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.
The epsin family of endocytic adapter proteins are widely expressed, and interact with both proteins and lipids to regulate a variety of cell functions. However, the role of epsins in atherosclerosis ...is poorly understood. Here, we show that deletion of endothelial epsin proteins reduces inflammation and attenuates atherosclerosis using both cell culture and mouse models of this disease. In atherogenic cholesterol-treated murine aortic endothelial cells, epsins interact with the ubiquitinated endoplasmic reticulum protein inositol 1,4,5-trisphosphate receptor type 1 (IP3R1), which triggers proteasomal degradation of this calcium release channel. Epsins potentiate its degradation via this interaction. Genetic reduction of endothelial IP3R1 accelerates atherosclerosis, whereas deletion of endothelial epsins stabilizes IP3R1 and mitigates inflammation. Reduction of IP3R1 in epsin-deficient mice restores atherosclerotic progression. Taken together, epsin-mediated degradation of IP3R1 represents a previously undiscovered biological role for epsin proteins and may provide new therapeutic targets for the treatment of atherosclerosis and other diseases.
Neutrophil recruitment, mediated by β2 integrins, combats pyogenic infections but also plays a key role in ischemia-reperfusion injury and other inflammatory disorders. Talin induces allosteric ...rearrangements in integrins that increase affinity for ligands (activation). Talin also links integrins to actin and other proteins that enable formation of adhesions. Structural studies have identified a talin1 mutant (L325R) that perturbs activation without impairing talin's capacity to link integrins to actin and other proteins. Here, we found that mice engineered to express only talin1(L325R) in myeloid cells were protected from renal ischemia-reperfusion injury. Dissection of neutrophil function in vitro and in vivo revealed that talin1(L325R) neutrophils had markedly impaired chemokine-induced, β2 integrin-mediated arrest, spreading, and migration. Surprisingly, talin1(L325R) neutrophils exhibited normal selectin-induced, β2 integrin-mediated slow rolling, in sharp contrast to the defective slow rolling of neutrophils lacking talin1 or expressing a talin1 mutant (W359A) that blocks talin interaction with integrins. These studies reveal the importance of talin-mediated activation of integrins for renal ischemia-reperfusion injury. They further show that neutrophil arrest requires talin recruitment to and activation of integrins. However, although neutrophil slow rolling requires talin recruitment to integrins, talin-mediated integrin activation is dispensable.
A2A adenosine receptor (A2AAR) signaling negatively regulates inflammatory responses in many disease models, but the detailed mechanisms remain unclear. We used the selective A2AAR agonist, ATL313, ...to examine how A2AAR signaling affects human and murine neutrophil adhesion under flow. Treating neutrophils with ATL313 inhibited selectin-induced, β2 integrin-dependent slow rolling and chemokine-induced, β2 integrin-dependent arrest on ICAM-1. ATL313 inhibited selectin-induced β2 integrin extension, which supports slow rolling, and chemokine-induced hybrid domain "swing-out," which supports arrest. Furthermore, ATL313 inhibited integrin outside-in signaling as revealed by reduced neutrophil superoxide production and spreading on immobilized anti-β2 integrin Ab. ATL313 suppressed selectin-triggered activation of Src family kinases (SFKs) and p38 MAPK, chemokine-triggered activation of Ras-related protein 1, and β2 integrin-triggered activation of SFKs and Vav cytoskeletal regulatory proteins. ATL313 activated protein kinase A and its substrate C-terminal Src kinase, an inhibitor of SFKs. Treating neutrophils with a protein kinase A inhibitor blocked the actions of ATL313. In vivo, ATL313-treated neutrophils rolled faster and arrested much less frequently in postcapillary venules of the murine cremaster muscle after TNF-α challenge. Furthermore, ATL313 markedly suppressed neutrophil migration into the peritoneum challenged with thioglycollate. ATL313 did not affect A2AAR-deficient neutrophils, confirming its specificity. Our findings provide new insights into the anti-inflammatory mechanisms of A2AAR signaling and the potential utility of A2AAR agonists in inflammatory diseases.
