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

• Published in: British journal of pharmacology Vol. 138; no. 3; pp. 501 - 511
• Main Authors: Phillips, Darren C, Woollard, Kevin J, Griffiths, Helen R
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-02-2003; Nature Publishing
• Subjects: adhesion; Anti-Inflammatory Agents - administration & dosage; Anti-Inflammatory Agents - pharmacology; Antigens, Surface - metabolism; Apoptosis; Biological and medical sciences; Bones, joints and connective tissue. Antiinflammatory agents; Cell Adhesion - drug effects; Cell Cycle - drug effects; Dose-Response Relationship, Drug; Endothelium, Vascular - cytology; Endothelium, Vascular - physiology; Flow Cytometry; glutathione; Glutathione - metabolism; growth arrest; Humans; Immunomodulators; immunosuppression; inflammation; Jurkat Cells; Medical sciences; Methotrexate; Methotrexate - administration & dosage; Methotrexate - pharmacology; Monocytes - drug effects; Monocytes - physiology; Peroxides - metabolism; Pharmacology. Drug treatments; reactive oxygen species; Reactive Oxygen Species - metabolism; rheumatoid arthritis; T-Lymphocytes - drug effects; T-Lymphocytes - metabolism; Time Factors; U937 Cells; Umbilical Veins - cytology; Endothelial cell; Oxygen; Monocyte; glutathione; Cell cell interaction; apoptosis; immunosuppression; Adhesion; In vitro; rheumatoid arthritis; Biological activity; Free radical; Non steroidal antiinflammatory agent; inflammation; Blood vessel; Established cell line; T-Lymphocyte; reactive oxygen species; growth arrest; Circulatory system; Immunosuppressive agent; Intracellular; Mechanism of action; Methotrexate
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1038/sj.bjp.0705054

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).
Addition of MTX (100 nM–10 μM) 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).
MTX treatment of monocytes (10 nM–10 μM) 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 μM compared to controls (P<0.05).
MTX treatment significantly reduced monocyte adhesion to 5 h and 24 h LPS (1 μg 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.
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.
British Journal of Pharmacology (2003) 138, 501–511. doi:10.1038/sj.bjp.0705054