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

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Background: The mechanism by which low dose methotrexate (MTX) exerts its anti-inflammatory and immunosuppressive effect in rheumatoid arthritis (RA) patients is still debated. Recently it has been related to the induction of apoptosis.

Objective: We investigated the degree of apoptotic induction by MTX in lymphocytic (Jurkat T, EL4 T, and Raji B) and monocytic cell lines (U937 and THP1) and its relation to reactive oxygen species (ROS) generation, as a possible mechanism underlying the apoptotic events.

Methods: All cell types were incubated with a range of MTX concentrations (0.001, 0.01, 0.1, 1, and 10 μM) for up to 24 h. Cytotoxicity was assessed by Trypan Blue exclusion and MTT test; cell size and granularity by forward and side scatters (FSC, SSC). Apoptosis was measured by Annexin V test and FDA polarization; and mitochondrial ROS generation by DHR123 probe and by NAC inhibition.

Results: MTX significantly reduced cell viability and proliferation in all cell lines, being most effective in the Jurkat T lymphocytic line. The MTX cytotoxicity (at the optimal concentrations corresponding to low dose MTX therapy) was attributed to apoptosis, as suggested by morphological changes (shrinkage, increased granularity) and confirmed by Annexin V binding and FDA hyperpolarization. The apoptotic induction and the ROS generation (statistically correlated to apoptosis) were most pronounced in the Jurkat and EL4 T cell lines, and were partially inhibited by the antioxidant N-acetyl L-cysteine (NAC).

Conclusion: According to the present observations, MTX may most likely induce apoptosis through oxidative stress. The high susceptibility of T cell lines to MTX induced apoptosis may account for the beneficial effect of MTX treatment in rheumatoid arthritis, which is characterized by hyperproliferation of T cells.

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Correspondence to M. Deutsch.

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Received 29 June 2004; returned for revision 1 September 2004; returned for final revision 11 February 2005; accepted by M. J. Parnham 7 March 2005

This research was supported by the Horowitz Foundation

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Herman, S., Zurgil, N. & Deutsch, M. Low dose methotrexate induces apoptosis with reactive oxygen species involvement in T lymphocytic cell lines to a greater extent than in monocytic lines. Inflamm. res. 54, 273–280 (2005).

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