Sialyltransferase ST3Gal-IV controls CXCR2-mediated firm leukocyte arrest during inflammation

Recent in vitro studies have suggested a role for sialylation in chemokine receptor binding to its ligand (Bannert, N., S. Craig, M. Farzan, D. Sogah, N.V. Santo, H. Choe, and J. Sodroski. 2001. J. Exp. Med. 194:1661–1673). This prompted us to investigate chemokine-induced leukocyte adhesion in inflamed cremaster muscle venules of α2,3 sialyltransferase (ST3Gal-IV)-deficient mice. We found a marked reduction in leukocyte adhesion to inflamed microvessels upon injection of the CXCR2 ligands CXCL1 (keratinocyte-derived chemokine) or CXCL8 (interleukin 8). In addition, extravasation of ST3Gal-IV−/− neutrophils into thioglycollate-pretreated peritoneal cavities was significantly decreased. In vitro assays revealed that CXCL8 binding to isolated ST3Gal-IV−/− neutrophils was markedly impaired. Furthermore, CXCL1-mediated adhesion of ST3Gal-IV−/− leukocytes at physiological flow conditions, as well as transendothelial migration of ST3Gal-IV−/− leukocytes in response to CXCL1, was significantly reduced. In human neutrophils, enzymatic desialylation decreased binding of CXCR2 ligands to the neutrophil surface and diminished neutrophil degranulation in response to these chemokines. In addition, binding of α2,3-linked sialic acid–specific Maackia amurensis lectin II to purified CXCR2 from neuraminidase-treated CXCR2-transfected HEK293 cells was markedly impaired. Collectively, we provide substantial evidence that sialylation by ST3Gal-IV significantly contributes to CXCR2-mediated leukocyte adhesion during inflammation in vivo.

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Endothelial E- and P-selectin expression in iNOS- deficient mice exposed to polymicrobial sepsis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.2001.280.2.g291 ◽

2001 ◽

Vol 280 (2) ◽

pp. G291-G297 ◽

Cited By ~ 20

Author(s):

Cameron W. Lush ◽

Gediminas Cepinskas ◽

William J. Sibbald ◽

Peter R. Kvietys

Keyword(s):

Leukocyte Adhesion ◽

Cecal Ligation ◽

Leukocyte Rolling ◽

Wild Type ◽

Endothelial Adhesion Molecule ◽

Acute Peritonitis ◽

Leukocyte Accumulation ◽

Deficient Mice

In vitro, nitric oxide (NO) decreases leukocyte adhesion to endothelium by attenuating endothelial adhesion molecule expression. In vivo, lipopolysaccharide-induced leukocyte rolling and adhesion was greater in inducible NO synthase (iNOS)−/− mice than in wild-type mice. The objective of this study was to assess E- and P-selectin expression in the microvasculature of iNOS−/− and wild-type mice subjected to acute peritonitis by cecal ligation and perforation (CLP). E- and P-selectin expression were increased in various organs within the peritoneum of wild-type animals after CLP. This CLP-induced upregulation of E- and P-selectin was substantially reduced in iNOS−/− mice. Tissue myeloperoxidase (MPO) activity was increased to a greater extent in the gut of wild-type than in iNOS−/− mice subjected to CLP. In the lung, the reduced expression of E-selectin in iNOS−/− mice was not associated with a decrease in MPO. Our findings indicate that NO derived from iNOS plays an important role in sepsis-induced increase in selectin expression in the systemic and pulmonary circulation. However, in iNOS−/− mice, sepsis-induced leukocyte accumulation is affected in the gut but not in the lungs.

