METHOTREXATE INHIBITION OF INDUCIBLE NITRIC-OXIDE SYNTHASE IN MURINE LUNG EPITHELIAL-CELLS IN-VITRO

Nitric oxide (NO) is produced in lung epithelial cells by nitric oxide synthases (NOSs), which can enhance inflammation and edema formation. The inducible NOS (iNOS, type II NOS) has been shown to be increased in lung disorders such as asthma. Therapy for asthma includes antiinfl ammatory agents such as corticosteroids and antineoplastic agents such as methotrexate (MTX). We hypothesized that NO production by epitheli al cells in vitro would be attenuated by MTX, and that this effect wou ld be additive with corticosteroids. In order to test this hypothesis, cells from the murine lung epithelial-cell line LA-4 were cultured to confluence and stimulated to express iNOS and produce NO by cytomix, a combination of human tumor necrosis factor-alpha (TNF-alpha), human interleukin-1 beta (IL-1 beta) and murine interferon-gamma (IFN-gamma) . Nitrite and nitrite + nitrate were measured in the culture supernata nt fluids as an index of NO production, MTX caused a dose- and time-de pendent inhibition of nitrite and nitrite + nitrate (P < 0.05, all com parisons). Importantly, the inhibition of NO production by MTX (10(-3) M) was additive with dexamethasone (10(-5) to 10(-9) M), but cyclopho sphamide, bleomycin, and cytosine-beta-D-arabinofuranoside (Ara-C), ot her antineoplastic agents, caused no inhibition of NO production. To i nvestigate the mechanism of NO inhibition with MTX, we added tetrahydr obiopterin, which reversed the inhibition. MTX had no effect on the ex pression of iNOS on Western blotting or iNOS mRNA on Northern blotting . These data show that MTX inhibits NO production by iNOS in murine lu ng epithelial cells in vitro and that MTX produces added inhibition wi th corticosteroids, and suggest a potential strategy for reducing NO p roduction in vivo.