The studies outlined in this proposal will focus on the role of transforming growth factor-beta (TGF-beta) in the mechanisms which govern the immune functions of intestinal epithelial cells as related to an inflammatory response at the intestinal mucosa. As a model system, we will use the nontransformed rat intestinal epithelial cell line, IEC-6. We have previously found that TGF-beta could enhance secretory component (SC) and class I major histocompatibility antigen (MHC) expression on IEC-6 cells. TGF-beta was also found to enhance the secretion of the inflammatory cytokine interleukin-6 (IL-6) by IEC-6 cells, as were IL-1alpha, IL-1beta, and TNF-alpha (all inflammatory cytokines themselves) able to induce IL-6 secretion by these cells. Co-stimulation of the IEC-6 cells with TGF-beta and either IL-1 or TNF-alpha resulted in a synergistic enhancement of IL-6 secretion by the IEC-6 cells to levels much greater than that of both cytokines combined. Preliminary results indicate that TGF-beta induced the IEC-6 cells to become more sensitive to IL-1 stimulation, an effect which was partially due to an enhanced expression of IL-1 receptors induced by TGF-beta. However, high levels of both TGF-beta and IL-1beta tended to lower the synergistic effect on IL-6 responses by the IEC-8 cells. In this proposal, we will (10 investigate the effect of TGF-beta on the expression of mRNA for SC as well as the effect of other cytokines in combination with TGF-beta. (2) The regulation of IL-6 secretion by TGF-beta with or without other inflammatory cytokines will be determined using a cytokine specific bioassay, RNA and protein synthesis inhibitors, and mRNA analysis to monitor the induction of and secretion of IL-6. (3) The effect of TGF-beta on the expression of receptors for the inflammatory cytokines will be explored in order to yield insight as to a possible mechanism whereby TGF- beta could enhance the effectiveness of these cytokines to induce IL-6 secretion. (4) The mechanism by which high levels of TGF-beta limit the synergistic effect of TGF-beta and IL-1beta on IL-6 secretion will be studied to determine if IL-6 mRNA or protein synthesis and IL-1 receptor expression are altered by high levels of these cytokines. (5) The effect of TGF-beta on the secretion of other inflammatory cytokines such as IL-1 and TNF-alpha will be assessed by specific assays and mRNA analysis. Finally, (6) the effect of TGF-beta on IL-6 secretion (and possibly the secretion of other cytokines) by IEC-6 cells induced with bacterial lipopolysaccharide will be studied as a model of the response of IEC to bacterial agents. The proposed study should lend valuable insight as to the role of TGF-beta and the intestinal epithelial cell in mucosal inflammatory responses such as those seen with mucosal infections and inflammatory bowel disease.
