Inhibition of cytokine production by methotrexate. Studies in healthy volunteers and patients with rheumatoid arthritis
A. H. Gerards,
S. de Lathouder1,
E. R. de Groot1,
B. A. C. Dijkmans and
L. A. Aarden1
Department of Rheumatology, Free University Medical Centre Amsterdam, Amsterdam and 1Department of Immunopathology, Sanquin Research at CLB and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
Correspondence to:
L. A. Aarden, CLB, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. E-mail: L.Aarden{at}sanquin.nl
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Abstract
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Objectives. To analyse whether the beneficial effects of methotrexate in rheumatoid arthritis (RA) could be due to inhibition of inflammatory cytokine production.
Methods. Cytokine production was studied using whole blood (WB) and mononuclear cells (MNC) of healthy volunteers and RA patients. Cultures were stimulated with either bacterial products such as lipo-oligosaccharide (LOS) or Staphylococcus aureus Cowan I (SAC) to activate monocytes or with monoclonal antibodies to CD3 and CD28 to induce polyclonal T-cell activation. We analysed the effect of methotrexate on cytokine production in these systems.
Results. We showed that methotrexate inhibits production of cytokines induced by T-cell activation. Among the cytokines inhibited were interleukin 4 (IL-4), IL-13, IFN
, tumour necrosis factor-
(TNF
) and granulocytemacrophage colony-stimulating factor. Inhibition was seen at concentrations easily achieved in plasma of RA patients taking the drug. IL-8 production was hardly influenced by methotrexate. Furthermore, inhibition was dependent on the stimulus; IL-6, IL-8, IL-1ß and TNF
production induced by LOS or SAC was only slightly decreased by methotrexate. The addition of folinic acid or thymidine and hypoxanthine reversed the inhibitory effects of methotrexate on cytokine production. Concentrations of methotrexate required for inhibition varied between donors. Oral intake of 10 mg methotrexate by RA patients led to marked inhibition of cytokine production in blood drawn after 2 h.
Conclusions. Methotrexate turns out to be an efficient inhibitor of cytokine production induced by T-cell activation in freshly drawn blood. This is due to inhibition of the de novo synthesis of purines and pyrimidines. Cytokines produced by monocytes are hardly affected by methotrexate.
KEY WORDS: Methotrexate, Cytokines, Tumour necrosis factor, Rheumatoid arthritis, Whole blood culture
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Introduction
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Methotrexate has become the most frequently used anti-rheumatic drug [1, 2]. However, the exact mechanism of action in rheumatoid arthritis (RA) is not yet clarified [35]. After administration, the kidneys rapidly excrete methotrexate and only a small amount of the drug is transported into cells by folate receptors. Intracellular methotrexate and polyglutamated derivatives of methotrexate not only inhibit dihydrofolate reductase (DHFR) but also have marked affinity for other folate-dependent enzymes such as thymidylate synthase, AICAR (5-amino-imidazol-4-carboxamide ribonucleotide transformylase) and AICARFT (AICAR formyltransferase). The inhibition of these enzymes affects purine, pyrimidine and homocysteine metabolism and DNA synthesis [68]. Methotrexate polyglutamate levels in circulating erythrocytes and polymorphs correlate with clinical efficacy in RA [9].
Notwithstanding our knowledge of methotrexate as a folate antagonist, the mechanism by which weekly administered, low-dose methotrexate attenuates the disease process in RA patients remains elusive. Nesher and Moore [10] showed that methotrexate inhibits pokeweed mitogen-induced proliferation and immunoglobulin synthesis of peripheral blood cells via reduction of polyamine synthesis. Cronstein et al. [11] have put forward the interesting hypothesis that methotrexate may act via adenosine. Methotrexate increases adenosine levels by inhibition of AICAR. Adenosine is known to have anti-inflammatory properties [12, 13]. Indeed, in animal models it was shown that methotrexate inhibits neutrophil function via stimulation of adenosine release [11] and that it also affects leucocyte recruitment to inflamed tissue [14]. However, other experiments in animal models using adenosine agonists and antagonists, as well as measurement of purine and pyrimidine levels in blood of methotrexate-treated patients did not support the idea that methotrexate acts via adenosine [7,15].
