title: Acute and chronic suppression of leukotriene B4 synthesis ex vivo in neutrophils from patients with rheumatoid arthritis beginning treatment with methotrexate.
abstract: OBJECTIVE
  To compare the cumulative effects of oral methotrexate (MTX) therapy (after 6-8 weeks) with the acute effects (24 hours after a dose) on arachidonic acid metabolism by the 5-lipoxygenase (5-LO) pathway in neutrophils from patients with active rheumatoid arthritis (RA) who were beginning therapy with MTX.
  
  
  METHODS
  Neutrophils and monocytes were isolated from whole blood from 7 patients with RA, immediately before and 24 hours after their first weekly dose of 7.5 mg of MTX, and again after their dose at 6-8 weeks.
  
  
  RESULTS
  Total immunoreactive leukotriene B4 (LTB4) formation in neutrophils activated ex vivo with calcium ionophore A23187 was significantly suppressed (by 33%) before the 6-8-week dose, compared with the level before the first dose (mean +/- SEM 8.29 +/- 1.24 ng/10(6) cells at predose 6-8 weeks versus 12.29 +/- 2.13 ng/10(6) cells at predose 1; P = 0.03). Reductions were also observed after the first dose (27%; P = 0.07) and after the 6-8-week dose (43%; P = 0.05) compared with the respective predose levels. MTX treatment produced significant reductions in the total generation of 5-LO pathway products (5-hydroxyeicosatetraenoic acid + 6-trans-LTB4 + LTB4 + omega-oxidation products of LTB4) by calcium ionophore-activated neutrophils, as quantitated by integrated optical density after resolution on reverse-phase high-performance liquid chromatography. Decreases were observed after the first dose (26%; P = 0.025), immediately before the 6-8-week dose (23%; P = 0.05), and after the 6-8-week dose (47%; P = 0.0033) compared with levels before the first dose, and after the 6-8-week dose compared with the level before it (32%; P = 0.04). The generation of LTB4 by calcium ionophore-activated monocytes was not significantly affected by MTX therapy.
  
  
  CONCLUSIONS
  The significant decreases in the formation of omega-oxidation products of LTB4 and in the total generation of neutrophil 5-LO pathway products in the absence of a significant change in the release of 3H-arachidonic acid or the generation of platelet-activating factor suggest that the activity of the 5-LO enzyme in neutrophils is inhibited. We conclude that weekly oral MTX therapy in patients with active RA inhibits neutrophil 5-LO pathway product generation in a pattern consistent with inhibition of the activity of the 5-LO enzyme; an effect is observed after the first dose. The inhibition of 5-LO is cell-selective and cumulative, with a superimposed incremental inhibition observed after the weekly MTX dose.
authors.affiliation: Department of Medicine, Harvard Medical School, Boston, MA 02115.
authors.forename: R I
authors.initials: RI
authors.lastname: Sperling
authors.forename: A I
authors.initials: AI
authors.lastname: Benincaso
authors.forename: R J
authors.initials: RJ
authors.lastname: Anderson
authors.forename: J S
authors.initials: JS
authors.lastname: Coblyn
authors.forename: K F
authors.initials: KF
authors.lastname: Austen
authors.forename: M E
authors.initials: ME
authors.lastname: Weinblatt
mesh_terms: D000328:Adult; D000368:Aged; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism; D001172:Arthritis, Rheumatoid / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D006801:Humans; D007975:Leukotriene B4 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D008727:Methotrexate / Q000097:blood / Q000627:therapeutic use*; D008875:Middle Aged; D009000:Monocytes / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors
chemical_list: D007975:Leukotriene B4; D001094:Arachidonate 5-Lipoxygenase; D008727:Methotrexate