Our hypothesis is that adhesion molecule-dependent modulation of T cells and endothelia modulates not only adhesive functions, but also initiates specific protease induction, surface assembly, and activation which facilitates transmigration, as well as changes in adhesive properties of the T cells which affects residency of the cells at the site of inflammation. T cell transmigration through the endothelial cell layer and migration into the underlying and surrounding extracellular matrix is initiated by T cell adhesion to the endothelium, mediated by specific ligands resident on the surfaces of both the T cell [VLA-4 (a4B1)] and the endothelial cell (VCAM-1). We have demonstrated that engagement of this receptor/ligand pair evokes changes in MMP-2 expression and activation, consistent with the manifestation of an invasive phenotype in the adherent T cell population and an """"""""activated"""""""" phenotype in the endothelial cells. Resultant proteolysis of basement membrane and interstitial matrix components is thought to facilitate T cell extravasation out of the affected vessel and toward the site of inflammation and angiogenesis. In this proposal we will: 1) determine, compare and contrast the MT1-MMP/TIMP-2/MMP-2 ternary complex characteristics in T lymphocytes and endothelial cells following their stimulation. 2) continue our characterizations of the MT1-MMP and MMP-2 promoters and their respective pertinent transcription factors. 3) identify, characterize, compare and contrast the signal transduction pathways involved in MT1-MMP and MMP-2 induction, complex formation, activation and clustering in T lymphocytes and endothelial cells.
These aims will be accomplished with a combination of methodologies including an in vitro culture model utilizing antigen-specific murine T cell clones and lines; an in vivo adoptive transfer murine model of experimental allergic encephalomyelitis (EAE) and a variety of cellular and molecular biological techniques including cell culture, zymography, reverse zymography, immunoprecipitation, Western blotting, Northern blotting, transfection and stable expression of selected gene products, histology, immunohistochemistry, MALDI-TOF, DNA array analyses and the use of selected transgenic and knockout mice. These experiments will lead to a better understanding of T cell migration through and interaction with local extracellular matrix and the development of new and novel therapies directed at modulating selected proteinase/proteinase inhibitor cascade systems in the inflammatory processes of arthritis, vasculitis, and tissue rejection organ.
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