title: Methotrexate and cyclooxygenase metabolism in cultured human rheumatoid synoviocytes.
abstract: OBJECTIVE
  Our objective was to characterize the effect of methotrexate (MTX) on prostaglandin E2 (PGE2) synthesis in cultured human rheumatoid synovial cells. Prostaglandins (PG) are important mediators of inflammation and joint destruction in rheumatoid arthritis (RA). Two isoforms of cyclooxygenase (COX), the key enzyme in PG synthesis, have been characterized: a constitutively expressed form, COX-1, and an inducible form, COX-2. The mechanisms of action of low dose MTX in RA treatment are still poorly understood. As the clinical effects are often first noticed within a month of starting MTX therapy, an antiinflammatory action has been proposed.
  
  
  METHODS
  Adherent synovial cells were obtained by collagenase digestion of rheumatoid synovium, isolated from patients with RA undergoing synovectomy. Between passages 3 and 6, cultured synovial cells were incubated with or without MTX for 54 h, at various concentrations. Interleukin (IL)-1beta (1 ng/ml) was added or not for the last 6 h of incubation. Supernatants were harvested and assayed for PGE2 by enzyme immunoassay (EIA). Exogenous [1-14C]arachidonic acid metabolism of synoviocytes was analyzed by reverse phase high performance liquid chromatography (RP-HPLC). COX-1 and COX-2 mRNA expression was determined by total RNA extraction and reverse transcription polymerase chain reaction.
  
  
  RESULTS
  Cellular viability was not affected by MTX. EIA showed that MTX decreased IL-1beta induced PGE2 production by synoviocytes in a dose dependent manner. RP-HPLC analysis confirmed the inhibition of PGE2 and (12S)-12-hydroxy-5,8,10-heptadecatrienoic acid production. COX-1 and IL-1beta induced COX-2 mRNA expression were not inhibited by MTX.
  
  
  CONCLUSIONS
  MTX has an inhibitory effect on IL-1beta stimulated production of PGE2 by cultured human rheumatoid synoviocytes, without affecting either COX mRNA expression. Among various biochemical and immunologic events, MTX could have an antiinflammatory action by decreasing PGE2 release.
authors.affiliation: Department of Rheumatology, CHRU Dupuytren, and the University of Medicine of Limoges, France.
authors.forename: P
authors.initials: P
authors.lastname: Vergne
authors.forename: B
authors.initials: B
authors.lastname: Liagre
authors.forename: P
authors.initials: P
authors.lastname: Bertin
authors.forename: J
authors.initials: J
authors.lastname: Cook-Moreau
authors.forename: R
authors.initials: R
authors.lastname: Treves
authors.forename: J L
authors.initials: JL
authors.lastname: Beneytout
authors.forename: M
authors.initials: M
authors.lastname: Rigaud
mesh_terms: D018501:Antirheumatic Agents / Q000494:pharmacology*; D016718:Arachidonic Acid / Q000378:metabolism; D001172:Arthritis, Rheumatoid / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured / Q000187:drug effects; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000096:biosynthesis; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D006801:Humans; D007527:Isoenzymes / Q000187:drug effects* / Q000378:metabolism; D008565:Membrane Proteins; D008727:Methotrexate / Q000494:pharmacology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D013583:Synovial Membrane / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism
chemical_list: D018501:Antirheumatic Agents; D005493:Folic Acid Antagonists; D007527:Isoenzymes; D008565:Membrane Proteins; D012333:RNA, Messenger; D016718:Arachidonic Acid; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone; D008727:Methotrexate