The Pro-inflammatory Cytokines, IL-1ß and TNF-a, Specifically Alter Enterocyte Gene Expression
Abstracts
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The pro-inflammatory cytokines, Interleukin-1ß (IL-1ß) and Tumor Necrosis Factor-a (TNF-a), are thought to play a role in a variety of gut inflammatory conditions, including Crohn?s Disease and Ulcerative Colitis. These gut inflammatory conditions are always associated with villus atrophy and we have previously identified a molecular phenotype that occurs in the setting of villus atrophy, i.e., silencing of the brush border marker, intestinal alkaline phosphatase (IAP). In the present studies, we hypothesized that IL-1ß and TNF-a might directly affect IAP gene expression. METHODS: HT-29 cells were grown in standard media and treated with 5 mM sodium butyrate (NB) to induce a villus-like differentiation. The cells were exposed to the cytokines IL-1ß or TNF-a (10 ng/ml) prior to the differentiation stimulus in order to model the inflammatory conditions seen in vivo. Northern analyses were then performed using specific radiolabeled cDNA probes (IAP, villin, and the actin control). Transient transfections were performed using a Luciferase reporter construct containing a 2.5 kb segment of the human IAP 5' flanking region. RESULTS: Northern blots confirmed the marked induction in IAP expression after 24 hrs of NB treatment. However, in those cells pre-treated with the cytokines there was a significant decrease in IAP levels (37 % decrease with IL-1ß and 27 % with TNF-a, p < 0.01). These changes were specific for IAP, since other differentiation induced markers (e.g., villin) were unaffected by the cytokines. The transfection studies revealed that IAP-LUC was markedly induced by NB (5-fold), as we have previously shown. But, when the cells were pre-treated with the cytokines, the level of LUC activation was significantly diminished, 35 % and 50 % decreases with IL-1ß and TNF-a, respectively. CONCLUSIONS: The pro-inflammatory cytokines, IL-1ß and TNF-a, specifically inhibit IAP expression both at the mRNA level and in transfection studies using the human IAP gene promoter. These effects of the cytokines on IAP gene transcription might explain the changes in enterocyte phenotype seen in vivo in a variety of gut disease states. |
