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

Abstract

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:

Low-Dose Methotrexate: A Mainstay in the Treatment of Rheumatoid Arthritis

“…Based on in vitro studies, it has recently been reported that methotrexate reduces intracellular glutathione concentrations, and this leads to reversible inhibition of macrophage and lymphocyte function (Phillips et al, 2003). Although this is an interesting hypothesis and the in vitro data reported in this article generally sup-166 port this conclusion, it is unlikely to explain the antiinflammatory actions of methotrexate in patients with rheumatoid arthritis since intracellular glutathione levels are reduced to well below the methotrexate-induced levels already in the synovial cells of these patients (Maurice et al, 1997).…”

Low dose methotrexate induces apoptosis with reactive oxygen species involvement in T lymphocytic cell lines to a greater extent than in monocytic lines

“…It has been demonstrated also that a synthetic analogue of prostaglandin E 1 prevents the production of ROS in the intestinal mucosa of MTX treated rats [34]. Finally, Phillips et al reported that ROS generation by MTX plays an important role in cytostasis of monocytes and cytotoxicity in T-cells [30].…”

“…Various cytokines [29,30] and free radicals including ROS play a crucial role in the inflammatory and immunity processes involved in rheumatoid arthritis (RA) [31]. The earliest evidence for pro-oxidant properties of MTX was presented by Gressier et al [32].…”

“…Few attempts have been made to relate the immunosuppressive effect of MTX in RA patients to ROS production. [29,30]. The first related report was made by Babiak et al [33] who investigated the effect of MTX on the activity of enzymes involved in enzyme defense mechanisms against ROS in HeLa cells.…”

Ameliorative effect of gallic acid on methotrexate-induced hepatotoxicity and nephrotoxicity in rat

“…1 MTX is a cytotoxic agent used in the treatment of leukaemia and various malignancies, as well as in non-neoplastic diseases such as psoriasis, rheumatoid arthritis and as anti-inflammatory and immunosuppressive agent. [1][2][3] Earlier studies have revealed that MTX is converted mainly to metabolites connected to glutamate (MTX-glu) in cells and tissues. 4 The complex so formed is referred to as polyglutamated forms which is responsible for most biochemical activities of MTX.…”