Bojing Shao

Blocking neutrophil integrin activation prevents ischemia–reperfusion injury

Yago et al. find that neutrophils expressing a talin1 mutant that perturbs integrin activation fail to undergo chemokine-induced arrest, spreading, and migration, thus preventing ischemia–reperfusion injury.

Signal-dependent Slow Leukocyte Rolling Does Not Require Cytoskeletal Anchorage of P-selectin Glycoprotein Ligand-1 (PSGL-1) or Integrin αLβ2

Background: The cytoskeleton contributes to receptor-initiated signaling and has been linked to force-regulated integrin activation. Results: Disrupting cytoskeletal interactions of PSGL-1 or αLβ2 does not impair slow rolling of leukocytes on P-selectin and ICAM-1. Conclusion: The cytoskeleton is not required for PSGL-1-initiated signals to extend αLβ2. Significance: Signals can “prime” integrins without the cytoskeleton. In inflamed venules, neutrophils roll on P- or E-selectin, engage P-selectin glycoprotein ligand-1 (PSGL-1), and signal extension of integrin αLβ2 in a low affinity state to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Cytoskeleton-dependent receptor clustering often triggers signaling, and it has been hypothesized that the cytoplasmic domain links PSGL-1 to the cytoskeleton. Chemokines cause rolling neutrophils to fully activate αLβ2, leading to arrest on ICAM-1. Cytoskeletal anchorage of αLβ2 has been linked to chemokine-triggered extension and force-regulated conversion to the high affinity state. We asked whether PSGL-1 must interact with the cytoskeleton to initiate signaling and whether αLβ2 must interact with the cytoskeleton to extend. Fluorescence recovery after photobleaching of transfected cells documented cytoskeletal restraint of PSGL-1. The lateral mobility of PSGL-1 similarly increased by depolymerizing actin filaments with latrunculin B or by mutating the cytoplasmic tail to impair binding to the cytoskeleton. Converting dimeric PSGL-1 to a monomer by replacing its transmembrane domain did not alter its mobility. By transducing retroviruses expressing WT or mutant PSGL-1 into bone marrow-derived macrophages from PSGL-1-deficient mice, we show that PSGL-1 required neither dimerization nor cytoskeletal anchorage to signal β2 integrin-dependent slow rolling on P-selectin and ICAM-1. Depolymerizing actin filaments or decreasing actomyosin tension in neutrophils did not impair PSGL-1- or chemokine-mediated integrin extension. Unlike chemokines, PSGL-1 did not signal cytoskeleton-dependent swing out of the β2-hybrid domain associated with the high affinity state. The cytoskeletal independence of PSGL-1-initiated, αLβ2-mediated slow rolling differs markedly from the cytoskeletal dependence of chemokine-initiated, αLβ2-mediated arrest.

Elevated CXCL1 expression in gp130-deficient endothelial cells impairs neutrophil migration in mice

Neutrophils emigrate from venules to sites of infection or injury in response to chemotactic gradients. How these gradients form is not well understood. Some IL-6 family cytokines stimulate endothelial cells to express adhesion molecules and chemokines that recruit leukocytes. Receptors for these cytokines share the signaling subunit gp130. We studied knockout mice lacking gp130 in endothelial cells. Unexpectedly, gp130-deficient endothelial cells constitutively expressed more CXCL1 in vivo and in vitro, and even more upon stimulation with tumor necrosis factor-α. Mobilization of this increased CXCL1 from intracellular stores to the venular surface triggered β2 integrin-dependent arrest of neutrophils rolling on selectins but impaired intraluminal crawling and transendothelial migration. Superfusing CXCL1 over venules promoted neutrophil migration only after intravenously injecting mAb to CXCL1 to diminish its intravascular function or heparinase to release CXCL1 from endothelial proteoglycans. Remarkably, mice lacking gp130 in endothelial cells had impaired histamine-induced venular permeability, which was restored by injecting anti-P-selectin mAb to prevent neutrophil rolling and arrest. Thus, excessive CXCL1 expression in gp130-deficient endothelial cells augments neutrophil adhesion but hinders migration, most likely by disrupting chemotactic gradients. Our data define a role for endothelial cell gp130 in regulating integrin-dependent adhesion and de-adhesion of neutrophils during inflammation.

Circulating soluble P-selectin must dimerize to promote inflammation and coagulation in mice

Leukocyte adhesion to P-selectin on activated platelets and endothelial cells induces shedding of the P-selectin ectodomain into the circulation. Plasma soluble P-selectin (sP-selectin) is elevated threefold to fourfold in patients with cardiovascular disease. Circulating sP-selectin is thought to trigger signaling in leukocytes that directly contributes to inflammation and thrombosis. However, sP-selectin likely circulates as a monomer, and in vitro studies suggest that sP-selectin must dimerize to induce signaling in leukocytes. To address this discrepancy, we expressed the entire ectodomain of mouse P-selectin as a monomer (sP-selectin) or as a disulfide-linked dimer fused to the Fc portion of mouse immunoglobulin G (sP-selectin-Fc). Dimeric sP-selectin-Fc, but not monomeric sP-selectin, triggered integrin-dependent adhesion of mouse leukocytes in vitro. Antibody-induced oligomerization of sP-selectin or sP-selectin-Fc was required to trigger formation of neutrophil extracellular traps. Injecting sP-selectin-Fc, but not sP-selectin, into mice augmented integrin-dependent adhesion of neutrophils in venules, generated tissue factor-bearing microparticles, shortened plasma-clotting times, and increased thrombus frequency in the inferior vena cava. Furthermore, transgenic mice that overexpressed monomeric sP-selectin did not exhibit increased inflammation or thrombosis. We conclude that elevated plasma sP-selectin is a consequence rather than a cause of cardiovascular disease.