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Leukocyte antiadhesive actions of annexin 1: ALXR- and FPR-related anti-inflammatory mechanisms

Blood ◽

10.1182/blood-2002-11-3411 ◽

2003 ◽

Vol 101 (10) ◽

pp. 4140-4147 ◽

Cited By ~ 138

Author(s):

Felicity N. E. Gavins ◽

Simon Yona ◽

Ahmad M. Kamal ◽

Roderick J. Flower ◽

Mauro Perretti

Keyword(s):

Ischemia Reperfusion ◽

Human Neutrophils ◽

Lipoxin A4 ◽

Annexin 1 ◽

Cell Incubation ◽

High Concentrations ◽

Adherent Leukocytes ◽

Deficient Mice

Abstract Recent investigations conducted with human neutrophils have indicated an involvement for the receptor for formylated peptides, termed FPR, and its analog FPRL1 (or ALXR because it is the receptor for the endogenous ligand lipoxin A4) in the in vitro inhibitory actions of the glucocorticoid-regulated protein annexin 1 and its peptidomimetics. To translate these findings in in vivo settings, we have used an ischemia/reperfusion (I/R) procedure to promote leukocyte-endothelium interactions in the mouse mesenteric microcirculation. In naive mice, the annexin 1 mimetic peptide Ac2-26 (20 to 100 μg administered intravenously prior to reperfusion) abolished I/R-induced cell adhesion and emigration, but not cell rolling. In FPR-deficient mice, peptide Ac2-26 retained significant inhibitory actions (about 50% of the effects in naive mice), and these were blocked by an FPR antagonist, termed butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe, or Boc2. In vitro, neutrophils taken from these animals could be activated at high concentrations of formyl-Met-Leu-Phe (30 μM; fMLP), and this effect was blocked by cell incubation with peptide Ac2-26 (66 μM) or Boc2 (100 μM). FPR-deficient neutrophils expressed ALXR mRNA and protein. Both ALXR agonists, lipoxin A4 and peptide Ac2-26, provoked detachment of adherent leukocytes in naive as well as in FPR-deficient mice, whereas the CXC chemokine KC or fMLP were inactive. The present findings demonstrate that endogenous regulatory autocoids such as lipoxin A4 and annexin 1–derived peptides function to disengage adherent cells during cell-cell interactions.

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Urokinase Receptor (CD87) Regulates Leukocyte Recruitment via β2 Integrins In Vivo

Journal of Experimental Medicine ◽

10.1084/jem.188.6.1029 ◽

1998 ◽

Vol 188 (6) ◽

pp. 1029-1037 ◽

Cited By ~ 179

Author(s):

Andreas E. May ◽

Sandip M. Kanse ◽

Leif R. Lund ◽

Roland H. Gisler ◽

Beat A. Imhof ◽

...

Keyword(s):

Leukocyte Adhesion ◽

Close Association ◽

Phosphatidyl Inositol ◽

Urokinase Receptor ◽

Β2 Integrin ◽

And Migration ◽

Deficient Mice ◽

Β2 Integrins

The urokinase receptor (CD87; uPAR) is found in close association with β2 integrins on leukocytes. We studied the functional consequence of this association for leukocyte adhesion and migration. In vivo, the β2 integrin–dependent recruitment of leukocytes to the inflamed peritoneum of uPAR-deficient mice was significantly reduced as compared with wild-type animals. In vitro, β2 integrin–mediated adhesion of leukocytes to endothelium was lost upon removal of uPAR from the leukocyte surface by phosphatidyl-inositol–specific phospholipase C. Leukocyte adhesion was reconstituted when soluble intact uPAR, but not a truncated form lacking the uPA-binding domain, was allowed to reassociate with the cell surface. uPAR ligation with a monoclonal antibody induced adhesion of monocytic cells and neutrophils to vascular endothelium by six- to eightfold, whereas ligation with inactivated uPA significantly reduced cell-to-cell adhesion irrespective of the β2 integrin–stimulating pathway. These data indicate that β2 integrin–mediated leukocyte–endothelial cell interactions and recruitment to inflamed areas require the presence of uPAR and define a new phenotype for uPAR-deficient mice. Moreover, uPAR ligation differentially modulates leukocyte adhesion to endothelium and provides novel targets for therapeutic strategies in inflammation-related vascular pathologies.

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Neutrophil α-defensins promote thrombosis in vivo by altering fibrin formation, structure, and stability

Blood ◽

10.1182/blood-2018-07-861237 ◽

2019 ◽

Vol 133 (5) ◽

pp. 481-493 ◽

Cited By ~ 10

Author(s):

Rami Abu-Fanne ◽

Victoria Stepanova ◽

Rustem I. Litvinov ◽

Suhair Abdeen ◽

Khalil Bdeir ◽

...

