Int J Colorectal Dis (2008) 23:305–317 DOI 10.1007/s00384-007-0396-6 ORIGINAL ARTICLE NF-κB-dependent synergistic regulation of CXCL10 gene expression by IL-1β and IFN-γ in human intestinal epithelial cell lines Sunil Yeruva & Giuliano Ramadori & Dirk Raddatz Accepted: 17 October 2007 / Published online: 28 November 2007 # Springer-Verlag 2007 Abstract Background and aims Little is known about the intestinal epithelial expression and secretion of CXCL10 (IP-10), a chemokine involved in recruiting T cells and monocytes. We aimed to study CXCL10 gene expression and regula- tion by the pro-inflammatory cytokines interleukin (IL)-1β, interferon (IFN)-γ and tumour necrosis factor (TNF)-α in intestinal epithelial cell lines. Materials and methods CXCL10 expression and secretion kinetics were assessed in Caco-2, HT-29 and DLD1 human colon epithelial cells, treated with IL-1β, TNF-α, IFN-γ alone or in combination with each other by real-time polymerase chain reaction (PCR), Northern blotting and enzyme-linked immunoabsorbent assay (ELISA). Transient transfections with TGL-IP10 (CXCL10 promoter) and TGL-IP10-κB2 mutant promoter and gelshifts and super- shifts for nuclear factor (NF)-κB were also performed. S. Yeruva : G. Ramadori : D. Raddatz Centre for Internal Medicine, Department of Gastroenterology and Endocrinology, University of Göttingen, Goettingen, Germany D. Raddatz (*) Department of Gastroenterology and Endocrinology, University clinic of Göttingen, Robert Koch Strasse 40, 37075 Goettingen, Germany e-mail: draddat@gwdg.de Present address: S. Yeruva Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Carl-Neuberg Strasse-1, 30625 Hannover, Germany Results Real-time PCRs and ELISA experiments revealed that IL-1β was the strongest and earliest inducer of CXCL10 messenger ribonucleic acid (mRNA) expression and protein secretion in Caco-2 cell line, whereas INF-γ had a delayed kinetics. There was a strong synergistic effect of either TNF-α or IL-1β with IFN-γ both on CXCL10 mRNA expression and protein secretion in all three cell lines. Real-time PCR and ELISA experiments using a specific NF- κB inhibitor and transfection experiments with a NF-κB- binding defective CXCL10 promoter construct revealed that the induction of CXCL10 by IL-1β and its synergism with IFN-γ is NF-κB dependent. Conclusion These data demonstrate that in colonic epithe- lial cells, depending on the cellular context and utilizing the NF-κB pathway, IL-1β alone and/or in synergism with IFN-γ may play a major role in the induction of CXCL10. Keywords Intestinal epithelial cells . Nuclear factor kappa B . Inflammatory bowel disease . Cytokines and chemokines . Colon cancer Introduction Intestinal epithelial cells (IECs) form the first immunologic barrier of the organism while the villus surface is continuously in contact with various agents. IEC gene expression is tightly regulated to induce appropriate responses to pathogens and to avoid deleterious reactions to microbial flora. When stimulated with pro-inflammatory cytokines or infected with microbial pathogens, IECs may secrete chemokines, a set of low-molecular weight cyto- kines, directing migration and activation of leucocytes, which play a major role in the perpetuation of inflammatory processes [1–5]. The final composition of leucocytes