An obligate role for membrane-associated neutral sphingomyelinase activity in orienting chemotactic migration of human neutrophils.
For polymorphonuclear neutrophils (PMNs) to orient migration to chemotactic gradients, weak external asymmetries must be amplified into larger internal signaling gradients. Lipid mediators, associated with the plasma membrane and within the cell, participate in generating these gradients. This study examined the role in PMN chemotaxis of neutral sphingomyelinase (N-SMase), a plasma membrane-associated enzyme that converts sphingomyelin to ceramide. A noncompetitive N-SMase inhibitor, GW4869 (5 mM, 5 minutes), did not inhibit PMN motility (as percentage of motile cells, or mean cell velocity), but it abrogated any orientation of movement toward the source of the chemotaxin, formylmethionylleucylphenylanaline (FMLP) (net displacement along the gradient axis in micrometers, or as percentage of total migration distance). This defect could be completely reversed by treatment with lignoceric ceramide (5 μg/ml, 15 minutes). Immunolocalization studies demonstrated that N-SMase (1) distributes preferentially toward the leading edge of some elongated cells, (2) is associated with the plasma membrane, (3) is more than 99.5% localized to the cytofacial aspect of the plasma membrane, (4) is excluded from pseudopodial extensions, and (5) increases rapidly in response to FMLP. Morphologically, the inhibition of N-SMase limited cellular spreading and the extension of sheet-like pseudopods. Elongated PMNs demonstrated a polarized distribution of GTPases, with Rac 1/2 accumulated at, and RhoA excluded from, the front of the cell. This polarity was negated by N-SMase inhibition and restored by lignoceric ceramide. We conclude that N-SMase at the cytofacial plasma membrane is an essential element for the proper orientation of PMNs in FMLP gradients, at least in part by polarizing the distribution of Rac 1/2 and RhoA GTPases.
Sitrin
Robert G
RG
Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, 48109-5642, USA. rsitrin@umich.edu
Sassanella
Timothy M
TM
Petty
Howard R
HR
D000814:Aniline Compounds / Q000494:pharmacology; D001597:Benzylidene Compounds / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D016764:Cell Polarity; D002518:Ceramides / Q000378:metabolism / Q000494:pharmacology; D002634:Chemotaxis, Leukocyte / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008855:Microscopy, Electron, Scanning; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D013107:Sphingolipids / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000502:physiology*; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D000097766:RAC2 GTP-Binding Protein
D000814:Aniline Compounds; D001597:Benzylidene Compounds; D002518:Ceramides; D004791:Enzyme Inhibitors; D009240:N-Formylmethionine Leucyl-Phenylalanine; D013107:Sphingolipids; D013108:Sphingomyelin Phosphodiesterase; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; D000097766:RAC2 GTP-Binding Protein; C468773:GW 4869