In view of the efficacy of anti-tumour necrosis factor (anti-TNF) treatment in RA, inhibition of cytokine production is another candidate mechanism for methotrexate. Down-regulation of inflammatory cytokines such as TNF
and interleukin 1ß (IL-1ß) in rheumatoid synovium has been observed during treatment with methotrexate [16, 17]. In addition, plasma levels of various inflammatory cytokines are decreased during methotrexate treatment [1820]. Recently, it was shown that methotrexate treatment results in a decreased number of T cells capable of TNF
production, whereas the number of T cells producing IL-10 after polyclonal activation increased [21]. Methotrexate possibly suppresses TNF
-induced NF-
B activation [22]. Surprisingly, reports on in vitro effects of methotrexate on cytokine production are scarce. Available data demonstrate little or no effect of methotrexate on IL-1ß or TNF
production in vitro [3, 18, 2327]. Only a very high dose of a liposomal preparation of methotrexate reduced TNF
production in peripheral blood-derived monocytes [28].
There is no effect of methotrexate on TNF
production in lipopolysaccharide (LPS)-stimulated whole blood cultures [18] or on IL-1 production of LPS-stimulated MNC [24]. Seitz et al. [29] noticed an enhanced in vitro production of IL-10 by MNC of RA patients treated with methotrexate. Recently, it was shown that methotrexate inhibits TNF
production in primed T cells, cultured for an extended period in the presence IL-2 [30]. In contrast, no effect of methotrexate in primary cultures of activated T cells was observed [30, 31]. There is no unanimity about effects of methotrexate on T cells. Some authors claim that methotrexate selectively kills activated T cells and fibroblasts by apoptosis [32, 33], and induces apoptosis in synovium [34]. Fairbanks et al. [32] found that methotrexate is cytostatic and not cytotoxic, halting proliferation at the G1 phase of the cell cycle, by inhibition of amidophosphoribosyltransferase.
In short, the studies on methotrexate appear to be inconclusive regarding the effect on T cells, and although inflammatory cytokines diminish during methotrexate therapy, this effect was not seen in in vitro tests. The purpose of the present study is to assess whether methotrexate has an effect on T-cell-mediated production of inflammatory cytokines in vitro.
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Materials and methods
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Blood samples were collected from a total of 20 healthy volunteers and 10 RA patients using 4-ml evacuated blood collection tubes (Greiner, Alphen a/d Rijn, The Netherlands), containing sodium heparin. Whole blood (WB) cultures were performed in flat-bottom microtitre plates (Nunc, Kamstrup, Denmark) by a method previously described in detail [20]. Heparinized venous blood was used and cultured at a final 1:10 dilution at a final heparin concentration of 15 U/ml. In experiments performed with RA patients, whole blood was cultured at a final 1:4 dilution. All cultures were carried out in endotoxin-free Iscoves modified Dulbeccos medium (IMDM, BioWhittaker, Verviers, Belgium), supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml), 0.1% endotoxin-free fetal calf serum (FCS), 50 mM 2-mercaptoethanol and 15 U/ml sodium heparin. Cultures were performed in duplicate except for experiments presented in Table 1 and Fig 6. It was essential to screen the batch of blood collection tubes as well as medium and FCS for absence of stimulatory material.
An aliquot of 200 µl of diluted blood was stimulated with a combination of endotoxin-free anti-CD3 (CLB.T3/4.E, 1 µg/ml, Sanquin, Amsterdam, The Netherlands) and anti-CD28 (CLB.CD28/1, 1 µg/ml, Sanquin) or with LOS (100 pg/ml, derived from Neisseria meningitidis, a kind gift of Dr J. Poolman, RIVM, Bilthoven, The Netherlands) or with SAC (Pansorbin, 1:4000, Calbiochem, La Jolla, CA). Cultures were incubated for 1 day (SAC and LOS) or 3 days (anti-CD3/anti-CD28) unless otherwise indicated.
Methotrexate was obtained from AHP Pharma, Hoofddorp, The Netherlands. Folinic acid, folic acid, hypoxanthine and thymidine were obtained from Sigma (Sigma-Aldrich, Steinheim, Germany). Stock solutions of folinic acid (3 mg/ml), folic acid (3 mg/ml), hypoxanthine (100 mM) and thymidine (100 mM) were prepared in H2O.
