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Inhibition of tumour necrosis factor alpha production by activated T cells of rheumatoid arthritis patients by novel anti-folate drugs: an ex vivopilot study

  • JW van der Heijden1,
  • AH Gerards2,
  • R Oerlemans1,
  • WF Lems1,
  • RJ Scheper3,
  • LA Aarden4,
  • BAC Dijkmans1 and
  • G Jansen1
Arthritis Research & Therapy20057(Suppl 1):P84

Received: 11 January 2005

Published: 17 February 2005


Rheumatoid Arthritis PatientInhibit Tumour Necrosis FactorAICARSulphasalazineReduce Folate Carrier


The folate antagonist methotrexate (MTX) is the 'anchor-drug' in the treatment of patients with rheumatoid arthritis (RA) [1]. The main target of MTX in intracellular folate metabolism is dihydrofolate reductase (DHFR) but several other targets have been described (e.g. thymidylate synthase [TS] and 5-amino-imidazole-4-carboxamide ribonucleotide [AICAR]). At present the exact mechanism of action of MTX in RA still remains elusive [1]. Despite the potent anti-rheumatic capacity of MTX many patients (at least 50%) become resistant to MTX during long-lasting therapy. However, little is known about the mechanisms of resistance against MTX in RA patients [2].

From the field of oncology, where MTX is used against haematological malignancies, new anti-folate drugs were developed to circumvent MTX resistance [3]. These new folate antagonists have the following characteristics: are better transported through the reduced folate carrier, are retained intracellular more efficiently by polyglutamylation via folylpolyglutamate synthetase, and/or have other targets in the folate pathway besides DHFR (e.g. TS).


To investigate whether two new-generation anti-folate drugs, PT523 (DHFR-inhibitor) and ZD1694 (TS-inhibitor), have equal or better anti-inflammatory capacity compared with MTX based on their capacity to inhibit tumour necrosis factor alpha (TNF-α) production by activated T cells.


Whole blood from 11 RA patients and six healthy volunteers was incubated ex vivo with MTX, PT523, ZD1694 and, as a control, the DMARD sulphasalazine (SSZ) after T-cell stimulation with α-CD3/CD28. Inhibition of TNF-α production was measured after 72 hours by ELISA [4].

The IC-50 values (defined as the drug concentration exerting 50% inhibition of TNF-α production) are used as a value for the anti-inflammatory capacity of the drug (Table 1).
Table 1

Characteristics of drugs and mean IC-50 values (± standard deviation)


Mean IC-50 for TNF-α inhibition



Reduced folate carrier affinity (cell uptake)

Folylpolyglutamate synthetase affinity (cell retention)

Rheumatoid arthritis patients (n = 11)

Controls (n = 6)





78 (77) nM

57 (27) nM





5.8 (3.2) nM

6.4 (8.3) nM





16 (26) nM

4.8 (3.0) nM





278 (216) μM

191 (29) μM


Both PT523 and D1694 turned out to inhibit TNF-α production by activated T cells much more efficiently than MTX (5–15 times). For comparison, the DMARD SSZ is effective at much higher concentration (μM range). The inhibition of TNF-α production by the anti-folate drugs does not seem to be a result of (apoptotic) cell death of T cells whereas SSZ induces apoptosis of T cells (data not shown).


In an ex vivo setting, two novel anti-folate drugs designed to circumvent MTX resistance proved to be very effective in inhibiting TNF-α production by activated T cells from RA patients and healthy volunteers. Future experiments are designed to evaluate ex vivo anti-folate sensitivity profiles for 'MTX-responders' and 'MTX-non-responders' to investigate whether these novel generation of antifolate drugs can be useful in cases of clinical failure on MTX.



This study is supported by the Dutch Arthritis Association (Grant NRF-03-I-40).

Authors’ Affiliations

Department of Rheumatology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands
Vlietland Hospital, Schiedam, The Netherlands
Department of Pathology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands
Department of Immunopathology, Sanquin-Research, Amsterdam, The Netherlands


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© BioMed Central Ltd 2005