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 antiinflammatory agents such as corticosteroids and antineoplastic agents such as methotrexate (MTX). We hypothesized that NO production by epithelial cells in vitro would be attenuated by MTX, and that this effect would 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-1beta (IL-1beta) and murine interferon-gamma (IFN-gamma). Nitrite and nitrite + nitrate were measured in the culture supernatant fluids as an index of NO production. MTX caused a dose- and time-dependent inhibition of nitrite and nitrite + nitrate (P < 0.05, all comparisons). Importantly, the inhibition of NO production by MTX (10(-3) M) was additive with dexamethasone (10(-5) to 10(-9) M), but cyclophosphamide, bleomycin, and cytosine-beta-D-arabinofuranoside (Ara-C), other antineoplastic agents, caused no inhibition of NO production. To investigate the mechanism of NO inhibition with MTX, we added tetrahydrobiopterin, which reversed the inhibition. MTX had no effect on the expression of iNOS on Western blotting or iNOS mRNA on Northern blotting. These data show that MTX inhibits NO production by iNOS in murine lung epithelial cells in vitro and that MTX produces added inhibition with corticosteroids, and suggest a potential strategy for reducing NO production in vivo.