MECNHANISMS OF CELL ADHESION AND TRAFFICKING IN EXPERIMENTAL ILEITIS

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Project 4, headed by Dr. Klaus Ley, studies the mechanisms of intestinal T cell adhesion and inflammatory cell trafficking in SAMP1/YitFc (SAMP) mice. The original project was designed to test the hypothesis that: 1) SAMP mice express adhesion molecules relevant to the trafficking of T cells, neutrophils, and other leukocytes, 2) leukocytes and/or endothelial adhesion molecules are required for the development of ileitis in recipients of CD4+ T cells transferred from SAMP mice, and 3) CD4+ T cells use specific adhesion molecules to home to the terminal ileum of SAMP mice and cause disease. In the past four years, important progress has been made with respect to the discovery that the endothelial adhesion molecules VCAM-1, ICAM-1, MAdCAM-1, P-selectin, and PNAd are significantly upregulated in inflamed areas of the SAMP ileum. Therapeutic studies have shown that blocking alpha 4 integrins, a combination of VCAM-1 and ICAM-1, or a combination of MAdCAM-1 and L-selectin significantly ameliorates disease severity in the adoptive transfer model. The finding that L-selectin is important in promoting inflammation represented a novel and unexpected observation regarding this molecule, since L-selectin is thought to be expressed on naive T cells. However, L-selectin inhibition may limit the recruitment of inflammatory myeloid cells, as well as naive, memory, and regulatory T cells, or a combination thereof. In addition, we discovered that B cells play a pathogenic role in the SAMP model of ileitis. For the next funding period, Project 4 will focus on L-selectin and one of its ligands, PSGL-1, based on two recent discoveries: 1) demonstration of the importance of L-selectin in mediating ileitis, and 2) preliminary evidence that PSGL-1 is expressed in endothelial cells of lamina propria microvessels in the terminal ileum. Based on these discoveries, this project will first test whether the L-selectin ligand PSGL-1 is expressed in inflamed endothelial cells of the ileal lamina propria and submucosa using immunostaining, laser capture microdissection, and real-time RT-PCR. The functional role of the PSGL-1/L-selectin pathway of adhesion will be studied using monoclonal antibodies to block PSGL-1, as well as by crossing SAMP mice with existing PSGL-1 deficient mice using speed congenics. Finally, the nature of L-selectin expressing cells that contribute to ileitis severity in the SAMP adoptive transfer model will be studied. These studies are designed to understand the mechanisms of trafficking of pathogenic, naive and regulatory lymphocytes in the inflamed ileum of SAMP mice and to provide a rational basis for ameliorating disease severity by single or combined blockade of intestinal-specific adhesion molecules in patients with Crohn's disease.