epithelial cell line, were treated with one of the NF-KB inhibitors, tosyl-lys-chloro methylketone (TLCK), genistein, or pyrrolidine dithiocarbamate (PDTC), or w ith N-acetyl-leu-leu-norleucinal (LLnL), a proteasome inhibitor known to bl ock the degradation of I kappa B, the cytosolic inhibitor of NF-kappa B, Fo llowing this treatment, the Caco-2 cells were stimulated with IL-1 beta, an d C3 levels in the culture medium were measured after 24, h by ELISA. C3 mR NA levels were determined after 4 h by Northern blot analysis. In other exp eriments, Caco-8 cells were transfected with a mutant I kappa B alpha in wh ich serines 32 and 36 were substituted by alanine. This mutation prevents I kB alpha phosphorylation and subsequent NF-kappa B nuclear translocation. A fter transfection, the cells were stimulated with IL-1 beta, and C3 levels in the culture medium were measured after 24 h. Cytosolic I kappa B alpha w as determined by Western blot analysis. Results. TLCK, genistein, and LLnL each inhibited LL-1 beta-induced C3 prod uction in a dose-dependent fashion. These responses were associated with de creased C3 mRNA levels. In contrast, PDTC did not influence Ca production o r C3 mRNA in the Caco-2 cells. Transfection of the Caco-2 cells with the Se r 32/36 mutant IkB alpha resulted in maintained I kappa B alpha levels and decreased IL-beta-induced C3 production. Conclusions. TL-lp stimulated C3 production in the enterocyte may be regula ted by NF-KB. (C) 1999 Academic Press.