We have recently made the surprising discovery that adenosine mediates the antiinflammatory effects of methotrexate (MTX), one of the most effective agents used in the treatment of rheumatoid arthritis. Our observations suggest that MTX increases adenosine concentrations at inflammed sites by inhibiting 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase thereby increasing intracellular concentrations of AICAR. Intracellular accumulation of AICAR increases extracellular adenosine concentration by an unknown mechanism. To determine how MTX diminishes inflammation we propose to: 1. Examine the biochemical mechanism by which MTX increases extracellular adenosine concentrations of MTX (and its polyglutamates) that accumulate in human fibroblasts and endothelial cells, the concentrations of AICAR, inosine and adenine nucleotides in MTX-treated cells and the direct effect of MTX treatment on the activity of the enzymes most directly involved in regulation of adenosine release: adenosine deaminase, adenosine kinase and AMP deaminase. We will determine the concentration of nucleotides in the amount of adenosine release from and activity of adenosine-regulating enzymes in peripheral blood mononuclear cells of patients with RA before and 6 weeks after onset of treatment with MTX. 2. Examine signal transduction pathway at leukocyte adenosine A/2/A receptors. Although cAMP has been thought to mediate the effects of adenosine receptor ligation we have found evidence to indicate that adenosine inhibits leukocyte function via the cAMP- independent activation of a protein phosphatase (most likely protein phosphatase 1, pp1). To better understand signal transduction at adenosine receptors we will study adenosine receptor-stimulated protein phosphatase activity directly, adenosine-mediated translocation of protein phosphatases within the cell, effects of adenosine receptor occupancy on association of pp1 with regulatory proteins and molecular identification of pp1 regulatory proteins and substrates in the neutrophil. 3. Test the complementary hypothesis that the antiinflammatory effects of MTX result, in part, from inhibition of transmethylation reactions required for post- translational modification of proteins and lipids by examining transmethylation reactions directly. 4. Study the mechanism by which MTX inhibits chronic inflammation in animal models (collagen arthritis) and patients with rheumatoid arthritis. We will study the effect of monoclonal antiadenosine receptor antibodies on MTX inhibition of inflammation in appropriate animal models. To show evidence for adenosine-mediated suppression of inflammation in patients treated with MTX we will compare adenosine receptor expression on leukocytes from peripheral blood to those obtained from synovial fluid to establish whether there has been ligand-mediated down-regulation of adenosine receptors. We have designed these studies to improve our understanding of the antiinflammatory properties of MTX and we believe that the information gained from these studies will permit the development of more specific and less toxic therapeutic regimens for use in the therapy of rheumatoid arthritis and other chronic inflammatory diseases.
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