Keyword(s):

Inferior Vena ◽

Vena Cava ◽

Human Neutrophils ◽

Wild Type ◽

Intrinsic Pathway ◽

Antimicrobial Proteins ◽

Fiber Density ◽

Neutrophil Degranulation

Abstract Inflammation and thrombosis are integrated, mutually reinforcing processes, but the interregulatory mechanisms are incompletely defined. Here, we examined the contribution of α-defensins (α-defs), antimicrobial proteins released from activated human neutrophils, on clot formation in vitro and in vivo. Activation of the intrinsic pathway of coagulation stimulates release of α-defs from neutrophils. α-Defs accelerate fibrin polymerization, increase fiber density and branching, incorporate into nascent fibrin clots, and impede fibrinolysis in vitro. Transgenic mice (Def++) expressing human α-Def-1 developed larger, occlusive, neutrophil-rich clots after partial inferior vena cava (IVC) ligation than those that formed in wild-type (WT) mice. IVC thrombi extracted from Def++ mice were composed of a fibrin meshwork that was denser and contained a higher proportion of tightly packed compressed polyhedral erythrocytes than those that developed in WT mice. Def++ mice were resistant to thromboprophylaxis with heparin. Inhibiting activation of the intrinsic pathway of coagulation, bone marrow transplantation from WT mice or provision of colchicine to Def++ mice to inhibit neutrophil degranulation decreased plasma levels of α-defs, caused a phenotypic reversion characterized by smaller thrombi comparable to those formed in WT mice, and restored responsiveness to heparin. These data identify α-defs as a potentially important and tractable link between innate immunity and thrombosis.

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Involvement of SNAP-23 and syntaxin 6 in human neutrophil exocytosis

Blood ◽

10.1182/blood.v96.7.2574 ◽

2000 ◽

Vol 96 (7) ◽

pp. 2574-2583 ◽

Cited By ~ 91

Author(s):

Belén Martı́n-Martı́n ◽

Svetlana M. Nabokina ◽

Juan Blasi ◽

Pedro A. Lazo ◽

Faustino Mollinedo

Keyword(s):

Human Neutrophil ◽

Coiled Coil ◽

Neutrophil Activation ◽

Human Neutrophils ◽

Binding Studies ◽

Specific Granules ◽

Neutrophil Degranulation ◽

Carboxy Terminal

Abstract To understand the molecular basis of exocytosis in human neutrophils, the role of syntaxin 6 and SNAP-23 in neutrophil degranulation was examined. Human syntaxin 6 was cloned and identified as a 255-amino acid protein with a carboxy-terminal transmembrane region and two coiled-coil domains. Syntaxin 6 was localized mainly in the plasma membrane of human resting neutrophils, whereas SNAP-23 was located primarily in the mobilizable tertiary and specific granules. SNAP-23 was translocated to the cell surface, colocalizing with syntaxin 6, on neutrophil activation. In vitro binding studies established that SNAP-23 binds to syntaxin 6. Coimmunoprecipitation assays indicated that SNAP-23 interacts with syntaxin 6 in vivo, and this interaction was dramatically increased on neutrophil activation. Antibodies against SNAP-23 inhibited Ca++ and GTP-γ-S–induced exocytosis of CD67-enriched specific granules, but they hardly affected exocytosis of the CD63-enriched azurophilic granules, when introduced into electropermeabilized neutrophils. Anti–syntaxin 6 antibodies prevented exocytosis of both CD67- and CD63-enriched granules in electropermeabilized neutrophils. These data show that syntaxin 6 and SNAP-23 are involved in human neutrophil exocytosis, demonstrating that vesicle SNAP receptor-target SNAP receptor (v-SNARE– t-SNARE) interactions modulate neutrophil secretion. Syntaxin 6 acts as a target for secretion of specific and azurophilic granules, whereas SNAP-23 mediates specific granule secretion.

