Methotrexate, a folate antagonist, is a potent antiinflammatory agent when used weekly in low concentrations. We examined the hypothesis that the antiphlogistic effects of methotrexate result from its capacity to promote intracellular accumulation of 5-aminoimidazole4-carboxamide ribonucleotide (AICAR) that, under conditions of cell injury, increases local adenosine release. We now present the first evidence to establish this mechanism of action in an in vivo model of inflammation, the murine air pouch model. Mice were injected intraperitoneally with either methotrexate or saline for 34 wk during induction of air pouches. Pharmacologically relevant doses of methotrexate increased splenocyte AICAR content, raised adenosine concentrations in exudates from carrageenan-inflamed air pouches, and markedly inhibited leukocyte accumulation in inflamed air pouches. The methotrexate-mediated reduction in leukocyte accumulation was partially reversed by injection of adenosine deaminase (ADA) into the air pouch, completely reversed by a specific adenosine A2 receptor antagonist, 3,7-dimethyl-l-propargylxanthine (DMPX), but not affected by an adenosine Al receptor antagonist, 8-cyclopentyl-dipropylxanthine. Neither ADA nor DMPX affected leukocyte accumulation in the inflamed pouches of animals treated with either saline or the potent antiinflammatory steroid dexamethasone. These results indicate that methotrexate is a nonsteroidal antiinflammatory agent, the antiphlogistic action of which is due to increased adenosine release at inflamed sites. (J. Clin. Invest. 1993Invest. . 92:2675Invest. -2682
To examine the role of complement components as regulators of the expression of endothelial adhesive molecules in response to immune complexes (ICs), we determined whether ICs stimulate both endothelial adhesiveness for leukocytes and expression of E-selectin and intercellular and vascular cell adhesion molecules 1 (ICAM-1 and VCAM-1). We found that ICs [bovine serum albumin (BSA)-anti-BSA] stimulated endothelial cell adhesiveness for added leukocytes in the presence of complement-sufficient normal human serum (NHS) but not in the presence of heatinactivated serum (HIS) or in tissue culture medium alone. Depletion of complement component C3 or C8 from serum did not prevent enhanced endothelial adhesiveness stimulated by ICs. In contrast, depletion of complement component Clq markedly inhibited IC-stimulated endothelial adhesiveness for leukocytes. When the heat-labile complement component Clq was added to HIS, the capacity of ICs to stimulate endothelial adhesiveness for leukocytes was completely restored. Further evidence for the possible role of Clq in mediating the effect of ICs on endothelial cells was the discovery of the presence of the 100-to 126-kDa Clq-binding protein on the surface of endothelial cells (by cytofluorography) and of message for the 33-kDa Clq receptor in resting endothelial cells (by reverse transcription-PCR). Inhibition of protein synthesis by cycloheximide blocked endothelial adhesiveness for leukocytes stimulated by either interleukin 1 or ICs in the presence of NHS. After stimulation with ICs in the presence of NHS, endothelial cells expressed increased numbers of adhesion molecules (E-selectin, ICAM-1, and VCAM-1). Endothelial expression of adhesion molecules mediated, at least in part, endothelial adhesiveness for leukocytes, since leukocyte adhesion was blocked by monoclonal antibodies directed against E-selectin. These studies show that ICs stimulate endothelial cells to express adhesive proteins for leukocytes in the presence of a heat-labile serum factor. That factor appears to be Clq.Vascular endothelium plays a central role in inflammation by expressing specific adhesion molecules including P-selectin, E-selectin, and intercellular and vascular cell adhesion molecules 1 (ICAM-1 and VCAM-1) that attract and localize leukocytes to inflamed sites (reviewed in ref. 1). The recent observation that endothelial expression of E-selectin is required for development of inflammatory injury after intravascular injection of immune complexes (ICs) suggests an active role for endothelium in IC-mediated vasculitis (2). However,
Objective. The acute antiinflammatory effects of methotrexate are mediated, at least in part, by increased extracellular adenosine concentrations at inflamed sites. This observation suggests that other agents that increase extracellular adenosine concentrations might also reduce inflammation. Since adenosine can be rapidly taken up by cells, phosphorylated by adenosine kinase, and maintained intracellularly as adenine nucleotides, we investigated whether a potent inhibitor of adenosine kinase, GP-1-515, could increase exudate adenosine concentration and thereby diminish inflammation in the murine air pouch model of inflammation.Methods. We studied the effect of various oral doses of GP-1-515 on carrageenan-induced inflammation in air pouches induced on BALB/c mice. Adenosine concentration in pouch exudates was determined by high performance liquid chromatography, and intensity of inflammation was determined by leukocyte counts in the exudate fluid.Results. There was a greater