306 present in the inflamed intestine is most likely due to both secreted chemokines and the relative expression of chemo- kine cell surface receptors on different cell types. Chemo- kine production by colonic epithelial cells is thought to contribute to the characteristic increased infiltration of selected populations of leucocyte cells in inflammatory bowel disease (IBD) [6, 7]. CXCL10 is a CXC chemokine, known to be secreted by endothelial cells [8], keratinocytes [9], eosinophils [10] and neutrophils [11]. It is also constitutively expressed by normal human colon epithelium [12] and chemoattracts activated T cells as well as monocytes by binding to the CXCR3 receptors present on the respective cells [13, 14]. While CXCL10 up-regulation may occur in acute inflam- mation in response to pathogens, it is also known to be permanently over-expressed in IBD patients [15, 16]. Blocking CXCL10 could prevent mice from acute colitis, suggesting this chemokine to play an important role in IBD pathogenesis [17]. Moreover, CXCL10 is believed to possess anti-tumoural properties by promoting anti-tumour T cell immunity [18, 19]. Knowledge of its regulation in colonic cell lines may therefore give also insight into the pathobiology of colorectal cancer. Most previous studies on chemokine expression in IEC concentrated on CXCL8 (interleukin [IL]-8), a neutrophil chemoattractant [20, 21], but there were only few studies regarding the expression and secretion of CXCL10 in IECs [12]. Although it is well known that interferon (IFN)-γ is a major inducer of CXCL10 and that CXCL10 expression may be enhanced by pathogens and other inflammatory cytokines, the relative contribution of cytokines on CXCL10 induction, their synergistic action and the kinetics of cytokine-induced CXCL10 expression in intestinal epithelial cells has not been fully elucidated. Especially, the role of IL- 1β in CXCL10 gene regulation in IECs and also in other cell types has not been fully evaluated so far. Intestinal mucosa is composed of cell populations in continuous change from a proliferative and undifferentiated stage in the basal parts of crypts to mature surface villus epithelial cells [22, 23]. Little is known about the alteration of immunological functions as IECs mature. It has been shown that cellular differentiation affects IEC responsiveness to IL-1β [24]. In the present study, we aimed to elucidate the role of the pro-inflammatory cytokines IL-1β, tumour necrosis factor (TNF)-α and IFN-γ, which have established role in IBD pathogenesis, in inducing CXCL10 gene expression in IEC lines. Especially, the role of IL-1β, one of the most abundantly expressed cytokines, both in normal and inflamed mucosa [25], in inducing CXCL10 expression by IECs and its possible signalling mechanisms was given importance. To consider possible influences of cellular differentiation on cytokine responsiveness, we performed studies on three different colonic epithelial cell lines. Our Int J Colorectal Dis (2008) 23:305–317 findings will get an insight into the role of intestinal epithelium in immune responses and in evaluating pathways, which might be targets of present and future pharmacotherapy of IBD and possibly colorectal cancer. Materials and methods Biological reagents The recombinant cytokines IL-1β, TNF-α and IFN-γ were purchased from Roche applied science (Mannheim, Ger- many). An enzyme-linked immunosorbent assay (ELISA) kit for CXCL10 was purchased from RnD systems (Weisbaden, Germany). The nuclear factor (NF)-κB inhib- itor BAY11-7082 was purchased from Calbiochem (San Diego, CA). Cell culture and stimulation protocols The human colon adenocarcinoma cell lines Caco-2, HT-29 and DLD1 were obtained from DSMZ (Braunschweig, Germany). Caco-2 cells were grown in Eagle’s minimal essential medium (BioWhittacker, Germany) containing 20% foetal calf serum (FCS) supplemented with 100 U/ml each of penicillin and streptomycin and 1% non-essential amino acids at 37°C and 5% CO2. HT29 and DLD1 were grown in Roswell Park Memorial Institute medium con- taining 10% FCS and 100 U/ml penicillin and streptomycin at 37°C and 5% CO2. Caco-2, HT29 and DLD-1 cells were plated into six-well plates at a density of 5 × 105 cells per well for real-time polymerase chain reactions (PCRs), ELISA, Northern blotting and electrophoresis mobility shift assay (EMSA) experiments unless until stated and grown till they reached 70–80% confluence. These cells were then stimulated with IL-1β (0.1, 0.5, 1, 2, 10 and 50 ng/ml), TNF-α (2, 10 and 50 ng/ml) and IFN-γ (2, 10 and 50 ng/ml) based on the type of experiments. RNA isolation, real-time PCR Ribonucleic acid (RNA) was isolated by using Qiagen RNeasy mini kit according to the manufacturer’s protocol. The RNA concentrations were determined photometrically using a Gene Quant RNA/deoxyribonucleic acid (DNA) calculator (Pharmacia, Freiburg, Germany). RNA was subse- quently used for real-time PCR and Northern blot analysis. Reverse transcription of messenger RNA (mRNA) was performed using 1 μg of total cellular RNA. To determine the mRNA expression of CXCL10, real-time PCR was carried out using gene-specific primers for human CXCL10 (Invi- trogen GmbH, Karlsruhe) forward 5′-CCA GAA TCG AAG GCC ATC AA-3′, reverse 5′-CAT TTC CTT GCT AAC TGC