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Markus Sperandio,
David Frommhold,
Inna Babushkina,
Lesley G Ellies,
Timothy S Olson,
Michael L Smith,
Benedikt Fritzsching,
Eva Pauly, David F Smith,
Rainer Nobiling,
Otwin Linderkamp,
Jamey D Marth,
Klaus Ley
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ABSTRACT: L-selectin belongs to the C-type lectin family of glycoproteins and is constitutively expressed on most leukocytes. L-selectin mediates leukocyte rolling in inflamed microvessels and high endothelial venules (HEV) via binding to specific carbohydrate structures on selectin ligands. Previous studies using sialidase treatment suggested a role of sialic acid residues in L-selectin-dependent rolling. To investigate the role of the alpha2,3-sialyltransferase (ST3Gal)-IV on L-selectin ligand activity in vivo, we studied leukocyte rolling in inflamed venules of the cremaster muscle and in Peyer's patch HEV of ST3Gal-IV-deficient mice and littermate control mice. In cremaster muscle venules with or without TNF-alpha treatment, L-selectin-dependent rolling was almost completely abolished in ST3Gal-IV(-/-) mice. In both models, L-selectin interacts with P-selectin glycoprotein ligand-1 (PSGL-1) presented by adherent leukocytes and leukocyte fragments, but not with endothelial L-selectin ligands. In contrast, L-selectin-dependent rolling in Peyer's patch HEV, which is mediated by unknown endothelial L-selectin ligands, was not impaired in the absence of ST3Gal-IV. Our in vivo data show that PSGL-1, the molecule responsible for L-selectin-mediated leukocyte interactions in inflammation, is dependent on ST3Gal-IV, while alpha2,3-sialylation by ST3Gal-IV is not necessary for L-selectin ligand activity on high endothelial cells of Peyer's patch HEV.
European Journal of Immunology 01/2007; 36(12):3207-15. · 5.10 Impact Factor
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ABSTRACT: During inflammation, leukocytes roll along the wall of postcapillary venules scanning the surface for immobilized CXCL1, a chemokine that triggers firm adhesion by activating CXCR2 on the neutrophil. PI-3K are signaling molecules important in cellular processes, ranging from cellular differentiation to leukocyte migration. PI-3Kgamma can be activated directly by the betagamma dimer of heterotrimeric G proteins coupled to CXCR2. Here, we used in vivo and ex vivo intravital microscopy models to test the role of PI-3Kgamma in leukocyte arrest. PI-3Kgamma null mice showed an 80% decrease in CXCL1-induced leukocyte adhesion in venules of the exteriorized mouse cremaster muscle. In wild-type mice, rolling leukocytes showed rapid and sustained adhesion, but in PI-3Kgamma(-/-) mice, adhesion was not triggered at all or was transient, suggesting that absence of PI-3Kgamma interferes with integrin bond strengthening. Wild-type mice reconstituted with PI-3Kgamma null bone marrow showed a 50% decrease in CXCL1-induced leukocyte adhesion. In a blood-perfused micro-flow chamber, leukocytes from PI-3Kgamma(-/-) mice showed a defect in adhesion on a P-selectin/ICAM-1/CXCL1 substrate, indicating that leukocyte PI-3Kgamma was required for adhesion. The adhesion defect in PI-3Kgamma(-/-) mice was as severe as that in mice lacking LFA-1, the major integrin responsible for neutrophil adhesion. We conclude that the gamma isoform of PI-3K must be functional in leukocytes to allow efficient adhesion from rolling in response to chemokine stimulation.
Journal of Leukocyte Biology 01/2007; 80(6):1491-9. · 4.99 Impact Factor
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Markus Sperandio M.D,
David Frommhold,
Inna Babushkina,
Lesley G. Ellies,
Timothy S. Olson,
Michael L. Smith,
Benedikt Fritzsching,
Eva Pauly, David F. Smith,
Rainer Nobiling,
Otwin Linderkamp,
Jamey D. Marth,
Klaus Ley,
Markus Sperandio
[show abstract]
[hide abstract]
ABSTRACT: L-selectin belongs to the C-type lectin family of glycoproteins and is constitutively expressed on most leukocytes. L-selectin mediates leukocyte rolling in inflamed microvessels and high endothelial venules (HEV) via binding to specific carbohydrate structures on selectin ligands. Previous studies using sialidase treatment suggested a role of sialic acid residues in L-selectin-dependent rolling. To investigate the role of the α2,3-sialyltransferase (ST3Gal)-IV on L-selectin ligand activity in vivo, we studied leukocyte rolling in inflamed venules of the cremaster muscle and in Peyer's patch HEV of ST3Gal-IV-deficient mice and littermate control mice. In cremaster muscle venules with or without TNF-α treatment, L-selectin-dependent rolling was almost completely abolished in ST3Gal-IV–/– mice. In both models, L-selectin interacts with P-selectin glycoprotein ligand-1 (PSGL-1) presented by adherent leukocytes and leukocyte fragments, but not with endothelial L-selectin ligands. In contrast, L-selectin-dependent rolling in Peyer's patch HEV, which is mediated by unknown endothelial L-selectin ligands, was not impaired in the absence of ST3Gal-IV. Our in vivo data show that PSGL-1, the molecule responsible for L-selectin-mediated leukocyte interactions in inflammation, is dependent on ST3Gal-IV, while α2,3-sialylation by ST3Gal-IV is not necessary for L-selectin ligand activity on high endothelial cells of Peyer's patch HEV.
