Ceramide Generation in Situ Alters Leukocyte Cytoskeletal Organization and β2-Integrin Function and Causes Complete Degranulation*

Ceramide levels increase in activated polymorphonuclear neutrophils, and here we show that endogenous ceramide induced degranulation and superoxide generation and increased surface β2-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 Ca2+ flux and complete degranulation of both primary and secondary granules and increased surface β2-integrin expression. These integrins were in a nonfunctional state, and subsequent activation with platelet-activating factor or formyl-methionyl-leucyl-phenylalanine induced β2-integrin-dependent homotypic aggregation. However, these cells were completely unable to adhere to surfaces via β2-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 β2-integrin-mediated adhesive events.