The production of cytokines was measured in the supernatant of the cell cultures in four serial dilutions. Supernatant was harvested at indicated times and tested directly by enzyme-linked immunosorbent assay (ELISA) in various dilutions or stored at -20°C until use. IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-12p40, IL-13, TNF
and interferon-
(IFN
) were measured with ELISA kits (PeliKine-compact, Sanquin) according to the protocol and have been described previously [20, 31, 35]. The granulocytemacrophage colony-stimulating factor (GM-CSF) ELISA was performed via the same protocol. The GM-CSF antibodies were a kind gift from Dr G. Trinchieri (the Wistar Institute, Philadelphia, PA). In this assay the coating antibody was anti-GM-CSF 9.1 (used at 2 µg/ml), the biotinylated antibody was anti-GM-CSF 16.3 (0.1 µg/ml). Recombinant GM-CSF (Sandoz, Basel, Switzerland) was used for the preparation of a standard curve.
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Results
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In vitro cytokine production in WB cultures
To assess cytokine production, WB cultures were stimulated with SAC. Table 1 shows a representative cytokine profile of a normal donor. It appeared that monokines (IL-1ß, IL-6, IL-8, IL-12 and TNF
) are readily secreted into the supernatant. However, T-cell cytokines were not produced (IL-2, IL-13), or were in minor quantities only (GM-CSF, IFN
, Table 1). With LOS similar results were obtained (not shown). Stimulation with a combination of anti-CD3 and anti-CD28 results in production of IL-2, IL-4, IL-13, GM-CSF, IFN
and TNF
. Surprisingly, IL-8 is also elevated (Table 1). Polyclonal T-cell stimulation of WB cultures leads to production of T-cell cytokines and IL-8.
Inhibition of cytokine production by methotrexate
The next step was to study the influence of methotrexate on LOS-, SAC- or anti-CD3/anti-CD28-activated WB cultures. Addition of methotrexate to T-cell stimulated cultures results in major inhibition of all cytokines tested, except IL-8. Even at high doses of methotrexate, IL-8 production is not affected, whereas dose-dependent inhibition of the other cytokines is similar (Fig. 1). Figure 2 shows the inhibition of cytokine production of each donor. SAC-induced production of IL-6, IL-8, TNF
, IL-1ß and IL-12 is not influenced by as much as 2 µg/ml methotrexate (not shown). LOS-induced cytokine production is slightly inhibited by high-dose methotrexate (Fig. 3). Similar results were obtained using MNC or purified T cells. However, effects seen in purified cells were less profound and more variable than those in WB cultures (not shown).

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FIG. 2. Inhibition of T-cell-stimulated cytokine production by methotrexate of individual donors. WB cultures of eight different blood donors were stimulated with anti-CD3 and anti-CD28. Supernatants were harvested at day 4. Each dot represents cytokine production of a donor in the presence of 2 µg/ml methotrexate expressed as the percentage of production in the absence of methotrexate. The range of cytokine production for the donors in the absence of methotrexate is described in the legend of Fig. 1.
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FIG. 3. Effect of methotrexate on LOS-induced cytokine production. WB cultures of eight different blood donors were stimulated with LOS (100 pg/ml). Each dot represents cytokine production in the presence of methotrexate (2 µg/ml) expressed as the percentage of production in the absence of methotrexate. The production range for IL-6 was 18003860 pg/ml, IL-8 was 465026 100 pg/ml, TNF was 2201100 pg/ml and IL-1ß was 5802160 pg/ml.
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These experiments show that methotrexate inhibits in vitro cytokine production (except IL-8) after T-cell stimulation in WB, MNC and T cells, and not after stimulation with SAC or LOS.
Interference of methotrexate with folate metabolism
We then analysed whether this in vitro effect of methotrexate was due to interference with the folate metabolism. To evaluate the effects of methotrexate on folate metabolism, amethopterin, a stereoisomer of methotrexate incapable of inhibiting folate-dependent enzymes, was tested in WB cultures. Amethopterin was about 1000-fold less active then methotrexate in inhibiting anti-CD3/anti-CD28-induced cytokine production (not shown). We then investigated whether inhibition by methotrexate can be reversed by folinic acid or by folic acid. Indeed, folinic acid reverses the inhibition by methotrexate (Fig. 4), whereas high doses of folic acid had no effect (not shown). The effect of folinic acid on methotrexate-treated cultures is significant (95% confidence interval 4878%, P < 0.001, paired t-test on normalized data). Inhibition of cytokine production by methotrexate can also be reversed by addition of hypoxanthine and thymidine to the WB culture (Fig. 5). In some donors addition of thymidine alone was sufficient. So it seems that methotrexate interferes with the folate metabolism and thereby with the synthesis of purines and pyrimidines.