European Journal of Immunology 11/2006; 36(12):3207 - 3215. · 5.10 Impact Factor
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ABSTRACT: To study rolling of mouse neutrophils on E-selectin and ICAM-1 in an ex vivo flow chamber system.
The authors developed a small autoperfused flow chamber (20 x 200-microm cross section) that allows direct visualization of cells with and without fluorescent labeling and does not require recirculation of blood.
Neutrophils rolled on E-selectin alone, but were unable to interact with immobilized ICAM-1. When ICAM-1 was co-immobilized with E-selectin, the number of cells that rolled was doubled, but no significant firm adhesion was observed. This phenomenon was specific for E-selectin, and no enhancement of rolling was observed when P-selectin was immobilized with ICAM-1. The increased neutrophil rolling seen on E-selectin and ICAM-1 substrates required beta2 integrins. Treating mice with antibodies to the beta2 integrins LFA-1 and Mac-1 showed that LFA-1 was primarily responsible for mediating rolling on ICAM-1 in this model. Increased rolling on E-selectin and ICAM-1 was significantly reduced following administration of a specific p38 mitogen-activated protein kinase (MAPK) inhibitor.
The data show that neutrophil rolling on E-selectin leads to partial activation of LFA-1, enabling LFA-1-dependent rolling on ICAM-1. This mechanism is likely to amplify and accelerate neutrophil recruitment in inflammation.
Microcirculation 04/2006; 13(2):99-109. · 2.57 Impact Factor
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ABSTRACT: In models of acute lung injury, CXC chemokine receptor 2 (CXCR2) mediates migration of polymorphonuclear leukocytes (PMNs) into the lung. Since CXCR2 ligands, including CXCL1 and CXCL2/3, are chemotactic for PMNs, CXCR2 is thought to recruit PMNs by inducing chemotactic migration. In a model of PMN recruitment to the lung, aerosolized bacterial LPS inhalation induced PMN recruitment to the lung in wild-type mice, but not in littermate CXCR2-/- mice. Surprisingly, lethally irradiated wild-type mice reconstituted with CXCR2-/- BM still showed about 50% PMN recruitment into bronchoalveolar lavage fluid and into lung interstitium, but CXCR2-/- mice reconstituted with CXCR2-/- BM showed no PMN recruitment. Conversely, CXCR2-/- mice reconstituted with wild-type BM showed a surprisingly large defect in PMN recruitment, inconsistent with a role of CXCR2 on PMNs alone. Cell culture, immunohistochemistry, flow cytometry, and real-time RT-PCR were used to show expression of CXCR2 on pulmonary endothelial and bronchial epithelial cells. The LPS-induced increase in lung microvascular permeability as measured by Evans blue extravasation required CXCR2 on nonhematopoietic cells. Our data revealed what we believe to be a previously unrecognized role of endothelial and epithelial CXCR2 in LPS-induced PMN recruitment and lung injury.
Journal of Clinical Investigation 04/2006; 116(3):695-702. · 15.39 Impact Factor
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ABSTRACT: Chemokines participate in various processes of monocyte recruitment including monocyte arrest and migration. Our group and others have demonstrated that growth-related oncogene (GRO)-alpha (CXCL1) can support monocyte arrest in models of inflammation. Here we employed a parallel plate-flow chamber and Transwell reconstitution assay to test whether GRO family chemokines were sufficient for Mono Mac 6 (a human monocytic cell line) and isolated human monocyte recruitment. Our study shows that 1) GRO-alpha, -beta (CXCL2), and -gamma (CXCL3) all act as arrest chemokines for monocyte adhesion on vascular cell adhesion molecule (VCAM)-1 under flow in the presence of P-selectin; 2) CXCR2 is the functional receptor for GRO-family chemokines in monocyte arrest; however, CXCR2 is not an arrest chemokine receptor in general, since epithelial neutrophil-activating peptide ENA-78 failed to arrest monocytes; 3) GRO-alpha, -beta, and -gamma all fail to increase intracellular free Ca2+ or mediate monocyte chemotaxis; and 4) signaling through G alpha(i) protein, phosphoinositide 3-kinase, and actin polymerization but not Ca2+ mobilization or the mitogen-activated kinases p38 and MAPK/extracellular signal-related kinase are necessary for GRO-alpha-mediated Mono Mac 6 cell arrest under flow. We conclude that the GRO-family chemokines are specialized monocyte-arrest chemokines. Their role in monocyte recruitment in inflammation can be inhibited by blocking CXCR2 function or downstream signaling events.
