NF-kappaB-dependent synergistic regulation of CXCL10 gene expression by IL-1beta and IFN-gamma in human intestinal epithelial cell lines.
OBJECTIVE
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 regulation by the pro-inflammatory cytokines interleukin (IL)-1beta, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha in intestinal epithelial cell lines.


METHODS
CXCL10 expression and secretion kinetics were assessed in Caco-2, HT-29 and DLD1 human colon epithelial cells, treated with IL-1beta, TNF-alpha, IFN-gamma 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-kappaB2 mutant promoter and gelshifts and supershifts for nuclear factor (NF)-kappaB were also performed.


RESULTS
Real-time PCRs and ELISA experiments revealed that IL-1beta was the strongest and earliest inducer of CXCL10 messenger ribonucleic acid (mRNA) expression and protein secretion in Caco-2 cell line, whereas INF-gamma had a delayed kinetics. There was a strong synergistic effect of either TNF-alpha or IL-1beta with IFN-gamma both on CXCL10 mRNA expression and protein secretion in all three cell lines. Real-time PCR and ELISA experiments using a specific NF-kappaB inhibitor and transfection experiments with a NF-kappaB-binding defective CXCL10 promoter construct revealed that the induction of CXCL10 by IL-1beta and its synergism with IFN-gamma is NF-kappaB dependent.


CONCLUSIONS
These data demonstrate that in colonic epithelial cells, depending on the cellular context and utilizing the NF-kappaB pathway, IL-1beta alone and/or in synergism with IFN-gamma may play a major role in the induction of CXCL10.
Centre for Internal Medicine, Department of Gastroenterology and Endocrinology, University of Göttingen, Goettingen, Germany.
Sunil
S
Yeruva
Giuliano
G
Ramadori
Dirk
D
Raddatz
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D015152:Blotting, Northern; D054357:Chemokine CXCL10 / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics*; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D053583:Interleukin-1beta / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000473:pathology; D016328:NF-kappa B / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D012334:RNA, Neoplasm / Q000235:genetics*; D014407:Tumor Cells, Cultured
D014408:Biomarkers, Tumor; C114753:CXCL10 protein, human; D054357:Chemokine CXCL10; D053583:Interleukin-1beta; D016328:NF-kappa B; D012334:RNA, Neoplasm; D007371:Interferon-gamma