concentration of adenosine in the pouch exudates of animals treated with GP-1-515 than of those treated with saline (P < 0.002). GP-1-515 inhibited, in a dose-dependent manner (P < 0.01), leukocyte accumulation in the murine air pouch in response to carrageenan. Inhibition of inflammation by GP-1-515 in this model depended upon increased adenosine concentration in the inflamed pouch since injection of adenosine deaminase into the air pouch with the carrageenan completely reversed the antiinflammatory effects of GP-1-515 at all doses of GP-1-515 tested. Moreover, as previously demonstrated, the antiinflammatory effects of adenosine were mediated via occupancy of adenosine A, receptors, since the specific adenosine A, receptor antagonist 3,7-dimethyl-lpropargylxanthine, but not the A, receptor antagonist 8-cyclopentyl-dipropylxanthine, completely reversed the antiinflammatory effects of GP-1-515. GP-1-515 also decreased tumor necrosis factor a levels in the air pouch exudates by 51%, most likely as a result of the direct action of adenosine on macrophages. Conclusion.These results indicate that the antiinflammatory actions of GP-1-515 are mediated by adenosine. The development of agents that promote adenosine release at sites of inflammation is a novel strategy for the treatment of inflammatory diseases such as rheumatoid arthritis.In recent studies we have demonstrated that the antiinflammatory action of methotrexate (MTX), a commonly used agent for the treatment of rheumatoid arthritis, is mediated, at least in part, by increased release of adenosine at sites of inflammation. Treatment of mice with MTX is associated with intracellular accumulation of 5-aminoimidazolecarboxamidoribonucleotide (AICAR) which, when it accumulates intracellularly, promotes adenosine release by a mechanism that is not fully understood. The adenosine released at sites of inflammation diminishes inflammation via occupancy of specific A, receptors on inflammatory cells, since antagonism of A*, but not A,, receptors reverses the antiinflammatory effects of MTX in an ...
Adenosine modulates generation of superoxide anion by neutrophils via occupancy of specific adenosine A 2A receptors. However, the intracellular signal transduction pathways by which occupancy of neutrophil adenosine A 2A receptors inhibits superoxide anion generation (O 2 . ) are not well understood. We, therefore, tested the hypothesis that signaling at polymorphonuclear leukocyte (PMN) adenosine receptors proceeds via activation of a serine/threonine protein phosphatase (pp subcellular fractions demonstrated the presence of pp1␣ and pp1␥1 but not pp1␥ 2 isotypes in both cytosol and plasma membrane but not in azurophil or specific granules. We conclude from these studies that signal transduction by adenosine in PMN proceeds via a novel pathway: cAMP-independent activation of a serine/threonine protein phosphatase in the plasma membrane.Adenosine, an autacoid released by many different cell types, regulates a variety of stimulated neutrophil functions including production of superoxide anion generation (1, 2),  2 -integrin-and L-selectin-mediated adhesion to endothelial cells (3, 4), and phagocytosis (5). Adenosine regulates these neutrophil functions by coupling with specific cell surface receptors (2) on the neutrophil (reviewed in Ref. 6).Four different types of adenosine receptors (A 1 , A 2A , A 2B , and A 3 ) have been described, both at the molecular level and by pharmacological analyses (7). Based on pharmacologic data, it appears that the A 2A receptor on neutrophils mediates the inhibition of neutrophil superoxide anion generation, adhesion, and phagocytosis (6). Before their resolution at the molecular level, adenosine A 2 receptors were believed to modulate cellular function via activation of adenylyl cyclase, with cAMP as their intracellular messenger. As expected, occupancy of adenosine A 2 receptors stimulated the accumulation of cAMP in neutrophils, but unexpectedly, cAMP proved not to be the second messenger for inhibition of stimulated superoxide anion generation (8 -10).Because adenosine A 2 receptor occupancy had previously been shown to increase cytosolic protein phosphatase activity in bovine adrenal chromaffin (PC12) cells (11), we tested the hypothesis that occupancy of neutrophil adenosine receptors stimulates an increase in the serine/threonine protein phosphatase activity of the plasma membrane where it would be situated to modulate function of the neutrophil NADPH-oxidase. We found that the protein phosphatase 1 (pp1) 1 inhibitor calyculin A completely reversed the effects of adenosine receptor occupancy on stimulated neutrophil generation of superoxide anion. Moreover, adenosine receptor occupancy stimulated an increase in plasma membrane-associated protein phosphatase activity, most likely pp1, and that activation of this phosphatase is independent of cAMP. , N-formylmethionyl-leucyl-phenylalanine (FMLP), ATP, phosphorylase b, phosphorylase kinase, bovine serum albumin, Ficoll type 70, cytochalasin B, caffeine, cytochrome c, superoxide dismutase, and Brij were supplied by Sigma. K...
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