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...

In inflamed venules, leukocytes use Pselectin glycoprotein ligand-1 (PSGL-1) to roll on P-selectin and E-selectin and to activate integrin ␣L␤2 (lymphocyte function-associated antigen-1, LFA-1) to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Studies in cell lines have suggested that PSGL-1 requires its cytoplasmic domain to localize in membrane domains, to support rolling on Pselectin, and to signal through spleen tyrosine kinase (Syk). We generated "⌬CD" mice that express PSGL-1 without the cytoplasmic domain. Unexpectedly, neutrophils from these mice localized PSGL-1 normally in microvilli, uropods, and lipid rafts. ⌬CD neutrophils expressed less PSGL-1 on their surfaces because of inefficient export from the endoplasmic reticulum. Limited digestion of wild-type neutrophils with O-sialoglycoprotein endopeptidase was used to reduce the PSGL-1 density to that on ⌬CD neutrophils. At matched PSGL-1 densities, both ⌬CD and wild-type neutrophils rolled similarly on P-selectin. However, ⌬CD neutrophils rolling on P-selectin did not trigger Sykdependent activation of LFA-1 to slow rolling on ICAM-1. These data demonstrate that the PSGL-1 cytoplasmic domain is dispensable for leukocyte rolling on P-selectin but is essential to activate ␤2 integrins to slow rolling on ICAM-1. IntroductionDuring inflammation, leukocytes tether to and roll on the vessel wall. They then roll more slowly until they arrest. Finally, they crawl through or between endothelial cells into the underlying tissues. Interactions of selectins with glycosylated ligands mediate tethering and rolling. Interactions of ␤2 integrins with ligands, such as intercellular adhesion molecule-1 (ICAM-1), mediate slow rolling and arrest. 1,2 These interactions occur in blood flow, which exerts force on adhesive bonds that affects bond lifetimes. 3,4 Furthermore, engagement of adhesion receptors transmits signals that intersect with chemokine receptor signals to influence the adhesion cascade. 1 Binding of integrin cytoplasmic domains to signaling and cytoskeletal proteins is critical for integrin function. 1,5 Interactions of selectin cytoplasmic domains with cytosolic proteins also contribute to their adhesive properties. E-selectin and P-selectin are expressed on activated endothelial cells and/or platelets, whereas L-selectin is expressed on the microvilli of leukocytes. 2 The cytoplasmic domains of E-selectin and P-selectin interact with clathrin-coated pits. These interactions cluster E-selectin and P-selectin on the endothelial cell surface, enhancing leukocyte rolling under flow. 6,7 The cytoplasmic domain anchors L-selectin to the cytoskeleton by binding to ␣-actinin 8 and to the ezrin/radixin/ moesin (ERM) family. 9 Mutation of the ERM-binding site in the cytoplasmic domain shifts L-selectin out of microvilli onto the cell body of transfected cells and impairs tethering to L-selectin ligands under flow. 10 Removal of the ␣-actinin-binding site markedly impairs rolling of transfected cells on L-selectin ligands, and deletion of the cytoplas...

Colon mucus segregates the intestinal microbiota from host tissues, but how it organizes to function throughout the colon is unclear. In mice, we found that colon mucus consists of two distinct O-glycosylated entities of Muc2: a major form produced by the proximal colon, which encapsulates the fecal material including the microbiota, and a minor form derived from the distal colon, which adheres to the major form. The microbiota directs its own encapsulation by inducing Muc2 production from proximal colon goblet cells. In turn, O-glycans on proximal colon–derived Muc2 modulate the structure and function of the microbiota as well as transcription in the colon mucosa. Our work shows how proximal colon control of mucin production is an important element in the regulation of host-microbiota symbiosis.

Most platelet membrane proteins are modified by mucin-type core 1-derived glycans (O-glycans). However, the biological importance of O-glycans in platelet clearance is unclear. Here, we generated mice with a hematopoietic cell-specific loss of O-glycans (HC ). These mice lack O-glycans on platelets and exhibit reduced peripheral platelet numbers. Platelets from HC mice show reduced levels of α-2,3-linked sialic acids and increased accumulation in the liver relative to wild-type platelets. The preferential accumulation of HC platelets in the liver was reduced in mice lacking the hepatic asialoglycoprotein receptor [Ashwell-Morell receptor (AMR)]. However, we found that Kupffer cells are the primary cells phagocytosing HC platelets in the liver. Our results demonstrate that hepatic AMR promotes preferential adherence to and phagocytosis of desialylated and/or HC platelets by the Kupffer cell through its C-type lectin receptor CLEC4F. These findings provide insights into an essential role for core 1 O-glycosylation of platelets in their clearance in the liver.

Trousseau syndrome is classically defined as migratory, heparin-sensitive but warfarin-resistant microthrombi in patients with occult, mucinous adenocarcinomas. Injecting carcinoma mucins into mice generates platelet-rich microthrombi dependent on P-and L-selectin but not thrombin. Heparin prevents mucin binding to P-and L-selectin and mucininduced microthrombi. This model of Trousseau syndrome explains resistance to warfarin, which inhibits fluid-phase coagulation but not selectins. Here we found that carcinoma mucins do not generate microthrombi in mice lacking P-selectin glycoprotein ligand-1 (PSGL-1), the leukocyte ligand for P-and L-selectin. Furthermore, mucins did not activate platelets in blood from PSGL-1-deficient mice. Mucins induced microthrombi in radiation chimeras lacking endothelial P-selectin but not in chimeras lacking platelet Pselectin. Mucins caused leukocytes to release cathepsin G, but only if platelets were present. Mucins failed to generate microthrombi in cathepsin G-deficient mice. Mucins did not activate platelets in blood from mice lacking cathepsin G or protease-activated receptor-4 (PAR4), indicating that cathepsin G activates platelets through PAR4. Using knockout mice and blocking antibodies, we found that mucin-triggered cathepsin G release requires L-selectin and PSGL-1 on neutrophils, P-selectin on platelets, and Src family kinases in both cell types. IntroductionTrousseau syndrome is classically defined as a thrombotic event preceding or appearing concomitantly with a visceral malignancy. 1,2 The hallmarks are venous and arterial microthrombi with secondary microangiopathic hemolytic anemia. Notwithstanding its classic definition, Trousseau syndrome is sometimes used to describe any thrombotic complication associated with cancer. 2 Early studies of Trousseau syndrome emphasized the activation of fluid-phase coagulation. A cysteine protease in carcinoma extracts was reported to directly activate factor X. 3 Many tumors express tissue factor, 4 and tissue factor-bearing microparticles derived from platelets 5 or tumor cells 6,7 have been observed. However, heparin prevents recurrent thrombosis much more effectively than vitamin K antagonists such as warfarin. 1,[8][9][10][11][12][13] Although heparin might block tissue factor-triggered coagulation more effectively than warfarin, its clinical superiority implies that other mechanisms also contribute to Trousseau syndrome. The association of Trousseau syndrome with mucinous adenocarcinomas suggests a pathogenic role for tumor-secreted mucins. 1 Carcinoma cells express higher levels of mucins, 14 and elevated levels of these mucins and their fragments circulate in patients. 15,16 Mucins bearing highly sialylated Oglycans selectively resist clearance and circulate for long periods. 17 During inflammation or injury, interactions of selectins with their glycosylated ligands initiate adhesion of leukocytes to activated platelets and/or endothelial cells. 18 L-selectin is expressed on leukocytes. Thrombin and other agonists cause plat...