Interferon-Gamma Alone Triggers the Production of Nitric Oxide from Serum-Starved BNL CL.2, Murine Embryonic Liver Cells

Abstract

A previous study has demonstrated that both interferon-gamma (IFN-γ) and lipopolysaccharide (LPS) were needed to induce the production of nitric oxide (NO) in BNL CL.2 cells, murine embryonic liver cells. We here demonstrate that when BNL CL.2 cells were cultured with serum-free medium, they were induced to produce NO by the stimulation of IFN-γ alone. BNL CL.2 cells were cultured with serum-free or serum-containing medium for 1-3 days and then stimulated to synthesize NO by IFN-γ. Surprisingly, only serum-starved cells showed significant amount of nitrite accumulation and iNOS protein expression in response to IFN-γ in dose- and time-dependent manners, but serum-supplied cells did not. When the cells were stimulated with IFN-γ- tumor necrosis factor-α (TNF-α), or LPS in combinations, only the combination of IFN-γ and LPS produced more NO than that produced by IFN-γ alone. The production of NO by the cells stimulated with IFN-γ or IFN-γ plus LPS was blocked by the addition of N^G-monomethyl-L-arginine (N^GMMA), a NO synthesis inhibitor. To address the intracellular signal pathway responsible for the production of NO by the cells stimulated with IFN-γ alone or IFN-γ plus LPS, we examined the effects of several protein kinase inhibitors on the production of NO from the cells. The production of NO was significantly inhibited by protein tyrosine kinase (PTK) inhibitors, genistein and herbimycin A, but not by protein kinase A or C inhibitors. These results suggest that the deprivation of serum from BNL CL.2 cell culture medium might prime the cells to induce NO synthesis when the cells are triggered by IFN-γ and the involvement of PTK signal transduction pathway in the expression of inducible NO synthase gene in murine hepatoma cells.