Methotrexate and sulfasalazine promote adenosine release by a mechanism that requires ecto-5'-nucleotidase-mediated conversion of adenine nucleotides.

Abstract

We and others have shown that an increased extracellular concentration of adenosine mediates the antiinflammatory effects of methotrexate and sulfasalazine both in vitro and in vivo, but the mechanism by which these drugs increase extracellular adenosine remains unclear. The results of the experiments reported here provide three distinct lines of evidence that adenosine results from the ecto-5'-nucleotidase- mediated conversion of adenine nucleotides to adenosine. First, pretreatment of a human microvascular endothelial cell line (HMEC-1) with methotrexate increases extracellular adenosine after exposure of the pretreated cells to activated neutrophils; the ecto-5'-nucleotidase inhibitor alpha, beta-methylene adenosine-5'-diphosphate (APCP) abrogates completely the increase in extracellular adenosine. Second, there is no methotrexate-mediated increase in extracellular adenosine concentration in the supernate of cells deficient in ecto-5'-nucleotidase, but there is a marked increase in extracellular adenosine concentration in the supernates of these cells after transfection and surface expression of the enzyme. Finally, as we have shown previously, adenosine mediates the antiinflammatory effects of methotrexate and sulfasalazine in the murine air pouch model of inflammation, and injection of APCP, the ecto-5'-nucleotidase inhibitor, abrogates completely the increase in adenosine and the decrement in inflammation in this in vivo model. These results not only show that ecto-5'-nucleotidase activity is a critical mediator of methotrexate- and sulfasalazine-induced antiinflammatory activity in vitro and in vivo but also indicate that adenine nucleotides, released from cells, are the source of extracellular adenosine.

IFN‐β regulates CD73 and adenosine expression at the blood–brain barrier

“…CD73 modulates the extent of immune and inflammatory responses in vitro and in vivo. These effects are mediated via its enzymatic capability to hydrolyse adenosine monophosphate 5 0 -AMP into adenosine, an immunomodulatory purine compound [16][17][18].We have recently shown that CD73 is upregulated on vascular endothelium in a time-and dose-dependent manner after treatment with type-I IFN-a in vitro and in vivo [19], but how IFN-b affects CD73 in brain EC and in the context of MS has never been studied before. As adenosine, the end product of the CD73 catalytic reaction, has both anti-inflammatory and neuroprotective effects, we hypothesized that IFN-b might also exert some of its beneficial effects on MS-disease pathology by regulating CD73-mediated adenosine production.…”

“…CD73 modulates the extent of immune and inflammatory responses in vitro and in vivo. These effects are mediated via its enzymatic capability to hydrolyse adenosine monophosphate 5 0 -AMP into adenosine, an immunomodulatory purine compound [16][17][18].…”

Regulation of bone and cartilage by adenosine signaling

“…This enzyme modulates cartilage homeostasis and displays increased levels of activity in diseased cartilage [99,100]. Chondrocytes in osteoarthritic lesions show elevated 5NT activity relative to nonlesioned areas, and this causes increased extracellular adenosine levels, and in turn, higher intracellular concentrations via transporters.…”

Eficácia terapêutica do Metotrexato na Artrite Reumatóide depende da expressão de células T reguladoras CD39+?

“…Trabalhos também propõem que a eficácia terapêutica observada com a administração de baixas doses de MTX, é decorrente de sua capacidade em manter e aumentar os níveis de adenosina extracelular (Cronstein et al, 1993;Morabito et al, 1998 (Thiel et al, 1996), adesão (Cronstein et al, 1990), produção dos radicais de oxigênio (Cronstein et al, 1985), degranulação (Richter, 1992) e produção de TNF-α (Thiel & Chouker, 1995 Tregs, e conseqüentemente, ocasionar a geração de células T ativadas refratárias à supressão mediada pela adenosina (Bansard et al, 2009). …”

“…Dados na literatura sugerem que a eficácia terapêutica observada com a utilização do MTX foi decorrente de sua capacidade em manter os níveis extracelulares de adenosina elevados (Cronstein et al, 2005;Montesinos et al, 2007;Morabito et al, 1998). No presente estudo, nós demonstramos um mecanismo de refratariedade de pacientes com AR ao tratamento com MTX baseado no número de células Tregs circulantes e na contribuição deste subtipo leucocitário para a geração de adenosina extracelular pelas ectonucleotidases presentes em sua superfície.…”