Corticosteroids are the preeminent antiinflammatory agents although the molecular mechanisms that impart their efficacy have not been defined. The endothelium plays a critical role in inflammation by directing circulating leukocytes into extravascular tissues by expressing adhesive molecules for leukocytes [e.g., endothelial-leukocyte adhesion molecule 1 (ELAM-1) and intercellular adhesion molecule 1 (ICAM-1)]. We therefore determined whether corticosteroids suppress inflammation by inhibiting endothelial expression of adhesion molecules for neutrophils (polymorphonuclear leukocytes). Preincubation of endothelial cells with endotoxin [lipopolysaccharide (LPS), 1 microgram/ml] led to a 4-fold increase in subsequent adherence of polymorphonuclear leukocytes (P < 0.0001, n = 10) to endothelial cells, an increase that was markedly attenuated when endothelial cells were treated with dexamethasone (IC50 < 1 nM, P < 0.0001, n = 6 or 7) during preincubation with LPS. Moreover, the steroid receptor agonist cortisol (10 microM), but not its inactive metabolite tetrahydrocortisol (10 microM), diminished LPS-induced endothelial cell adhesiveness. Further evidence that the action of dexamethasone was mediated through ligation of corticosteroid receptors [human glucocorticoid receptors (hGRs)] was provided by experiments utilizing the steroid antagonist RU-486. RU-486 (10 microM), which prevents translocation of ligated hGR to the nucleus by inhibiting dissociation of hGR from heat shock protein 90, completely aborted the effect of dexamethasone on adhesiveness of endothelial cells (P < 0.0005, n = 3). Treatment of endothelial cells with LPS (1 microgram/ml) stimulated transcription of ELAM-1, as shown by Northern blot analysis, and expression of membrane-associated ELAM-1 and ICAM-1, as shown by quantitative immunofluorescence (both P < 0.001, n = 9). Dexamethasone markedly inhibited LPS-stimulated accumulation of mRNA for ELAM-1 and expression of ELAM-1 and ICAM-1 (IC50 < 10 nM, both P < 0.001, n = 4-9); inhibition of expression by dexamethasone was reversed by RU-486 (both P < 0.005, n = 4-6). As in the adhesion studies, cortisol but not tetrahydrocortisol inhibited expression of ELAM-1 and ICAM-1 (both P < 0.005, n = 3 or 4). In contrast, sodium salicylate (1 mM) inhibited neither adhesion nor expression of these adhesion molecules. These studies suggest that antagonism by dexamethasone of endotoxin-induced inflammation is a specific instance of the general biological principle that the glucocorticoid receptor is a hormone-dependent regulator of transcription.

Methotrexate, a folate antagonist, is a potent antiinflammatory agent when used weekly in low concentrations. We examined the hypothesis that the antiphlogistic effects of methotrexate result from its capacity to promote intracellular accumulation of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) that, under conditions of cell injury, increases local adenosine release. We now present the first evidence to establish this mechanism of action in an in vivo model of inflammation, the murine air pouch model. Mice were injected intraperitoneally with either methotrexate or saline for 3-4 wk during induction of air pouches. Pharmacologically relevant doses of methotrexate increased splenocyte AICAR content, raised adenosine concentrations in exudates from carrageenan-inflamed air pouches, and markedly inhibited leukocyte accumulation in inflamed air pouches. The methotrexate-mediated reduction in leukocyte accumulation was partially reversed by injection of adenosine deaminase (ADA) into the air pouch, completely reversed by a specific adenosine A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), but not affected by an adenosine A1 receptor antagonist, 8-cyclopentyl-dipropylxanthine. Neither ADA nor DMPX affected leukocyte accumulation in the inflamed pouches of animals treated with either saline or the potent antiinflammatory steroid dexamethasone. These results indicate that methotrexate is a nonsteroidal antiinflammatory agent, the antiphlogistic action of which is due to increased adenosine release at inflamed sites.

Methotrexate administered weekly in low doses is a mainstay in the therapy of rheumatoid arthritis. Although originally developed as a folate antagonist for the treatment of cancer, its mechanism of action in the therapy of rheumatoid arthritis remains less clear. Several mechanisms have been proposed including inhibition of T cell proliferation via its effects on purine and pyrimidine metabolism, inhibition of transmethylation reactions required for the prevention of T cell cytotoxicity, interference with glutathione metabolism leading to alterations in recruitment of monocytes and other cells to the inflamed joint, and promotion of the release of the endogenous anti-inflammatory mediator adenosine. These mechanisms of action and the role of methotrexate in the suppression of rheumatoid arthritis are reviewed.