AJP Heart and Circulatory Physiology 12/2005; 289(5):H1976-84. · 3.71 Impact Factor
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Yuqing Huo,
Lei Zhao,
Matthew Craig Hyman,
Pavel Shashkin,
Brian L Harry,
Tracy Burcin,
S Bradley Forlow,
Matthew A Stark, David F Smith,
Sean Clarke,
Suseela Srinivasan,
Catherine C Hedrick,
Domenico Praticò,
Joseph L Witztum,
Jerry L Nadler,
Colin D Funk,
Klaus Ley
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ABSTRACT: Mice lacking leukocyte type 12/15-lipoxygenase (12/15-LO) show reduced atherosclerosis in several models. 12/15-LO is expressed in a variety of cells, including vascular cells, adipocytes, macrophages, and cardiomyocytes. The purpose of this study was to determine which cellular source of 12/15-LO is important for atherosclerosis.
Bone marrow from 12/15-LO-/-/apoE-/- mice was transplanted into apoE-/- mice and vice versa. Deficiency of 12/15-LO in bone marrow cells protected apoE-/- mice fed a Western diet from atherosclerosis to the same extent as complete absence of 12/15-LO, although plasma 8,12-iso-iPF2alpha-IV, a measure of lipid peroxidation, remained elevated. 12/15-LO-/-/apoE-/- mice regained the severity of atherosclerotic lesion typical of apoE-/- mice after replacement of their bone marrow cells with bone marrow from apoE-/- mice. Peritoneal macrophages obtained from wild-type but not 12/15-LO-/- mice caused endothelial activation in the presence of native LDL. Absence of 12/15-LO decreased the ability of macrophages to form foam cells when exposed to LDL.
We conclude that macrophage 12/15-LO plays a dominant role in the development of atherosclerosis by promoting endothelial inflammation and foam cell formation.
Circulation 11/2004; 110(14):2024-31. · 14.74 Impact Factor
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ABSTRACT: We studied whether circulating activated platelets and platelet-leukocyte aggregates cause the development of atherosclerotic lesions in apolipoprotein-E-deficient (Apoe(-/-)) mice. Circulating activated platelets bound to leukocytes, preferentially monocytes, to form platelet-monocyte/leukocyte aggregates. Activated platelets and platelet-leukocyte aggregates interacted with atherosclerotic lesions. The interactions of activated platelets with monocytes and atherosclerotic arteries led to delivery of the platelet-derived chemokines CCL5 (regulated on activation, normal T cell expressed and secreted, RANTES) and CXCL4 (platelet factor 4) to the monocyte surface and endothelium of atherosclerotic arteries. The presence of activated platelets promoted leukocyte binding of vascular cell adhesion molecule-1 (VCAM-1) and increased their adhesiveness to inflamed or atherosclerotic endothelium. Injection of activated wild-type, but not P-selectin-deficient, platelets increased monocyte arrest on the surface of atherosclerotic lesions and the size of atherosclerotic lesions in Apoe(-/-) mice. Our results indicate that circulating activated platelets and platelet-leukocyte/monocyte aggregates promote formation of atherosclerotic lesions. This role of activated platelets in atherosclerosis is attributed to platelet P-selectin-mediated delivery of platelet-derived proinflammatory factors to monocytes/leukocytes and the vessel wall.
Nature Medicine 02/2003; 9(1):61-7. · 22.46 Impact Factor
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Markus Sperandio,
David Frommhold,
Inna Babushkina,
Lesley G Ellies,
Timothy S Olson,
Michael L Smith,
Benedikt Fritzsching,
Eva Pauly, David F Smith,
Rainer Nobiling,
Otwin Linderkamp,
Jamey D Marth,
Klaus Ley
[show abstract]
[hide abstract]
ABSTRACT: L-selectin belongs to the C-type lectin family of glycoproteins and is constitutively expressed on most leukocytes. L-selectin mediates leukocyte rolling in inflamed microvessels and high endothelial venules (HEV) via binding to specific carbohydrate structures on selectin ligands. Previous studies using sialidase treatment suggested a role of sialic acid residues in L-selectin-dependent rolling. To investigate the role of the a2,3-sialyltransferase (ST3Gal)-IV on L-selectin ligand activity in vivo, we studied leukocyte rolling in inflamed venules of the cremaster muscle and in Peyer's patch HEV of ST3Gal-IV-deficient mice and littermate control mice. In cremaster muscle venules with or without TNF-a treatment, L-selectin-dependent rolling was almost completely abolished in ST3Gal-IV –/– mice. In both models, L-selectin interacts with P-selectin glycoprotein ligand-1 (PSGL-1) presented by adherent leukocytes and leukocyte fragments, but not with endothelial L-selectin ligands. In contrast, L-selectin-dependent rolling in Peyer's patch HEV, which is mediated by unknown endothelial L-selectin ligands, was not impaired in the absence of ST3Gal-IV. Our in vivo data show that PSGL-1, the molecule responsible for L-selectin-mediated leukocyte interactions in inflammation, is dependent on ST3Gal-IV, while a2,3-sialylation by ST3Gal-IV is not necessary for L-selectin ligand activity on high endothelial cells of Peyer's patch HEV.