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FIG. 4. Inhibition of cytokine production by methotrexate can be reversed by folinic acid. WB cultures of 10 donors were stimulated by anti-CD3 and anti-CD28 with and without methotrexate in the presence (white bar) or absence (black bar) of 40 µg/ml folinic acid. GM-CSF production is expressed as the percentage of production in the absence of methotrexate for each individual donor. GM-CSF production of the donors was 11 19036 765 pg/ml in the absence of methotrexate. Error bars indicate the S.E.M. of 10 donors.
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FIG. 5. Thymidine and hypoxanthine can reverse inhibition of cytokine production by methotrexate. WB cultures of five donors were stimulated with anti-CD3 and anti-CD28 in the absence or presence of methotrexate (100 ng/ml). GM-CSF production is measured in the absence of methotrexate (light grey bar). Together with methotrexate we added nothing (white bar), 50 µM thymidine (black bar), 100 µM hypoxanthine (dark grey bar), or a combination of thymidine and hypoxanthine (striped bar). Error bars represent the S.E.M. of duplicate cultures.
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Inhibition of cytokine production by methotrexate is a late phenomenon
To explore the effects of methotrexate in our WB system in more detail, we evaluated cytokine production at different time points. Inhibition of IFN
and TNF
production is only seen from day 3 on; similar results were seen for the other cytokines analysed, again with the exception of IL-8 (Fig. 6). In line with this late effect of methotrexate, we observed that inhibition by methotrexate was identical when addition of methotrexate was delayed until 24 h after the start of the culture (not shown).
Sensitivity of donors to methotrexate
We noticed that different donors needed different amounts of methotrexate to suppress cytokine production in WB cultures. To quantify this notion we determined the concentration of methotrexate required for 50% inhibition (ID50) for each cytokine and in every individual. Doseresponse curves of seven donors were analysed. Figure 7 shows that TNF
and IFN
in each donor are similarly affected by methotrexate, and the same is true for the other cytokines (not shown). In addition, this experiment shows that between donors there is considerable variation in sensitivity to methotrexate.
Methotrexate therapy leads to ex vivo inhibition of cytokine production
Methotrexate effectively inhibits cytokine production with an ID50 between 5 and 25 ng/ml (Fig. 7). Such levels are easily achieved in plasma, a couple of hours after oral application of methotrexate. To investigate whether plasma methotrexate levels are sufficient to inhibit cytokine production, we analysed WB cultures of 10 methotrexate-naive RA patients just before and 2 h after their first administration of methotrexate (10 mg, orally). Indeed, 2 h after methotrexate administration the mean IFN
production in WB cultures was reduced from 21 to 5.8 ng/ml (Fig. 8), which corresponds to a mean ratio of 0.28 (95% confidence interval: 0.140.53; P < 0.002 by paired t-test on log-transformed data). The antagonistic effect of folinic acid was highly significant (P < 0.002 by paired t-test on log-transformed ratios). Similar results were obtained when GM-CSF was measured (not shown). As expected no change in IL-8 production was seen (not shown).

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FIG. 8. Effect of oral uptake of methotrexate on ex vivo cytokine production. Blood was obtained from 10 RA patients just before (t = 0) and 2 h after (t = 2) the first oral application of methotrexate (10 mg). WB of 10 RA patients was stimulated with anti-CD3 and anti-CD28 in the absence or presence of 40 µg/ml folinic acid (FA), without additional methotrexate.
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Discussion
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The in vitro inhibition of T-cell cytokine production by methotrexate in freshly isolated human blood cells has not been reported before. WB cultures were predominantly used for this analysis. There are advantages in using WB cultures. The presence of erythrocytes protects against too much stress caused by oxygen radicals. Indeed, WB cultures differ from isolated MNC. In WB cultures there is no background IL-8 production whereas, after stimulation, IL-12 and IFN
production per cell is much higher than in MNC [35]. Methotrexate also inhibits cytokine production by purified T cells, but this inhibition is less profound and more variable. Probably the higher activity of the salvage pathway as a result of the availability of nucleotides derived from dying cells and/or FCS added to the culture is responsible for this effect. We observed that activation of T cells in WB leads to production of a variety of T-cell cytokines and of IL-8. This IL-8 production in WB cultures is surprising because isolated T cells produce very little IL-8 after anti-CD3 and anti-CD28 stimulation. Most likely, activated T cells indirectly stimulate other cells such as monocytes or neutrophils.
