The anti-inflammatory actions of methotrexate are critically dependent upon the production of reactive oxygen species

Br J Pharmacol. 2003 Feb;138(3):501-11. doi: 10.1038/sj.bjp.0705054.


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.

MeSH terms

  • Anti-Inflammatory Agents / administration & dosage
  • Anti-Inflammatory Agents / pharmacology*
  • Antigens, Surface / metabolism
  • Apoptosis
  • Cell Adhesion / drug effects
  • Cell Cycle / drug effects
  • Dose-Response Relationship, Drug
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / physiology
  • Flow Cytometry
  • Glutathione / metabolism
  • Humans
  • Jurkat Cells
  • Methotrexate / administration & dosage
  • Methotrexate / pharmacology*
  • Monocytes / drug effects
  • Monocytes / physiology
  • Peroxides / metabolism
  • Reactive Oxygen Species / metabolism*
  • T-Lymphocytes / drug effects
  • T-Lymphocytes / metabolism
  • Time Factors
  • U937 Cells
  • Umbilical Veins / cytology


  • Anti-Inflammatory Agents
  • Antigens, Surface
  • Peroxides
  • Reactive Oxygen Species
  • Glutathione
  • Methotrexate