The primary goal of this research is to delineate the mechanisms by which CD73 (ecto-5'-nucleotidase) controls inflammatory responses using our newly developed CD73 gene-targeted mice. CD73 regulates adenosine receptor signaling through its ability to produce ligand (adenosine) from AMP. We will focus initially on two model systems where adenosine receptors are known to be important: the carrageenan-treated air pouch (Aim I) and LPS-induced septic shock (Aim II). CD73-/- mice show reduced accumulation of neutrophils in inflamed air pouches, suggesting a generalized defect in neutrophil activation or migration. Pre-treatment of CD73+ mice with methotrexate increases adenosine levels in the air pouches and correlates with a marked reduction in leukocyte influx. The anti-inflammatory action of methotrexate is dependent upon CD73. In the second model, wild type mice are protected from the lethal effects of LPS by the adenosine kinase inhibitor GP-1-515 that increases local adenosine levels. CD73-/- mice, however, are refractory to the protective effects of GP-1-515. Thirdly, we will investigate the ability of CD73-/- mice to mount a delayed type hypersensitivity (DTH) response (Aim III). CD39-/- mice are defective both in DTH responses and the ability to generate extracellular AMP, the substrate for CD73, leading to our hypothesis that CD73-generated adenosine is required for this type of inflammatory response. Fourth, we will determine whether CD73 is responsible for the generation of adenosine causing inflammatory lung disease in adenosine deaminase (ADA)-deficient mice by crossing them to our CD73 knock-outs (Aim IV). In all four inflammatory models (Aims I-IV), bone marrow chimeras will be used to determine whether the critical CD73-expressing cells are of hematopoietic or non-hematopoietic origin. Finally, we will determine whether the glycosyl phosphatidylinositol (GPI) membrane anchor of CD73 is required for its inflammation modulating capability by producing mice expressing a transmembrane form of CD73 and studying their ability to regulate inflammatory responses (Aim V). Overall, these studies will provide a clearer understanding of the function of CD73 and other GPI-anchored proteins and may suggest new therapeutic interventions in inflammatory diseases.
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