E-selectin engages PSGL-1 and CD44 through a common signaling pathway to induce integrin αLβ2-mediated slow leukocyte rolling

Abstract

In inflamed venules, neutrophils rolling on E-selectin induce integrin ␣ L ␤ 2 -dependent slow rolling on intercellular adhesion molecule-1 by activating Src family kinases (SFKs), DAP12 and Fc receptor-␥ (FcR␥), spleen tyrosine kinase (Syk), and p38. E-selectin signaling cooperates with chemokine signaling to recruit neutrophils into tissues. Previous studies identified P-selectin glycoprotein ligand-1 (PSGL-1) as the essential Eselectin ligand and Fgr as the only SFK that initiate signaling to slow rolling. In contrast, we found that E-selectin engagement of PSGL-1 or CD44 triggered slow rolling through a common, lipid raftdependent pathway that used the SFKs Hck and Lyn as well as Fgr. We identified the Tec kinase Bruton tyrosine kinase as a key signaling intermediate between Syk and p38. E-selectin engagement of PSGL-1 was dependent on its cytoplasmic domain to activate SFKs and slow rolling. Although recruiting phosphoinositide-3-kinase to the PSGL-1 cytoplasmic domain was reported to activate integrins, Eselectin-mediated slow rolling did not require phosphoinositide-3-kinase. Studies in mice confirmed the physiologic significance of these events for neutrophil slow rolling and recruitment during inflammation. Thus, E-selectin triggers common signals through distinct neutrophil glycoproteins to induce ␣ L ␤ 2 -dependent slow rolling. (Blood. 2010;116(3):485-494) IntroductionCirculating leukocytes enter inflamed tissues through sequential adhesive and signaling events. 1 Neutrophils first tether to and roll on P-and E-selectin expressed on activated endothelial cells. 2 They roll on P-selectin through interactions with P-selectin glycoprotein ligand-1 (PSGL-1) 3,4 and on E-selectin through interactions with PSGL-1, CD44, and E-selectin ligand-1. [5][6][7] Rolling neutrophils encounter immobilized chemokines that signal through G␣ i proteincoupled receptors. These signals activate integrins ␣ L ␤ 2 and ␣ M ␤ 2 to their high-affinity states, enabling interactions with intercellular adhesion molecule-1 (ICAM-1) that promote arrest, adhesion strengthening, intraluminal crawling, and transendothelial migration. 1 Importantly, E-selectin directly triggers signals in rolling neutrophils that cooperate with chemokine signals to maximize neutrophil recruitment during inflammation. 8 In autoperfused whole blood, neutrophils rolling on immobilized E-selectin activate ␣ L ␤ 2 to an intermediate-affinity state, which slows rolling on coimmobilized ICAM-1. 8 In vivo, neutrophils roll slowly on E-selectin expressed in tumor necrosis factor-␣ (TNF-␣)-stimulated postcapillary venules. 9 Injecting anti-␣ L ␤ 2 antibody increases rolling velocities, documenting the physiologic importance of integrins for slow rolling in vivo. 8,10 Integrin-dependent slow rolling prolongs transit times in inflamed venules 11 and enhances neutrophil recruitment. 8 In autoperfused whole blood, it was reported that integrinmediated slow rolling on E-selectin and ICAM-1 is eliminated in neutrophils lacking PSGL-1 but only marginally impaired in ne...

Integrin Regulation during Leukocyte Recruitment

“…The other pathway is PI3Kg dependent (22). However, McEver and colleagues (19) found E-selectin-mediated rolling to be independent of PI3K by using in vitro assays. These differences can be explained by the fact that isolated neutrophils were used.…”

“…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). Syk activates a protein complex of the Src homology 2 domaincontaining leukocyte phosphoprotein of 76 kDa (SLP76) and the adhesion and degranulation-promoting adaptor protein (ADAP) (20).…”

“…However, striking differences enable distinct activation of outside-in signaling events: in neutrophils, the Src kinases Hck and Fgr are necessary for outside-in-mediated adhesion strengthening but not for GPCR-mediated integrin activation (33). In contrast to this signaling pathway, only Fgr or Hck and Lyn are required for LFA-1 activation following E-selectin engagement (18,19), showing that the different Src family kinases expressed in neutrophils have distinct functions. In Jurkat T cells, crosslinking of LFA-1 results in formation of an actin cloud that is dependent on the presence of the Src kinase Lyn (60).…”

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).…”

“…# p , 0.05. their N-glycan-mediated binding (52). In contrast to Galnt1 2/2 mice, T-syn 2/2 mice also exhibited impaired Syk phosphorylation after E-selectin engagement (50). These partially divergent results suggest that other O-glycosylated ligands, which are not affected by ppGalNAcT-1, participate in downstream signaling after Eselectin engagement.…”