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

Metabolism of macrophage (MO) membrane phospholipids produces key mediators of inflammation and major second messengers that modulate inflammatory responses during sepsis. Sphingomyelin is a major class of phospholipid that releases ceramide and sphingosine. This study was designed to investigate the involvement of sphingomyelin metabolites in MO activation by lipopolysaccharide (LPS). Rabbit alveolar MO were obtained by bronchoalveolar lavage and exposed to C6-ceramide, a cell-permeable analogue of natural ceramide, or sphingosine in the presence of Escherichia coli LPS (100 ng/mL). Tumor necrosis factor (TNF) mRNA expression was measured by Northern blot assays. Total nuclear extract was harvested for the measurement of nuclear factor KB (NFkappaB) with electrophoretic mobility shift assays. MO TNF production was measured by L929 bioassays. C-6 ceramide did not have any effects on MO TNF production or TNF mRNA expression 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 comparison, sphingosine inhibited TNF mRNA expression as well as TNF production of LPS-stimulated MO. LPS-induced MO NFkappaB activity was also reduced by sphingosine. Our data indicate that ceramide alone has no effect on macrophage activity, but its metabolite sphingosine down-regulates MO activation induced by LPS stimulation. Therefore, the sphingomyelin pathway is involved in the regulation of MO activation.