title: Enhanced phagocytosis through inhibition of de novo ceramide synthesis.
abstract: Fcgamma receptors are important mediators of the binding of IgG to and induction of phagocytosis in neutrophils. COS-1 cells provide a potentially useful model for studying these receptors because transfection with the FcgammaRIIA renders these cells phagocytic. During FcgammaRIIA-mediated phagocytosis in COS-1 cells, endogenous ceramide levels increased 52% by 20 min (p < 0.01). Phospholipase D activity increased by 62% (p < 0.01). Correspondingly, the phagocytic index increased by 3.7-fold by 20 min. Two inhibitors of ceramide formation were used to assess the consequences of reduced ceramide generation. l-Cycloserine, an inhibitor that blocks serine palmitoyltransferase activity, lowered both sphingosine and ceramide levels. Under these conditions, the phagocytic index increased 100% in the presence of 2 mm l-cycloserine. The formation of ceramide resulting from the N-acylation of dihydrosphingosine or sphingosine by ceramide synthase is inhibited by the fungal toxin fumonisin B(1). When cells were treated with 5-50 microm fumonisin B(1), the cellular level of ceramide decreased in a concentration-dependent manner, while simultaneously the phagocytic index increased by 52%. Concomitantly, three indirect measures of FcgammaRIIA activity were altered with the fall in ceramide levels. Syk phosphorylation, phospholipase D activity, and mitogen-activated protein (MAP) kinase phosphorylation were increased at 30 min. When Syk phosphorylation was blocked with piceatannol and cells were similarly challenged, phosphatidylinositol 3-kinase activation was blocked, but no changes in either ceramide accumulation or MAP kinase activation were observed. Ceramide formation and MAP kinase activation are therefore not dependent on Syk kinase activity in this system. These results indicate that COS-1 cells provide a useful model for the recapitulation of sphingolipid signaling in the study of phagocytosis. Ceramide formed by de novo synthesis may represent an important mechanism in the regulation of phagocytosis.
authors.affiliation: Department of Pediatrics, Division of Hematology Oncology, University of Michigan, Ann Arbor 48109, USA.
authors.forename: Vania
authors.initials: V
authors.lastname: Hinkovska-Galcheva
authors.forename: Laurence
authors.initials: L
authors.lastname: Boxer
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Mansfield
authors.forename: Alan D
authors.initials: AD
authors.lastname: Schreiber
authors.forename: James A
authors.initials: JA
authors.lastname: Shayman
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000502:physiology; D019556:COS Cells; D002518:Ceramides / Q000096:biosynthesis*; D004792:Enzyme Precursors / Q000378:metabolism; D037341:Fumonisins / Q000633:toxicity; D047908:Intracellular Signaling Peptides and Proteins; D009504:Neutrophils / Q000276:immunology; D010587:Phagocytosis; D010739:Phospholipase D / Q000502:physiology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D017452:Receptors, IgG / Q000502:physiology; D012756:Sheep; D000072377:Syk Kinase
chemical_list: D015703:Antigens, CD; D002518:Ceramides; D004792:Enzyme Precursors; C080568:Fc gamma receptor IIA; D037341:Fumonisins; D047908:Intracellular Signaling Peptides and Proteins; D017452:Receptors, IgG; C056933:fumonisin B1; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; D010739:Phospholipase D