title: Ceramide generation in situ alters leukocyte cytoskeletal organization and beta 2-integrin function and causes complete degranulation.
abstract: Ceramide levels increase in activated polymorphonuclear neutrophils, and here we show that endogenous ceramide induced degranulation and superoxide generation and increased surface beta(2)-integrin expression. Ceramide accumulation reveals a bifurcation in integrin function, as it abolished agonist-induced adhesion to planar surfaces, yet had little effect on homotypic aggregation. We increased cellular ceramide content by treating polymorphonuclear neutrophils with sphingomyelinase C and controlled for loss of sphingomyelin by pretreatment with sphingomyelinase D to generate ceramide phosphate, which is not a substrate for sphingomyelinase C. Pretreatment with the latter enzyme blocked all the effects of sphingomyelinase C. Ceramide generation caused a Ca(2+) flux and complete degranulation of both primary and secondary granules and increased surface beta(2)-integrin expression. These integrins were in a nonfunctional state, and subsequent activation with platelet-activating factor or formyl-methionyl-leucyl-phenylalanine induced beta(2)-integrin-dependent homotypic aggregation. However, these cells were completely unable to adhere to surfaces via beta(2)-integrins. This was not due to a defect in the integrins themselves because the active conformation could be achieved by cation switching. Rather, ceramide affected cytoskeletal organization and inside-out signaling, leading to affinity maturation. Cytochalasin D induced the same disparity between aggregation and surface adhesion. We conclude that ceramide affects F-actin rearrangement, leading to massive degranulation, and reveals differences in beta(2)-integrin-mediated adhesive events.
authors.affiliation: Department of Medicine, University of Utah, Salt Lake City, Utah 8411, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Feldhaus
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Weyrich
authors.forename: Guy A
authors.initials: GA
authors.lastname: Zimmerman
authors.forename: Thomas M
authors.initials: TM
authors.lastname: McIntyre
mesh_terms: D000199:Actins / Q000378:metabolism; D018821:CD18 Antigens / Q000378:metabolism*; D015550:Cell Degranulation; D002478:Cells, Cultured; D002518:Ceramides / Q000096:biosynthesis*; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000648:ultrastructure*; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure*; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism
chemical_list: D000199:Actins; D018821:CD18 Antigens; D002518:Ceramides; D013481:Superoxides; D015638:Cytochalasin D; D009240:N-Formylmethionine Leucyl-Phenylalanine; D013108:Sphingomyelin Phosphodiesterase