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Rescue of cleft palate inMsx1-deficient mice by transgenicBmp4reveals a network of BMP and Shh signaling in the regulation of mammalian palatogenesis

Development ◽

10.1242/dev.129.17.4135 ◽

2002 ◽

Vol 129 (17) ◽

pp. 4135-4146 ◽

Cited By ~ 18

Author(s):

Zunyi Zhang ◽

Yiqiang Song ◽

Xiang Zhao ◽

Xiaoyun Zhang ◽

Cesar Fermin ◽

...

Keyword(s):

Cell Proliferation ◽

Cleft Palate ◽

Genetic Regulation ◽

Homeobox Gene ◽

In Vitro Assays ◽

Transgenic Expression ◽

Environmental Perturbations ◽

Deficient Mice

Cleft palate, the most frequent congenital craniofacial birth defects in humans, arises from genetic or environmental perturbations in the multi-step process of palate development. Mutations in the MSX1 homeobox gene are associated with non-syndromic cleft palate and tooth agenesis in humans. We have used Msx1-deficient mice as a model system that exhibits severe craniofacial abnormalities, including cleft secondary palate and lack of teeth, to study the genetic regulation of mammalian palatogenesis. We found that Msx1 expression was restricted to the anterior of the first upper molar site in the palatal mesenchyme and that Msx1 was required for the expression of Bmp4 and Bmp2 in the mesenchyme and Shh in the medial edge epithelium (MEE) in the same region of developing palate. In vivo and in vitro analyses indicated that the cleft palate seen in Msx1 mutants resulted from a defect in cell proliferation in the anterior palatal mesenchyme rather than a failure in palatal fusion. Transgenic expression of human Bmp4 driven by the mouse Msx1 promoter in the Msx1–/– palatal mesenchyme rescued the cleft palate phenotype and neonatal lethality. Associated with the rescue of the cleft palate was a restoration of Shh and Bmp2 expression, as well as a return of cell proliferation to the normal levels. Ectopic Bmp4 appears to bypass the requirement for Msx1 and functions upstream of Shh and Bmp2 to support palatal development. Further in vitro assays indicated that Shh (normally expressed in the MEE) activates Bmp2 expression in the palatal mesenchyme which in turn acts as a mitogen to stimulate cell division. Msx1 thus controls a genetic hierarchy involving BMP and Shh signals that regulates the growth of the anterior region of palate during mammalian palatogenesis. Our findings provide insights into the cellular and molecular etiology of the non-syndromic clefting associated with Msx1 mutations.

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Involvement of SNAP-23 and syntaxin 6 in human neutrophil exocytosis

Blood ◽

10.1182/blood.v96.7.2574.h8002574_2574_2583 ◽

2000 ◽

Vol 96 (7) ◽

pp. 2574-2583 ◽

Cited By ~ 1

Author(s):

Belén Martı́n-Martı́n ◽

Svetlana M. Nabokina ◽

Juan Blasi ◽

Pedro A. Lazo ◽

Faustino Mollinedo

Keyword(s):

Human Neutrophil ◽

Coiled Coil ◽

Neutrophil Activation ◽

Human Neutrophils ◽

Binding Studies ◽

Specific Granules ◽

Neutrophil Degranulation ◽

Carboxy Terminal

To understand the molecular basis of exocytosis in human neutrophils, the role of syntaxin 6 and SNAP-23 in neutrophil degranulation was examined. Human syntaxin 6 was cloned and identified as a 255-amino acid protein with a carboxy-terminal transmembrane region and two coiled-coil domains. Syntaxin 6 was localized mainly in the plasma membrane of human resting neutrophils, whereas SNAP-23 was located primarily in the mobilizable tertiary and specific granules. SNAP-23 was translocated to the cell surface, colocalizing with syntaxin 6, on neutrophil activation. In vitro binding studies established that SNAP-23 binds to syntaxin 6. Coimmunoprecipitation assays indicated that SNAP-23 interacts with syntaxin 6 in vivo, and this interaction was dramatically increased on neutrophil activation. Antibodies against SNAP-23 inhibited Ca++ and GTP-γ-S–induced exocytosis of CD67-enriched specific granules, but they hardly affected exocytosis of the CD63-enriched azurophilic granules, when introduced into electropermeabilized neutrophils. Anti–syntaxin 6 antibodies prevented exocytosis of both CD67- and CD63-enriched granules in electropermeabilized neutrophils. These data show that syntaxin 6 and SNAP-23 are involved in human neutrophil exocytosis, demonstrating that vesicle SNAP receptor-target SNAP receptor (v-SNARE– t-SNARE) interactions modulate neutrophil secretion. Syntaxin 6 acts as a target for secretion of specific and azurophilic granules, whereas SNAP-23 mediates specific granule secretion.

