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

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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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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Indigenous enteric eosinophils control DCs to initiate a primary Th2 immune response in vivo

Journal of Experimental Medicine ◽

10.1084/jem.20131800 ◽

2014 ◽

Vol 211 (8) ◽

pp. 1657-1672 ◽

Cited By ~ 74

Author(s):

Derek K. Chu ◽

Rodrigo Jimenez-Saiz ◽

Christopher P. Verschoor ◽

Tina D. Walker ◽

Susanna Goncharova ◽

...

Keyword(s):

Lymph Nodes ◽

Eosinophil Peroxidase ◽

Th2 Immune Response ◽

Intestinal Immune System ◽

Eosinophil Activation ◽

And Migration ◽

Deficient Mice ◽

Draining Lymph Nodes

Eosinophils natively inhabit the small intestine, but a functional role for them there has remained elusive. Here, we show that eosinophil-deficient mice were protected from induction of Th2-mediated peanut food allergy and anaphylaxis, and Th2 priming was restored by reconstitution with il4+/+ or il4−/− eosinophils. Eosinophils controlled CD103+ dendritic cell (DC) activation and migration from the intestine to draining lymph nodes, events necessary for Th2 priming. Eosinophil activation in vitro and in vivo led to degranulation of eosinophil peroxidase, a granule protein whose enzymatic activity promoted DC activation in mice and humans in vitro, and intestinal and extraintestinal mouse DC activation and mobilization to lymph nodes in vivo. Further, eosinophil peroxidase enhanced responses to ovalbumin seen after immunization. Thus, eosinophils can be critical contributors to the intestinal immune system, and granule-mediated shaping of DC responses can promote both intestinal and extraintestinal adaptive immunity.

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BMP activity controlled by BMPER regulates the proinflammatory phenotype of endothelium

Blood ◽

10.1182/blood-2011-03-339762 ◽

2011 ◽

Vol 118 (18) ◽

pp. 5040-5049 ◽

Cited By ~ 47

Author(s):

Thomas Helbing ◽

René Rothweiler ◽

Elena Ketterer ◽

Lena Goetz ◽

Jennifer Heinke ◽

...

Keyword(s):

Vascular Endothelial Cells ◽

Ex Vivo ◽

Leukocyte Adhesion ◽

Vascular Inflammation ◽

Bmp Signaling ◽

In Vivo Models ◽

In Vivo Experiments ◽

And Migration

Abstract The endothelium plays a pivotal role in vascular inflammation. Here we study bone morphogenetic protein (BMP) signaling in endothelial inflammation and in particular the role of BMPER, an extracellular BMP modulator that is important in vascular development and angiogenesis. Using the BMP antagonist dorsomorphin or BMP2 as an agonist we show that BMP signaling is essential for the inflammatory response of vascular endothelial cells as demonstrated by intravital microscopy. We found that BMPER is decreased in inflammation similar to vascular protective genes like KLF2 and eNOS. Using in vitro and in vivo models we show that BMPER is down-regulated through the TNFα-NFκB-KLF2 signaling pathway. Functionally, lack of BMPER induced by siRNA or in BMPER+/− mice confers a proinflammatory endothelial phenotype with reduced eNOS levels and enhanced expression of adhesion molecules leading to increased leukocyte adhesion and extravasation in ex vivo and in vivo experiments. Vice versa, addition of BMPER exerts endothelium protective functions and antagonizes TNFα induced inflammation. Mechanistically, we demonstrate that these effects of BMPER are dependent on BMP signaling because of enhanced NFκB activity. In conclusion, the BMP modulator BMPER is a new protective regulator of vascular inflammation that modulates leukocyte adhesion and migration in vitro and in vivo.

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The Vav binding site of the non–receptor tyrosine kinase Syk at Tyr 348 is critical for β2 integrin (CD11/CD18)–mediated neutrophil migration

Blood ◽

10.1182/blood-2005-12-030387 ◽

2006 ◽

Vol 108 (12) ◽

pp. 3919-3927 ◽

Cited By ~ 55

Author(s):

Jurgen Schymeinsky ◽

Anca Sindrilaru ◽

David Frommhold ◽

Markus Sperandio ◽

Ronald Gerstl ◽

...

