title: The anti-inflammatory actions of methotrexate are critically dependent upon the production of reactive oxygen species.
abstract: 1 The mechanism of action by which methotrexate (MTX) exerts its anti-inflammatory and immunosuppressive effects remains unclear. The aim of this study is to investigate the hypothesis that MTX exerts these effects via the production of reactive oxygen species (ROS). 2 Addition of MTX (100 nM-10 micro M) to U937 monocytes induced a time and dose dependent increase in cytosolic peroxide [peroxide](cyt) from 6-16 h. MTX also caused corresponding monocyte growth arrest, which was inhibited (P<0.05) by pre-treatment with N-acetylcysteine (NAC; 10 mM) or glutathione (GSH; 10 mM). In contrast, MTX induction of [peroxide](cyt) in Jurkat T cells was more rapid (4 h; P<0.05), but was associated with significant apoptosis at 16 h at all doses tested (P<0.05) and was significantly inhibited by NAC or GSH (P<0.05). 3 MTX treatment of monocytes (10 nM-10 micro M) for 16 h significantly reduced total GSH levels (P<0.05) independently of dose (P>0.05). However, in T-cells, GSH levels were significantly elevated following 30 nM MTX treatment (P<0.05) but reduced by doses exceeding 1 micro M compared to controls (P<0.05). 4 MTX treatment significantly reduced monocyte adhesion to 5 h and 24 h LPS (1 micro g ml(-1)) activated human umbilical vein endothelial cells (HUVEC; P<0.05) but not to resting HUVEC. Pre-treatment with GSH prevented MTX-induced reduction in adhesion. 5 In conclusion, ROS generation by MTX is important for cytostasis in monocytes and cytotoxicity T-cells. Furthermore, MTX caused a reduction in monocyte adhesion to endothelial cells, where the mechanism of MTX action requires the production of ROS. Therefore its clinical efficacy can be attributed to multiple targets.
authors.affiliation: Molecular Biosciences Group, Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET.
authors.forename: Darren C
authors.initials: DC
authors.lastname: Phillips
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Woollard
authors.forename: Helen R
authors.initials: HR
authors.lastname: Griffiths
mesh_terms: D000893:Anti-Inflammatory Agents / Q000008:administration & dosage / Q000494:pharmacology*; D000954:Antigens, Surface / Q000378:metabolism; D017209:Apoptosis; D002448:Cell Adhesion / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology; D005434:Flow Cytometry; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008727:Methotrexate / Q000008:administration & dosage / Q000494:pharmacology*; D009000:Monocytes / Q000187:drug effects / Q000502:physiology; D010545:Peroxides / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D020298:U937 Cells; D014471:Umbilical Veins / Q000166:cytology
chemical_list: D000893:Anti-Inflammatory Agents; D000954:Antigens, Surface; D010545:Peroxides; D017382:Reactive Oxygen Species; D005978:Glutathione; D008727:Methotrexate