Regulation of tight junction formation in intestinal epithelial cells by the IL-15 ligand-receptor system

Abstract

Background: Human intestinal epithelial cells express IL-15 as well as IL-15 receptors consisting of the IL-15 receptor ct, IL-2 receptor 13 and 7c subunits. However the functional role of the IL-15 ligand receptor system in the regulation of intestinal epithelial cell function is unknown but may modulate the intestinal epithelial barrier. We assessed the regulation of protein levels and cellular distribution of the tight junction associated proteins ZO-1, ZO-2, and occludin in intestinal epithelial ceils by an IL-15 and IL-2 receptor 13 chain dependent pathway. Methods: The human intestinal epithelial cell line T-84, a model of intestinal crypt epithelium, which lacks the expression of the IL-2 receptor 13 chain constitutively but expresses IL-15, was stably transfected with the IL-2 receptor 13 chain to substitute a complete IL-15 receptor complex. Formation of tight junctions was determined by the measurment of transepithelial electrical resistance (TER). Expression of ZO-1, ZO-2, occludin, and E-cadherin as well as phosphotyrosine kinase activity was assessed by Western blotting of membrane and cytosol associated proteins. Cell proliferation was determined by 3H Thymidine uptake and MTS assays. Results: Expression of the IL-2 receptor 13 chain in T-84 cells resulted in a 3 fold increase of steady state TER within 4 four days after replating above the levels reached by the parental T-84 cells. The development TER in IL-2 receptor 13 chain transfected T-84 cells could be further enhanced by exogenous IL-15 and blocked by addition of anti IL-2 receptor antibodies. In contrast cell proliferation of IL-2 receptor 13 chain transfected and parental T-84 ceils was not altered. IL-2 receptor 13 chain transfected T-84 ceils demonstrated strong upregulation of tyrosine phosphorylation of a number of proteins with molecular masses of 120-100 and 60-40 kDa. The protein levels of ZO-1 and phosphorylated and unphosphorylated Occludin were increased 10 fold in the membrane associated protein fractions of IL-2 receptor 13 chain transfected T-84 cells compared to parental cells. In contrast, the levels of ZO-2 in membrane associated fractions of the stable transfectans was decreased 3 fold. The protein levels of E-cadherin were not regulated by the expression of the IL-2 receptor 13 chain in T-84 cells. Conclusion: Reconstition of a complete IL-15 receptor complex in T-84 cells results in autocrine regulation of the tight junction associated proteins ZO-1, ZO-2 and Occludin leading to the marked increase of TER. IL-15 may be an important regulator of intestinal epithelial cell barrier function.