Keyword(s):

Bone Marrow ◽

Tyrosine Kinase ◽

Binding Site ◽

Leukocyte Adhesion ◽

Neutrophil Migration ◽

Nucleotide Exchange ◽

Edema Formation ◽

Β2 Integrin

Abstract Leukocyte adhesion via β2 integrins (CD11/CD18) activates the tyrosine kinase Syk. We found that Syk was enriched at the lamellipodium during N-formyl-Met-Leu-Phe–induced migration of neutrophil-like differentiated HL-60 cells. Here, Syk colocalized with Vav, a guanine nucleotide exchange factor for Rac and Cdc42. The enrichment of Syk at the lamellipodium and its colocalization with Vav were absent upon expression of a Syk kinase-dead mutant (Syk K402R) or a Syk mutant lacking the binding site of Vav (Syk Y348F). Live cell imaging revealed that both mutations resulted in excessive lamellipodium formation and severely compromised migration compared with control cells. Similar results were obtained upon down-regulation of Syk by RNA interference (RNAi) technique as well as in Syk–/– neutrophils from wild-type mice reconstituted with Syk–/– bone marrow. A pivotal role of Syk in vivo was demonstrated in the Arthus reaction, where neutrophil extravasation, edema formation, and hemorrhage were profoundly diminished in Syk–/– bone marrow chimeras compared with those in control animals. In the inflamed cremaster muscle, Syk–/– neutrophils revealed a defect in adhesion and migration. These findings indicate that Syk is critical for β2 integrin–mediated neutrophil migration in vitro and plays a fundamental role in neutrophil recruitment during the inflammatory response in vivo.

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A fundamental role of mAbp1 in neutrophils: impact on β2 integrin–mediated phagocytosis and adhesion in vivo

Blood ◽

10.1182/blood-2009-02-206169 ◽

2009 ◽

Vol 114 (19) ◽

pp. 4209-4220 ◽

Cited By ~ 32

Author(s):

Jürgen Schymeinsky ◽

Ronald Gerstl ◽

Ingrid Mannigel ◽

Katy Niedung ◽

David Frommhold ◽

...

Keyword(s):

Leukocyte Adhesion ◽

Green Fluorescence Protein ◽

Actin Binding ◽

Neutrophil Adhesion ◽

Flow Conditions ◽

Down Regulation ◽

Β2 Integrin ◽

Factor Α

Abstract The mammalian actin-binding protein 1 (mAbp1, Hip-55, SH3P7) is phosphorylated by the nonreceptor tyrosine kinase Syk that has a fundamental effect for several β2 integrin (CD11/CD18)–mediated neutrophil functions. Live cell imaging showed a dynamic enrichment of enhanced green fluorescence protein–tagged mAbp1 at the phagocytic cup of neutrophil-like differentiated HL-60 cells during β2 integrin–mediated phagocytosis of serum-opsonized Escherichia coli. The genetic absence of Syk or its pharmacologic inhibition using piceatannol abrogated the proper localization of mAbp1 at the phagocytic cup. The genetic absence or down-regulation of mAbp1 using the RNA interference technique significantly compromised β2 integrin–mediated phagocytosis of serum-opsonized E coli or Salmonella typhimurium in vitro as well as clearance of S typhimurium infection in vivo. Moreover, the genetic absence of mAbp1 almost completely abrogated firm neutrophil adhesion under physiologic shear stress conditions in vitro as well as leukocyte adhesion and extravasation in inflamed cremaster muscle venules of mice treated with tumor-necrosis factor α. Functional analysis showed that the down-regulation of mAbp1 diminished the number of β2 integrin clusters in the high-affinity conformation under flow conditions. These unanticipated results define mAbp1 as a novel molecular player in integrin biology that is critical for phagocytosis and firm neutrophil adhesion under flow conditions.