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ICAM-2 mediates neutrophil transmigration in vivo: evidence for stimulus specificity and a role in PECAM-1–independent transmigration

Blood ◽

10.1182/blood-2005-11-4683 ◽

2006 ◽

Vol 107 (12) ◽

pp. 4721-4727 ◽

Cited By ~ 77

Author(s):

Miao-Tzu Huang ◽

Karen Y. Larbi ◽

Christoph Scheiermann ◽

Abigail Woodfin ◽

Nicole Gerwin ◽

...

Keyword(s):

Direct Evidence ◽

Leukocyte Adhesion ◽

Neutrophil Migration ◽

Selective Reduction ◽

Leukocyte Transmigration ◽

Tnf Α ◽

Peritonitis Model ◽

Deficient Mice

AbstractICAM-2 has been implicated in leukocyte transmigration in vitro, but there is little in vivo evidence to support this. To address this, neutrophil migration was investigated in ICAM-2–deficient mice (KO) and in wild-type (WT) mice treated with an anti–ICAM-2 blocking monoclonal antibody (mAb) (3C4). In a peritonitis model, IL-1β–induced accumulation of neutrophils was significantly reduced in mice treated with 3C4 (51% inhibition) and in KO mice (41% inhibition). In contrast, TNF-α– or thioglycolate-induced responses were not suppressed in KO mice. Analysis of IL-1β–induced leukocyte responses in cremasteric venules of KO animals by intravital microscopy indicated a defect in transmigration (44% inhibition) but not rolling or adhesion. As found before, TNF-α–induced leukocyte transmigration was unaltered in the KO mice. WT mice treated with the anti–ICAM-2 mAb also exhibited a selective reduction in leukocyte transmigration in response to IL-1β while an anti–ICAM-1 mAb inhibited both leukocyte adhesion and transmigration. Interestingly, mAb 3C4 significantly suppressed IL-1β–induced neutrophil transmigration in PE-CAM-1 KO animals in the peritonitis model but not in the cremaster muscle. The findings provide direct evidence for the involvement of ICAM-2 in neutrophil transmigration in vivo, though this role appears to be stimulus specific. Furthermore, ICAM-2 appears capable of mediating PECAM-1–independent leukocyte transmigration.

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A in Vivo Assay for Standardizing Heparin Preparations

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646810 ◽

1979 ◽

Vol 41 (03) ◽

pp. 576-582

Author(s):

A R Pomeroy

Keyword(s):

In Vitro Assays ◽

British Pharmacopoeia ◽

In Vitro Method ◽

Standard Preparation ◽

Reliable Estimation ◽

Assay Procedure ◽

Accuracy And Precision ◽

Heparin Preparation

SummaryThe limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use.The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a “2 and 2 dose assay” procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation.The similarity of results obtained the in vivo method and the in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.

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Potentially Thrombogenic Materials in Factor IX Concentrates

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647857 ◽

1975 ◽

Vol 33 (03) ◽

pp. 617-631 ◽

Cited By ~ 47

Author(s):

H. S Kingdon ◽

R. L Lundblad ◽

J. J Veltkamp ◽

D. L Aronson

Keyword(s):

Human Plasma ◽

Antithrombin Iii ◽

Factor Ix ◽

In Vitro Assays ◽

Substantial Agreement ◽

Coefficient Of Correlation

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.

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