PSGL-1 engagement by E-selectin signals through Src kinase Fgr and ITAM adapters DAP12 and FcRγ to induce slow leukocyte rolling

Abstract

E-selectin binding to P-selectin glycoprotein ligand-1 (PSGL-1) can activate the β₂ integrin lymphocyte function-associated antigen-1 by signaling through spleen tyrosine kinase (Syk). This signaling is independent of Gα_i-protein–coupled receptors, results in slow rolling, and promotes neutrophil recruitment to sites of inflammation. However, the signaling pathways linking E-selectin engagement of PSGL-1 to Syk activation are unknown. To test the role of Src family kinases and immunoreceptor tyrosine-based activating motif (ITAM)–containing adaptor proteins, we used different gene-deficient mice in flow chamber, intravital microscopy, and peritonitis studies. E-selectin–mediated phosphorylation of Syk and slow rolling was abolished in neutrophils from fgr^−/− or hck^−/− lyn^−/− fgr^−/− mice. Neutrophils from Tyrobp^−/− Fcrg^−/− mice lacking both DAP12 and FcRγ were incapable of sustaining slow neutrophil rolling on E-selectin and intercellular adhesion molecule-1 and were unable to phosphorylate Syk and p38 MAPK. This defect was confirmed in vivo by using mixed chimeric mice. Gα_i-independent neutrophil recruitment into the inflamed peritoneal cavity was sharply suppressed in Tyrobp^−/− Fcrg^−/− mice. Our data demonstrate that an ITAM-dependent pathway involving the Src-family kinase Fgr and the ITAM-containing adaptor proteins DAP12 and FcRγ is involved in the initial signaling events downstream of PSGL-1 that are required to initiate neutrophil slow rolling.