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 Gαi-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. Gαi-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.

Integrin Regulation during Leukocyte Recruitment

“…E-selectin binding to PSGL-1 induces the phosphorylation of the ITAM containing adaptor proteins DAP12 and FcRg (18). This step is defective in mice lacking either the Src kinase Fgr or both Hck and Lyn (19): neutrophils from these mice fail to consecutively phosphorylate the spleen tyrosine kinase (Syk) (19).…”

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

“…As published recently, E-selectin engagement leads to the activation of LFA-1 via a Syk-dependent pathway (9,29,31,49,50). This pathway, together with chemokine-induced signaling, induces leukocyte adhesion and extravasation in the cremaster muscle after TNF-a application and neutrophil recruitment into the peritoneal cavity after thioglycollate injection (31, 34).…”

“…The Animal Care and Use Committees of the University of Münster approved all animal experiments. Chimeric mice were generated by performing bone marrow transplantation, as described previously (28,29). Briefly, unfractionated bone marrow cells isolated from gene-deficient mice were injected i.v.…”

“…Neutrophils were lysed with RIPA buffer (29). Lysates were boiled with sample buffer or incubated with Sepharose A/G beads and anti-Syk Ab (both from Santa Cruz Biotechnology) for 4 h on ice.…”

“…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.…”