title: The Src family kinases Hck and Fgr are dispensable for inside-out, chemoattractant-induced signaling regulating beta 2 integrin affinity and valency in neutrophils, but are required for beta 2 integrin-mediated outside-in signaling involved in sustained adhesion.
abstract: Neutrophil beta(2) integrins are activated by inside-out signaling regulating integrin affinity and valency; following ligand binding, beta(2) integrins trigger outside-in signals regulating cell functions. Addressing inside-out and outside-in signaling in hck(-/-)fgr(-/-) neutrophils, we found that Hck and Fgr do not regulate chemoattractant-induced activation of beta(2) integrin affinity. In fact, beta(2) integrin-mediated rapid adhesion, in static condition assays, and neutrophil adhesion to glass capillary tubes cocoated with ICAM-1, P-selectin, and a chemoattractant, under flow, were unaffected in hck(-/-)fgr(-/-) neutrophils. Additionally, examination of integrin affinity by soluble ICAM-1 binding assays and of beta(2) integrin clustering on the cell surface, showed that integrin activation did not require Hck and Fgr expression. However, after binding, hck(-/-)fgr(-/-) neutrophil spreading over beta(2) integrin ligands was reduced and they rapidly detached from the adhesive surface. Whether alterations in outside-in signaling affect sustained adhesion to the vascular endothelium in vivo was addressed by examining neutrophil adhesiveness to inflamed muscle venules. Intravital microscopy analysis allowed us to conclude that Hck and Fgr regulate neither the number of rolling cells nor rolling velocity in neutrophils. However, arrest of hck(-/-)fgr(-/-) neutrophils to >60 microm in diameter venules was reduced. Thus, Hck and Fgr play no role in chemoattractant-induced inside-out beta(2) integrin activation but regulate outside-in signaling-dependent sustained adhesion.
authors.affiliation: Department of Pathology, Section of General Pathology, University of Verona, Strada Le Grazie 8, 37134 Verona, Italy.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Giagulli
authors.forename: Linda
authors.initials: L
authors.lastname: Ottoboni
authors.forename: Elena
authors.initials: E
authors.lastname: Caveggion
authors.forename: Barbara
authors.initials: B
authors.lastname: Rossi
authors.forename: Clifford
authors.initials: C
authors.lastname: Lowell
authors.forename: Gabriela
authors.initials: G
authors.lastname: Constantin
authors.forename: Carlo
authors.initials: C
authors.lastname: Laudanna
authors.forename: Giorgio
authors.initials: G
authors.lastname: Berton
mesh_terms: D000818:Animals; D018821:CD18 Antigens / Q000378:metabolism / Q000502:physiology*; D002448:Cell Adhesion / Q000235:genetics / Q000276:immunology; D002464:Cell Migration Inhibition; D002478:Cells, Cultured; D002634:Chemotaxis, Leukocyte / Q000235:genetics / Q000276:immunology; D005260:Female; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018482:Muscle, Skeletal / Q000098:blood supply / Q000473:pathology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000276:immunology; D018375:Neutrophil Activation / Q000235:genetics / Q000276:immunology; D009504:Neutrophils / Q000201:enzymology* / Q000276:immunology* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D051572:Proto-Oncogene Proteins c-hck / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D014699:Venules / Q000276:immunology / Q000473:pathology; D019061:src-Family Kinases / Q000172:deficiency / Q000235:genetics / Q000502:physiology*
chemical_list: D018821:CD18 Antigens; D011518:Proto-Oncogene Proteins; D009240:N-Formylmethionine Leucyl-Phenylalanine; C496631:HCK protein, human; D051572:Proto-Oncogene Proteins c-hck; C053102:proto-oncogene proteins c-fgr; D019061:src-Family Kinases