314 Int J Colorectal Dis (2008) 23:305–317 Fig. 7 Transient transfection analysis of TGL-IP10 and TGL-IP10 κB2 mutant in IECs. TGL-IP-10 and TGL-IP10-κB2 Mut plasmid was transiently transfected into a Caco-2, b HT29 and c DLD1. 24 h after transfection, cells were stimulated with cytokines for 24 h, and promoter activity was monitored by luciferase assay. Data were normalised with renilla luciferase values. Data presented are the means ±SEM of three to five independent experiments done in duplicates for each sample. Significant differences vs controls (C) are indicated by an asterisk (p<0.05). Significant differences of wild type vs mutant are indicated by a number sign (p<0.05). N.S. is not significant interesting cytokine to study its effect on CXCL10 gene regulation. Although the synergistic effects of TNF-α + IFN-γ on CXCL10 gene expression and the signalling mechanisms involved were studied in astrocytes [26] and their synergistic effect on inducible nitric oxide synthase expression in IECs and macrophages [29] had been studied in detail, the synergistic action of these cytokines on CXCL10 gene expression and the signalling mechanisms involved have not been evaluated in IECs. Especially, the role of IL-1β alone and its synergism with both TNF-α and IFN-γ on CXCL10 gene expression in IECs has been given no attention. Previously, it has been shown that IECs may express elevated levels of CXCL10 [12], and its expression is increased in the mucosa of patients with IBD [16]. A critical role for CXCL10 in IBD has also been shown in animal models [30, 31]. Apart from this, CXCL10 has been shown to be expressed constitutively in colorectal carcino- mas and primary metastatic sites exerting anti-tumour activity by recruiting mononuclear cells via the CXCR3 receptor. However, at the same time, it has also been shown that CXCL10 promoted metastatic adhesion and tumour cell migration [32] so that care should be taken considering CXCL10 as an anti-tumoural therapy option. In light of these findings, we assessed whether stimulation of IECs in culture by inflammatory mediators could induce CXCL10 gene expression. All three IEC lines we used expressed CXCL10 mRNA in a concentration-dependent manner when stimulated with IFN-γ and TNF-α alone. These data support a vast body of evidence that demonstrates that IFN-γ can induce CXCL10 expression in a variety of cells types, including granulocytes, T lymphocytes, bronchial epithelial cells and keratinocytes [33, 34], while TNF-α is a potent inducer for CXCL10 in hepatocytes [17]. Importantly, in addition to these data, we could also show that Caco-2 cells stimulated with IL-1β up-regulate CXCL10 mRNA to a significant level, whereas in HT29 and DLD1 cells, induction of CXCL10 mRNA was less pronounced. A number of authors have reported functional synergy between TNF-α and IFN-γ in promoting gene expression for proteins such as RANTES, IFN regulatory factor 1 (IRF-1), MIG and intercellular adhesion molecule-1[35–38]