Bonds between adhesion molecules are often mechanically stressed. A striking example is the tensile force applied to selectin-ligand bonds, which mediate the tethering and rolling of flowing ...leukocytes on vascular surfaces. It has been suggested that force could either shorten bond lifetimes, because work done by the force could lower the energy barrier between the bound and free states ('slip'), or prolong bond lifetimes by deforming the molecules such that they lock more tightly ('catch'). Whereas slip bonds have been widely observed, catch bonds have not been demonstrated experimentally. Here, using atomic force microscopy and flow-chamber experiments, we show that increasing force first prolonged and then shortened the lifetimes of P-selectin complexes with P-selectin glycoprotein ligand-1, revealing both catch and slip bond behaviour. Transitions between catch and slip bonds might explain why leukocyte rolling on selectins first increases and then decreases as wall shear stress increases. This dual response to force provides a mechanism for regulating cell adhesion under conditions of variable mechanical stress.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Circulating neutrophils must avoid premature activation to prevent tissue injury. The leukocyte adhesion receptor L-selectin forms bonds with P-selectin glycoprotein ligand-1 (PSGL-1) on other ...leukocytes and with peripheral node addressin (PNAd) on high endothelial venules. Mechanical forces can strengthen (catch) or weaken (slip) bonds between biological molecules. How these mechanochemical processes influence function in vivo is unexplored. Here we show that mice expressing an L-selectin mutant (N138G) have altered catch bonds and prolonged bond lifetimes at low forces. Basal lymphocyte homing and neutrophil recruitment to inflamed sites are normal. However, circulating neutrophils form unstable aggregates and are unexpectedly primed to respond robustly to inflammatory mediators. Priming requires signals transduced through L-selectin N138G after it engages PSGL-1 or PNAd. Priming enhances bacterial clearance but increases inflammatory injury and enlarges venous thrombi. Thus, L-selectin mechanochemistry limits premature activation of neutrophils. Our results highlight the importance of probing how mechanochemistry functions in vivo.
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 â»/â» 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 â»/â» neutrophils or neuraminidase-treated neutrophils failed to activate tyrosine kinases when plated on immobilized antiâPSGL-1 or anti-CD44 F(abâ²) â. Furthermore, C1galt1 â»/â» neutrophils incubated with antiâPSGL-1 F(abâ²) â did not generate microparticles. In marked contrast, PSGL-1, CD43, and CD44 moved normally to the uropods of chemokine-stimulated C1galt1 â»/â» 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.
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.
In the earliest phase of inflammation, histamine and other agonists rapidly mobilize P-selectin to the apical membranes of endothelial cells, where it initiates rolling adhesion of flowing ...neutrophils. Clustering of P-selectin in clathrin-coated pits facilitates rolling. Inflammatory cytokines typically signal by regulating gene transcription over a period of hours. We found that neutrophils rolling on P-selectin secreted the cytokine oncostatin M (OSM). The released OSM triggered signals through glycoprotein 130 (gp130)–containing receptors on endothelial cells that, within minutes, further clustered P-selectin and markedly enhanced its adhesive function. Antibodies to OSM or gp130, deletion of the gene encoding OSM in hematopoietic cells, or conditional deletion of the gene encoding gp130 in endothelial cells inhibited neutrophil rolling on P-selectin in trauma-stimulated venules of the mouse cremaster muscle. In a mouse model of P-selectin–dependent deep vein thrombosis, deletion of OSM in hematopoietic cells or of gp130 in endothelial cells markedly inhibited adhesion of neutrophils and monocytes and the rate and extent of thrombus formation. Our results reveal a paracrine-signaling mechanism by which neutrophil-released OSM rapidly influences endothelial cell function during physiological and pathological inflammation.
Following inflammatory responses, a series of events leads to mobilization of neutrophils that adhere to P-selectin on the apical membranes of endothelial cells. The McEver laboratory finds the neutrophil-produced oncostatin M plays a critical role in this process, establishing a paracrine signaling mechanism between neutrophils and endothelial cells that influences endothelial-cell function.
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Leukocytes roll on P-selectin after its mobilization from secretory granules to the surfaces of platelets and endothelial cells. Tumor necrosis factor (TNF), IL-1β, and lipopolysaccharide increase ...synthesis of P-selectin in murine but not in human endothelial cells. To explore the physiological significance of this difference in gene regulation, we made transgenic mice bearing the human Selp gene and crossed them with mice lacking murine P-selectin (Selp(-/-)). The transgenic mice constitutively expressed human P-selectin in platelets, endothelial cells, and macrophages. P-selectin mediated comparable neutrophil migration into the inflamed peritoneum of transgenic and wild-type (WT) mice. Leukocytes rolled similarly on human or murine P-selectin on activated murine platelets and in venules of the cremaster muscle subjected to trauma. However, TNF increased murine P-selectin in venules, slowing rolling and increasing adhesion, whereas it decreased human P-selectin, accelerating rolling and decreasing adhesion. Both P- and E-selectin mediated basal rolling in the skin of WT mice, but E-selectin dominated rolling in transgenic mice. During contact hypersensitivity, murine P-selectin messenger (m) RNA was up-regulated and P-selectin was essential for leukocyte recruitment. However, human P-selectin mRNA was down-regulated and P-selectin contributed much less to leukocyte recruitment. These findings reveal functionally significant differences in basal and inducible expression of human and murine P-selectin in vivo.
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.
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