Intestinal epithelial cells express an array of cytokine and chemokine receptors allowing these cells to be targets for immune mediators. Human intestinal epithelial cells regulate nutrient and elctrolyte transport, maintain barrier integrity, and can release proinflammatory cytokines and chemokines in response to inflammatory mediators or microbial infection, consistent with an integral role for these cells in initiating and regulating mucosal innate and acquired immunity. The overall hypothesis of this proposal is that the defined array of chemokine receptors constitutively expressed by human intestinal epithelial cells activates G-protein-coupled signaling pathways permitting these cells to be functional targets of chemokine signaling. Studies in Aim 1 will characterize the cellular localization, ligand binding, and receptor internalization of chemokine receptors expressed by human intestinal epithelial cells. Intestinal epithelial cells are polarized into apical and basolateral domains, typically reflected in apical surface proteins specialized for nutrient absorption and ion secretion and basolateral membrane proteins specialized for maintenance of the electrochemical gradient. These studies will use molecular and immunocytochemical techniques to define the apical and basolateral cellular localization, ligand binding characteristics, and receptor internalization/desensitization of chemokine receptors constitutively expressed by intestinal epithelial cells in vitro. Studies in Aim 2 will use a step-wise approach to define the G-protein linked intracellular signaling pathways activated by chemokine receptors expressed by human intestinal epithelial cells. Chemokine receptors are coupled to specific G-proteins and activate several intracellular signaling pathways in varying cell types. These studies will focus on defining the specific G-protein activated and the intracellular signaling pathways utilized by chemokine receptors constitutively expressed by intestinal epithelial cells. Together these studies will provide insights into the potential immune mediated regulation of epithelial cell functions, e.g. ion secretion, through the convergence of intracellular regulatory signaling pathways in chemokine receptor expressing intestinal epithelial cells. The expression and signaling of chemokine receptors by human intestinal epithelial cells indicates these cells may respond in an autocrine or paracrine manner to enteric chemokine receptor ligangs and is consistent with the notion that these cells have an essential role in the mucosal response to inflammation.