O-glycans direct selectin ligands to lipid rafts on leukocytes

Significance Leukocytes partition certain proteins into cholesterol- and sphingolipid-rich membrane regions (lipid rafts) that function as signaling platforms. Inflammatory stimuli cause leukocytes to elongate to form lamellipodia and uropods at opposite ends that facilitate migration. Many raft-associated proteins move to uropods. Proteins are typically thought to use their transmembrane and cytoplasmic domains to associate with rafts. Here, we found that some leukocyte adhesion proteins used carbohydrate modification (glycosylation) of their extracellular domains to associate with lipid rafts. These proteins required preassociation with rafts to transduce signals but, unexpectedly, not to move to uropods. These data define a mechanism for localizing proteins to critical membrane regions of leukocytes. Palmitoylated cysteines typically target transmembrane proteins to domains enriched in cholesterol and sphingolipids (lipid rafts). P-selectin glycoprotein ligand-1 (PSGL-1), CD43, and CD44 are O-glycosylated proteins on leukocytes that associate with lipid rafts. During inflammation, they transduce signals by engaging selectins as leukocytes roll in venules, and they move to the raft-enriched uropods of polarized cells upon chemokine stimulation. It is not known how these glycoproteins associate with lipid rafts or whether this association is required for signaling or for translocation to uropods. Here, we found that loss of core 1-derived O-glycans in murine C1galt1−/− neutrophils blocked raft targeting of PSGL-1, CD43, and CD44, but not of other glycosylated proteins, as measured by resistance to solubilization in nonionic detergent and by copatching with a raft-resident sphingolipid on intact cells. Neuraminidase removal of sialic acids from wild-type neutrophils also blocked raft targeting. C1galt1−/− neutrophils or neuraminidase-treated neutrophils failed to activate tyrosine kinases when plated on immobilized anti–PSGL-1 or anti-CD44 F(ab′)2. Furthermore, C1galt1−/− neutrophils incubated with anti–PSGL-1 F(ab′)2 did not generate microparticles. In marked contrast, PSGL-1, CD43, and CD44 moved normally to the uropods of chemokine-stimulated C1galt1−/− neutrophils. These data define a role for core 1-derived O-glycans and terminal sialic acids in targeting glycoprotein ligands for selectins to lipid rafts of leukocytes. Preassociation of these glycoproteins with rafts is required for signaling but not for movement to uropods.

Sialylation on O-glycans protects platelets from clearance by liver Kupffer cells

Significance Although many platelet glycoproteins, such as GPIbα and GPIIb/IIIa, are predominately modified by O-glycans, the biological importance of O-glycans in platelet homeostasis is unclear. Here, we report that platelets lacking O-glycans exhibit a reduced life-span and increased clearance in the liver due to defective sialylation. We found that Kupffer cells play a major role in clearing desialylated O-glycan–deficient platelets in cooperation with hepatocytes via the hepatic asialoglycoprotein receptor. These findings reveal how O-glycosylation regulates platelet homeostasis and clearance; they may also provide insights into the pathogenesis of disorders with thrombocytopenia such as sepsis and immune thrombocytopenia refractory to splenectomy. 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 C1galt1−/−). These mice lack O-glycans on platelets and exhibit reduced peripheral platelet numbers. Platelets from HC C1galt1−/− 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 C1galt1−/− 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 C1galt1−/− platelets in the liver. Our results demonstrate that hepatic AMR promotes preferential adherence to and phagocytosis of desialylated and/or HC C1galt1−/− 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.

Replacing the Promoter of the Murine Gene Encoding P-selectin with the Human Promoter Confers Human-like Basal and Inducible Expression in Mice

In humans and mice, megakaryocytes/platelets and endothelial cells constitutively synthesize P-selectin and mobilize it to the plasma membrane to mediate leukocyte rolling during inflammation. TNF-α, interleukin 1β, and LPS markedly increase P-selectin mRNA in mice but decrease P-selectin mRNA in humans. Transgenic mice bearing the entire human SELP gene recapitulate basal and inducible expression of human P-selectin and reveal human-specific differences in P-selectin function. Differences in the human SELP and murine Selp promoters account for divergent expression in vitro, but their significance in vivo is not known. Here we generated knockin mice that replace the 1.4-kb proximal Selp promoter with the corresponding SELP sequence (SelpKI). SelpKI/KI mice constitutively expressed more P-selectin on platelets and more P-selectin mRNA in tissues but only slightly increased P-selectin mRNA after injection of TNF-α or LPS. Consistent with higher basal expression, leukocytes rolled more slowly on P-selectin in trauma-stimulated venules of SelpKI/KI mice. However, TNF-α did not further reduce P-selectin-dependent rolling velocities. Blunted up-regulation of P-selectin mRNA during contact hypersensitivity reduced P-selectin-dependent inflammation in SelpKI/− mice. Higher basal P-selectin in SelpKI/KI mice compensated for this defect. Therefore, divergent sequences in a short promoter mediate most of the functionally significant differences in expression of human and murine P-selectin in vivo.