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Activation-induced apoptosis is a critical mechanism by which the immune system maintains tolerance to self antigens by the clonal deletion of autoreactive T and B cells.1 The involvement of apoptosis in the mechanism of cell death induced by high doses of methotrexate (MTX) was first demonstrated in Chinese hamster ovary cells.2 Fas mediated apoptosis may have a critical role in the regression of synovial hyperplasia in rheumatoid arthritis (RA).3 Cutolo et al demonstrated the influence of MTX on the proliferation and apoptosis of cultured human monocytic myeloid cells (THP-1).4 Genestier et al investigated the effect of low doses of MTX on human peripheral blood lymphocytes and showed that MTX could induce the in vitro apoptosis of mitogen activated lymphocytes CD4+ and CD8+ but not resting T cells. He also demonstrated that a single injection of MTX during low dose treatment in six patients with RA was sufficient to prime lymphocytes to apoptosis upon subsequent mitogenic activation ex vivo.5
The purpose of our investigation was to examine the influence of MTX on the apoptosis of in vitro phytohaemagglutinin (PHA) activated peripheral blood mononuclear cells (PBMC) from patients with RA subjected to MTX treatment, and its correlation with their clinical response.
PATIENTS AND METHODS
Investigations were carried out on 34 patients (aged 38–76 years) with active RA. MTX, 10–15 mg, was given orally once weekly. An assessment of these patients’ clinical response to MTX according to the American College of Rheumatology criteria was made 8 weeks after beginning MTX treatment.6 Patients who had improved ⩾20% compared with the baseline were defined as responders and patients with improvement <20% were classified as non-responders. In the concomitant treatment one non-steroidal anti-inflammatory drug and prednisone (up to 7,5 mg daily) was allowed.
PBMC were isolated according to Böyum before MTX administration, activated with PHA (5 μg/ml) for 72 hours, and then washed and incubated with MTX (0.1–100 µmol/l) in 96 well plates in duplicate.7 Cell death was evaluated using Hoechst 33342 (apoptotic cells) and ethidium bromide (necrotic cells). Three hundred cells from each well were counted and the mean percentage of apoptotic cells calculated.
RESULTS AND DISCUSSION
There were no significant differences in the percentage of the MTX-induced (0.1–100 µmol/l) apoptosis of PHA activated PBMC derived from MTX treated patients with clinical improvement compared with the patients without improvement (fig 1). These results did not differ when compared with the results in the MTX untreated group. However, as in Genestier’s study, MTX increased the specific apoptosis of PHA activated PBMC as compared with cells not activated with PHA (data not shown).5 Lack of differences in the in vitro MTX-induced apoptosis of PHA activated PBMC from MTX treated responding patients compared with the non-responders indicates that this property may not be a good predictive factor for optimising MTX treatment.
Low dose MTX in RA treatment seems to exert anti-inflammatory effects by acting at different levels of the pathophysiological cascade.8 Several studies have recently shown that low dose MTX may well induce antiproliferative effects on the immune cells owing to the inhibition of dihydrofolate reductase and folate dependent transmethylation as an independent mechanism of apoptosis. We did not confirm (unpublished data) that low dose treatment MTX was sufficient to prime PBMC to apoptosis upon subsequent PHA activation ex vivo. It is interesting that in Cutolo’s experiments MTX at a concentration of 50 μg/ml did not induce apoptosis and did not affect the proliferation of RA synovial macrophages.4
It seems that either the apoptosis of cells in the tissue, which is directly involved in the inflammatory process, is more important than that seen in peripheral blood lymphocytes, or that other mechanisms of the MTX action may be responsible for the clinical improvement in patients treated with low doses of MTX.
This study was supported by the Polish State Committee for Scientific Research (KBN), grant No 4 P05B 108 19.
Jerzy Swierkot and Ryszard Miedzybrodzki contributed equally to this work.
We thank Mrs Barbara Bubak MSc and Mrs Wiesława Kilian for their skilful assistance.