Ceramide generation in situ alters leukocyte cytoskeletal organization and beta 2-integrin function and causes complete degranulation

• Published in: The Journal of biological chemistry Vol. 277; no. 6; p. 4285
• Main Authors: Feldhaus, Michael J, Weyrich, Andrew S, Zimmerman, Guy A, McIntyre, Thomas M
• Format: Journal Article
• Language: English
• Published: United States 08.02.2002
• Subjects: Actins - metabolism; CD18 Antigens - metabolism; Cell Degranulation; Cells, Cultured; Ceramides - biosynthesis; Cytochalasin D - pharmacology; Cytoskeleton - ultrastructure; Humans; N-Formylmethionine Leucyl-Phenylalanine - pharmacology; Neutrophils - drug effects; Neutrophils - enzymology; Neutrophils - metabolism; Neutrophils - ultrastructure; Sphingomyelin Phosphodiesterase - metabolism; Superoxides - metabolism
• ISSN: 0021-9258

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.