Upon cell-cell contact, one of the mechanisms of immune cells to cause tissue damage is by delivery of lytic granules containing preformed effector molecules--perforin proteins and granzyme proteases. The presence of granzymes and perforin in the extracellular milieu not only reflects the presence of activated CTL and NK cells but also significantly contribute to inflammatory reaction. These protein enzymes can cause circular pore-like lesions on the membrane surface of endothelial cells and induce target cell death leading to local tissue damage and chronic vascular cell damage. Increased granzyme level is closely correlated with the inflammatory activity in autoimmune diseases (e.g. rheumatoid arthritis) and virally-infected lung and heart diseases. A clear understanding of the pathophysiology of inflammation mediated tissue damage would greatly facilitate management of this disease. The signal transduction pathways in effector lymphocytes, which trigger the redistribution of the lytic granules towards the target endothelial cells, are not well defined. Identification of key signaling molecules which specifically control this lytic process could enable pharmaceutical disruption of this process, thereby reducing the tissue damage mediated by activated lymphocytes. In this proposal, we will use a human primary pulmonary endothelial cell line, CRL- 2598, as the trigger to activate the lytic signal cascade in NK cells. Using biochemical and gene delivery approaches, we will directly test the hypothesis that the early signals via NK activation receptors and their associated adaptor proteins, DAP12 and DAP10, will play a specific role in control of granule movement in NK cells. Blocking of this initial step, at the level of the adaptor proteins, will inhibit granule exocytosis. More importantly, we will test our hypothesis on LGL leukemic patients with primary pulmonary hypertension (PPH) and determine if DAP10 and/or DAP12 critically control NK cell- mediated endothelial cell damage and death. A better understanding of the signaling pathways that control granule movement and exocytosis will offer new opportunities, e.g. design of DAP10 and DAP12 antagonists, for therapeutic intervention to specifically control lymphocyte-mediated tissue damage. ? ?
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