Skip to main content
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1998 Jan 15;101(2):295–300. doi: 10.1172/JCI1554

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

L Morabito 1, M C Montesinos 1, D M Schreibman 1, L Balter 1, L F Thompson 1, R Resta 1, G Carlin 1, M A Huie 1, B N Cronstein 1
PMCID: PMC508567  PMID: 9435300

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.

Full Text

The Full Text of this article is available as a PDF (165.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baggott J. E., Morgan S. L., Ha T. S., Alarcón G. S., Koopman W. J., Krumdieck C. L. Antifolates in rheumatoid arthritis: a hypothetical mechanism of action. Clin Exp Rheumatol. 1993 Mar-Apr;11 (Suppl 8):S101–S105. [PubMed] [Google Scholar]
  2. Baggott J. E., Vaughn W. H., Hudson B. B. Inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase, adenosine deaminase and 5'-adenylate deaminase by polyglutamates of methotrexate and oxidized folates and by 5-aminoimidazole-4-carboxamide riboside and ribotide. Biochem J. 1986 May 15;236(1):193–200. doi: 10.1042/bj2360193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barankiewicz J., Jimenez R., Ronlov G., Magill M., Gruber H. E. Alteration of purine metabolism by AICA-riboside in human B lymphoblasts. Arch Biochem Biophys. 1990 Nov 1;282(2):377–385. doi: 10.1016/0003-9861(90)90132-i. [DOI] [PubMed] [Google Scholar]
  4. Barankiewicz J., Ronlov G., Jimenez R., Gruber H. E. Selective adenosine release from human B but not T lymphoid cell line. J Biol Chem. 1990 Sep 15;265(26):15738–15743. [PubMed] [Google Scholar]
  5. Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl. 1968;97:77–89. [PubMed] [Google Scholar]
  6. Chabner B. A., Allegra C. J., Curt G. A., Clendeninn N. J., Baram J., Koizumi S., Drake J. C., Jolivet J. Polyglutamation of methotrexate. Is methotrexate a prodrug? J Clin Invest. 1985 Sep;76(3):907–912. doi: 10.1172/JCI112088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cronstein B. N., Eberle M. A., Gruber H. E., Levin R. I. Methotrexate inhibits neutrophil function by stimulating adenosine release from connective tissue cells. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2441–2445. doi: 10.1073/pnas.88.6.2441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cronstein B. N., Kimmel S. C., Levin R. I., Martiniuk F., Weissmann G. A mechanism for the antiinflammatory effects of corticosteroids: the glucocorticoid receptor regulates leukocyte adhesion to endothelial cells and expression of endothelial-leukocyte adhesion molecule 1 and intercellular adhesion molecule 1. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):9991–9995. doi: 10.1073/pnas.89.21.9991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cronstein B. N., Kramer S. B., Weissmann G., Hirschhorn R. Adenosine: a physiological modulator of superoxide anion generation by human neutrophils. J Exp Med. 1983 Oct 1;158(4):1160–1177. doi: 10.1084/jem.158.4.1160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cronstein B. N. Molecular therapeutics. Methotrexate and its mechanism of action. Arthritis Rheum. 1996 Dec;39(12):1951–1960. doi: 10.1002/art.1780391203. [DOI] [PubMed] [Google Scholar]
  11. Cronstein B. N., Naime D., Firestein G. The antiinflammatory effects of an adenosine kinase inhibitor are mediated by adenosine. Arthritis Rheum. 1995 Aug;38(8):1040–1045. doi: 10.1002/art.1780380804. [DOI] [PubMed] [Google Scholar]
