The inhibitory effects of roflumilast on lipopolysaccharide-induced nitric oxide production in RAW264.7 cells are mediated by heme oxygenase-1 and its product carbon monoxide

Heme oxygenase-1 (HO-1) is an enzyme that degrades heme into biliverdin, free iron, and carbon monoxide (CO). This enzyme is known to have cytoprotective and anti-inflammatory effects. In this study, we investigated whether roflumilast, a newly developed specific phosphodiesterase 4 (PDE4) inhibitor, mediates some of its anti-inflammatory effects by blocking nitric oxide (NO) and tumor necrosis factor alpha (TNF-alpha) via the induction of HO-1 expression in macrophages. The expression of iNOS and HO-1 was analyzed by western blot analysis. The production of NO and TNF-alpha was assayed by Greiss and ELISA, respectively. Roflumilast markedly suppressed LPS-induced NO and TNF-alpha production and these phenomena were correlated with the induction of HO-1 protein levels. Moreover, the inhibitory effects of roflumilast on NO production were abrogated by a HO-1 inhibitor and a CO scavenger. Tricarbonyldichlrororuthenium(II) dimer, a CO releasing molecule significantly suppressed NO production. These results suggested that roflumilast exerts its anti-inflammatory effects in macrophages through a novel mechanism that involves the action of HO-1 and its product, CO.