Spleen Tyrosine Kinase Syk Is Necessary for E-Selectin-Induced αLβ2 Integrin-Mediated Rolling on Intercellular Adhesion Molecule-1

Abstract

Engagement of neutrophils by E-selectin results in integrin activation. Here, we investigated primary mouse neutrophils in whole blood by using intravital microscopy and autoperfused flow chambers. Slow rolling on E-selectin coimmobilized with intercellular adhesion molecule-1 (ICAM-1) required P-selectin glycoprotein ligand (PSGL)-1, was dependent on alpha(L)beta(2) integrin (LFA-1), and required continuous E-selectin engagement. Slow rolling was abolished by pharmacological blockade of spleen tyrosine kinase (Syk) and was absent in Syk(-/-) bone-marrow chimeric mice. Treatment with tumor necrosis factor-alpha lowered rolling velocity further and induced CXC chemokine ligand-1 (CXCL1) and CXC chemokine receptor-2 (CXCR2)-dependent leukocyte arrest on E-selectin and ICAM-1. Arrest but not rolling was blocked by an allosteric inhibitor of LFA-1 activation. Neutrophil recruitment in a thioglycollate-induced peritonitis model was almost completely inhibited in Selplg(-/-) mice or Syk(-/-) bone-marrow chimeras treated with pertussis toxin. This identifies a second neutrophil-activation pathway that is as important as activation through G protein-coupled receptors (GPCRs).

Severe Impairment of Leukocyte Recruitment in ppGalNAcT-1–Deficient Mice

“…Two hours after TNF-a injection, leukocyte rolling is mediated by P-selectin and E-selectin expressed on inflamed endothelial cells of the cremaster muscle (9,33). Galnt1 2/2 mice had a reduced rolling flux fraction on E-and P-selectin compared with control mice 2 h after TNF-a injection ( Fig.…”

“…To investigate whether the signal-transduction cascade is disturbed after E-selectin binding to PSGL-1, we performed an E-selectin-engagement assay to demonstrate intact downstream signaling. We already showed that Syk is phosphorylated after stimulating neutrophils with E-selectin (9). Therefore, we checked Syk phosphorylation in Galnt1 2/2 and control mice.…”

“…injection of ketamine hydrochloride (125 mg/kg; Sanofi Winthrop Pharmaceuticals) and xylazine (12.5 mg/kg; TranquiVed, Phoenix Scientific). In the trauma model, the cremaster muscle was prepared for intravital imaging, as described previously (9,29). To investigate short-term inflammation, mice received an intrascrotal injection of 500 ng TNF-a (R&D Systems, Wiesbaden-Nordenstadt, Germany) in 0.3 ml saline 2 h before cremaster muscle exteriorization.…”

“…To test whether ppGalNAcT-1 is involved in chemokine-induced arrest, we conducted intravital microscopy of the untreated cremaster muscle (9). In this model, neutrophils roll in cremaster venules as the result of P-selectin expression on the endothelium but rarely adhere.…”

“…In venules of the cremaster muscle, the L-selectin-dependent rolling (130 mm/s) is faster than the velocity on P-selectin (40 mm/s) (8). E-selectin mediates slow rolling, with a velocity of 3-7 mm/s (7,9).…”