Macrophage TNF mRNA expression induced by LPS is regulated by sphingomyelin metabolites

Metabolism of macrophage (Mempty set) membrane phospholipids produces key m ediators of inflammation and major second messengers that modulate inflamma tory responses during sepsis, Sphingomyelin is a major class of phospholipi d that releases ceramide and sphingosine, This study was designed to invest igate the involvement of sphingomyelin metabolites in Mempty set activation by lipopolysaccharide (LPS). Rabbit alveolar Mempty set were obtained by b ronchoalveolar lavage and exposed to CG-ceramide, a cell-permeable analogue of natural ceramide, or sphingosine in the presence of Escherichia coil LP S (100 ng/mL). Tumor necrosis factor (TNF) mRNA expression was measured by Northern blot assays. Total nuclear extract was harvested for the measureme nt of nuclear factor kappa B (NF kappa B) With electrophoretic mobility shi ft assays. Mempty set TNF production was measured by L929 bioassays, C-6 ce ramide did not have any effects on Mempty set TNF production or TNF mRNA ex pression with or without LPS stimulation. Inhibition of ceramide metabolism with 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), or N-oleoyl- ethanolamine (NOE) also did not induce TNF mRNA or TNF production. In compa rison, sphingosine inhibited TNF mRNA expression as well as TNF production of LPS-stimulated Mempty set, LPS-induced Mempty set NF kappa B activity wa s also reduced by sphingosine. Our data indicate that ceramide alone has no effect on macrophage activity, but its metabolite sphingosine down-regulat es Mempty set activation induced by LPS stimulation. Therefore, the sphingo myelin pathway is involved in the regulation of Mempty set activation.