  12. Cronstein B. N., Naime D., Ostad E. The antiinflammatory mechanism of methotrexate. Increased adenosine release at inflamed sites diminishes leukocyte accumulation in an in vivo model of inflammation. J Clin Invest. 1993 Dec;92(6):2675–2682. doi: 10.1172/JCI116884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Decking U. K., Schlieper G., Kroll K., Schrader J. Hypoxia-induced inhibition of adenosine kinase potentiates cardiac adenosine release. Circ Res. 1997 Aug;81(2):154–164. doi: 10.1161/01.res.81.2.154. [DOI] [PubMed] [Google Scholar]
  14. Deussen A., Bading B., Kelm M., Schrader J. Formation and salvage of adenosine by macrovascular endothelial cells. Am J Physiol. 1993 Mar;264(3 Pt 2):H692–H700. doi: 10.1152/ajpheart.1993.264.3.H692. [DOI] [PubMed] [Google Scholar]
  15. Gadangi P., Longaker M., Naime D., Levin R. I., Recht P. A., Montesinos M. C., Buckley M. T., Carlin G., Cronstein B. N. The anti-inflammatory mechanism of sulfasalazine is related to adenosine release at inflamed sites. J Immunol. 1996 Mar 1;156(5):1937–1941. [PubMed] [Google Scholar]
  16. Gruber H. E., Hoffer M. E., McAllister D. R., Laikind P. K., Lane T. A., Schmid-Schoenbein G. W., Engler R. L. Increased adenosine concentration in blood from ischemic myocardium by AICA riboside. Effects on flow, granulocytes, and injury. Circulation. 1989 Nov;80(5):1400–1411. doi: 10.1161/01.cir.80.5.1400. [DOI] [PubMed] [Google Scholar]
  17. Gunning P., Leavitt J., Muscat G., Ng S. Y., Kedes L. A human beta-actin expression vector system directs high-level accumulation of antisense transcripts. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4831–4835. doi: 10.1073/pnas.84.14.4831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hirschhorn R., Roegner-Maniscalco V., Kuritsky L., Rosen F. S. Bone marrow transplantation only partially restores purine metabolites to normal in adenosine deaminase-deficient patients. J Clin Invest. 1981 Dec;68(6):1387–1393. doi: 10.1172/JCI110389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kitakaze M., Hori M., Morioka T., Takashima S., Minamino T., Sato H., Inoue M., Kamada T. Attenuation of ecto-5'-nucleotidase activity and adenosine release in activated human polymorphonuclear leukocytes. Circ Res. 1993 Sep;73(3):524–533. doi: 10.1161/01.res.73.3.524. [DOI] [PubMed] [Google Scholar]
  20. Madara J. L., Patapoff T. W., Gillece-Castro B., Colgan S. P., Parkos C. A., Delp C., Mrsny R. J. 5'-adenosine monophosphate is the neutrophil-derived paracrine factor that elicits chloride secretion from T84 intestinal epithelial cell monolayers. J Clin Invest. 1993 May;91(5):2320–2325. doi: 10.1172/JCI116462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Newby A. C., Holmquist C. A., Illingworth J., Pearson J. D. The control of adenosine concentration in polymorphonuclear leucocytes, cultured heart cells and isolated perfused heart from the rat. Biochem J. 1983 Aug 15;214(2):317–323. doi: 10.1042/bj2140317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Oritani K., Kincade P. W. Identification of stromal cell products that interact with pre-B cells. J Cell Biol. 1996 Aug;134(3):771–782. doi: 10.1083/jcb.134.3.771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Resta R., Hooker S. W., Laurent A. B., Shuck J. K., Misumi Y., Ikehara Y., Koretzky G. A., Thompson L. F. Glycosyl phosphatidylinositol membrane anchor is not required for T cell activation through CD73. J Immunol. 1994 Aug 1;153(3):1046–1053. [PubMed] [Google Scholar]
  24. Vincent M. F., Bontemps F., Van den Berghe G. Substrate cycling between 5-amino-4-imidazolecarboxamide riboside and its monophosphate in isolated rat hepatocytes. Biochem Pharmacol. 1996 Oct 11;52(7):999–1006. doi: 10.1016/0006-2952(96)00413-3. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES