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


CONCLUSIONS
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.
Herman
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S
The Biophysical Interdisciplinary Jerome Schottenstein Center for the Research and the Technology of the Cellome, Physics Department, Bar-Ilan University, Ramat-Gan 52900, Israel.
Zurgil
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N
Deutsch
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D000111:Acetylcysteine / Q000494:pharmacology; D017304:Annexin A5 / Q000494:pharmacology; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D000964:Antimetabolites, Antineoplastic / Q000008:administration & dosage / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis; D001172:Arthritis, Rheumatoid; D001681:Biological Assay; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D007166:Immunosuppressive Agents; D019169:Jurkat Cells; D007962:Leukocytes / Q000378:metabolism; D008727:Methotrexate / Q000008:administration & dosage / Q000494:pharmacology*; D009000:Monocytes / Q000187:drug effects / Q000473:pathology*; D018384:Oxidative Stress; D011419:Propidium / Q000494:pharmacology; D017382:Reactive Oxygen Species; D013601:T-Lymphocytes / Q000187:drug effects / Q000473:pathology*; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors; D020298:U937 Cells
D017304:Annexin A5; D000893:Anti-Inflammatory Agents; D000964:Antimetabolites, Antineoplastic; D000975:Antioxidants; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007166:Immunosuppressive Agents; D017382:Reactive Oxygen Species; D013778:Tetrazolium Salts; D013844:Thiazoles; D011419:Propidium; C022616:thiazolyl blue; D016650:Fluorescein-5-isothiocyanate; D000111:Acetylcysteine; D008727:Methotrexate