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THU0058 Apoptosis, sex hormones and adenosine deaminase levels in cultured monocytes (thp-1 cells) and synovial rheumatoid macrophages treated with methotrexate
  1. ME Cutolo,
  2. B Villaggio,
  3. A Sulli,
  4. A Bisso,
  5. C Pizzorni,
  6. M Briata,
  7. M Grosso,
  8. C Prete,
  9. L Felli,
  10. B Seriolo
  1. Department Internal Medicine Division Rheumatology, University of Genova, Genova, Italy

Abstract

Background Both testosterone (T) and methotrexate (MTX) seem to exert negative effects on cell growth that might partially explain some of their antiproliferative/antiinflammatory effects exerted in rheumatoid arthritis (RA).1,2

Objectives Therefore, we evaluated the T and MTX effects on cell apoptosis (apo) in cultured human myeloid monocytic cells (THP-1) differentiating into macrophages (M) and in cultured synovial M obtained from RA patients. Since extracellular adenosine increase seems implicated in the MTX action, we evaluated also the enzyme adenosine deaminase (ADA) levels. ADA is involved in adenosine metabolism.

Methods Synovial RA M as well as the THP-1 cells induced to differentiate into M (IFN gamma 500U), were cultured in presence of T (10–8M) and 17-beta estradiol (E2,10–8M) alone. The cells were also treated with the MTX concentrations present in vivo during RA therapy (= 50 microg/ml) again alone, or in association with T plus MTX or E2 plus MTX. After 24 h early and late apo were evaluated by immunofluorescence (annexin V and propidium iodide assay, respectively). ADA was evaluated on supernatants by spectrophotometry.

Results By considering the THP-1 cells, a significant induction of the late apo in T (17%) (p less 0.05), MTX (80%) alone, and T (72%), E2 (74%) plus MTX-treated cells was observed (all p < 0.001). No significant differences were observed during the early apo for all the treatments during the 24 hr. By evaluating the RA synovial M, a significant (p < 0.001) induction of the late apo (50%) was observed in the T plus MTX-treated M when compared with the alone MTX- or E2-treated M. The treatment of RA M with T alone was found to induce an evident cell apo (30%) (although non significant). ADA levels increased only in the supernatants from all the MTX-treated cells vs MTX-untreated controls, mainly in THP-1 cells.

Conclusion The T and MTX-induced apoptotic effects were more evident in cultured THP-1 cells than in cultured RA M. Therefore, immature differentiating monocytes (i.e. infiltrating monocytes) seem more sensible to the pharmacologic effects of MTX than differentiated cells (i.e. resident M). The significant increase of ADA levels found only in MTX-treated cells, might support the already suggested implication of adenosine level increase as part of the pharmacologic effects exerted by MTX.

References

  1. Cutolo M, Wilder R. Rheum Dis Clin North Am. 2000;26:825

  2. Cutolo M, et al. J Rheumatol. 2000;27:2551

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