We analysed some of the possible mechanisms by which methotrexate inhibits TNF
, IFN
, IL-2, IL-4, IL-13 and GM-CSF and not IL-8 production. It is unlikely that adenosine is involved in the effects seen in our cultures. We observed that adenosine or adenosine receptor agonists inhibit production of all cytokines, including IL-8 (not shown). In addition, adenosine antagonists had no effect on methotrexate inhibition. In 1990, Nesher and Moore [10] proposed that methotrexate might inhibit polyamine synthesis in MNC. In our system, addition of polyamines failed to restore cytokine production in methotrexate-inhibited cultures. Moreover, our observation that inhibition of cytokine production by methotrexate can be reversed by a combination of hypoxanthine and thymidine, shows that inhibition of purine and pyrimidine synthesis is the main mechanism by which cytokine production is inhibited. This observation is in agreement with the experiments by Genestier et al. [36]. They observed that methotrexate induces apoptosis in activated T cells, whereas non-activated T cells are not affected. We also have evidence that in our cultures methotrexate leads to apoptosis in activated T cells as analysed by Annexin-V staining (not shown). Probably a lack of thymidine and/or purines during the transition from the G1 to the S phase leads to p53-mediated cell death. Monocytes are probably not inhibited by methotrexate because they hardly proliferate upon stimulation with SAC or LOS. Why Fairbanks et al. [32], using very similar conditions to Genestier et al., did not find induction of apoptosis is not clear. Possibly, salvage of nucleotides derived from dying cells in the high-density cell culture could have influenced the outcome.
Recently, Hildner et al. [30] reported that cytokine production by long-term T-cell cultures was inhibited by methotrexate. However, they did not see an effect of low-dose methotrexate in primary cultures. This lack of effect can be ascribed to the choice to analyse cytokine production at day 2. We showed that inhibition of T-cell cytokines does not occur on day 2, but is found from day 3 onwards.
Oral intake of 10 mg methotrexate leads to peak plasma levels of methotrexate around 50100 ng/ml at 13 h. We observed that in WB cultures of RA patients 2 h after their first oral intake of methotrexate, plasma methotrexate levels are sufficient to inhibit cytokine production, even after diluting the blood four times.
The main question to be addressed is whether our findings have any relation to the clinical situation. Possibly, T cells are important targets for methotrexate, but it is conceivable that other cells, for example in the synovial tissue, are the primary targets. If T cells are important, studying in vitro effects of methotrexate on T cells could be relevant for understanding its in vivo action. This would be in line with the observation of Rudwaleit et al. [21] that during treatment with methotrexate the percentage of TNF
-producing T cells decreases. If the real targets are other cells in the body, the experiments with T cells or WB cultures can still be clinically relevant. Various membrane receptors are involved in transport of methotrexate, folic acid and folinic acid into the cell. Moreover, in the cell the ratio of enzymes involved in polyglutamation and deglutamation can vary. Finally, levels of purines and pyrimidines capable of salvaging the inhibition by methotrexate can differ from compartment to compartment and from individual to individual. In some donors, thymidine alone could reverse the inhibition of cytokine production by methotrexate. This is probably due to hypoxanthine release in the cultures by dying blood cells or by the presence of hypoxanthine in the plasma. Indeed HPLC analysis showed that up to 10 µM of free hypoxanthine could be present in WB supernatant after 1 day of culture. If the different sensitivity to methotrexate observed in our WB cultures is a reflection of (some of) these individual variations, then sensitivity of cytokine production to methotrexate could be useful in predicting clinical effectiveness of methotrexate in individual patients.
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Conclusions
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Methotrexate is a specific inhibitor of pro-inflammatory cytokines in WB cultures after T-cell stimulation. Inhibition is seen at methotrexate levels easily achieved in plasma after oral uptake of 10 mg methotrexate. The inhibition is due to interference with folate-dependent purine and pyrimidine synthesis. There is considerable variation between donors in sensitivity to these in vitro effects of methotrexate. This could reflect the in vivo situation in which some patients respond to lower doses of methotrexate than other patients.
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Conflict of interest
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The authors have declared no conflicts of interest.
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Submitted 28 November 2002;
revised version accepted 17 February 2003.