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Upregulation of Monocyte Urokinase Plasminogen Activator Receptor during Human Endotoxemia

Infection and Immunity ◽

10.1128/iai.68.4.2156-2160.2000 ◽

2000 ◽

Vol 68 (4) ◽

pp. 2156-2160 ◽

Cited By ~ 48

Author(s):

Pascale E. P. Dekkers ◽

Tessa ten Hove ◽

Anje A. te Velde ◽

Sander J. H. van Deventer ◽

Tom van der Poll

Keyword(s):

Plasminogen Activator ◽

Leukocyte Adhesion ◽

Urokinase Plasminogen Activator Receptor ◽

Necrosis Factor Alpha ◽

Human Endotoxemia ◽

Type Plasminogen Activator ◽

Upar Expression ◽

And Migration

ABSTRACT The receptor for urokinase-type plasminogen activator (uPAR) (CD87) plays an important role in leukocyte adhesion and migration. To assess the effect of endotoxin on cellular uPAR, uPAR expression was determined on leukocytes by fluorescence-activated cell sorter analysis in seven healthy subjects following intravenous injection of endotoxin (lot G; 4 ng/kg). Endotoxin induced a transient increase in uPAR expression on monocytes, reaching a 92% ± 46% increase over baseline expression after 6 h (P < 0.05). Endotoxin did not influence uPAR expression on granulocytes, while uPAR remained undetectable on lymphocytes. Endotoxin also increased soluble uPAR levels in plasma (P < 0.05). Stimulation of human whole blood with endotoxin or gram-positive stimuli in vitro also resulted in an upregulation of monocyte uPAR expression. Although tumor necrosis factor alpha (TNF) upregulated monocyte uPAR expression, anti-TNF did not influence the endotoxin-induced increase in monocyte uPAR expression. These data suggest that infectious stimuli may influence monocyte function in vivo by enhancing the expression of uPAR.

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Regulation of LFA-1–dependent inflammatory cell recruitment by Cbl-b and 14-3-3 proteins

Blood ◽

10.1182/blood-2007-07-103077 ◽

2008 ◽

Vol 111 (7) ◽

pp. 3607-3614 ◽

Cited By ~ 40

Author(s):

Eun Young Choi ◽

Valeria V. Orlova ◽

Susanna C. Fagerholm ◽

Susanna M. Nurmi ◽

Li Zhang ◽

...

Keyword(s):

Bone Marrow ◽

Inflammatory Cell ◽

Mononuclear Phagocytes ◽

Cell Recruitment ◽

Β2 Integrin ◽

Cytoplasmic Proteins ◽

Inflammatory Cell Recruitment ◽

And Migration

Abstract Inside-out signaling regulation of the β2-integrin leukocyte function–associated antigen-1 (LFA-1) by different cytoplasmic proteins, including 14-3-3 proteins, is essential for adhesion and migration of immune cells. Here, we identify a new pathway for the regulation of LFA-1 activity by Cbl-b, an adapter molecule and ubiquitin ligase that modulates several signaling pathways. Cbl-b−/− mice displayed increased macrophage recruitment in thioglycollate-induced peritonitis, which was attributed to Cbl-b deficiency in macrophages, as assessed by bone marrow chimera experiments. In vitro, Cbl-b−/− bone marrow–derived mononuclear phagocytes (BMDMs) displayed increased adhesion to endothelial cells. Activation of LFA-1 in Cbl-b–deficient cells was responsible for their increased endothelial adhesion in vitro and peritoneal recruitment in vivo, as the phenotype of Cbl-b deficiency was reversed in Cbl-b−/−LFA-1−/− mice. Consistently, LFA-1–mediated adhesion of BMDM to ICAM-1 but not VLA-4–mediated adhesion to VCAM-1 was enhanced by Cbl-b deficiency. Cbl-b deficiency resulted in increased phosphorylation of T758 in the β2-chain of LFA-1 and thereby in enhanced association of 14-3-3β protein with the β2-chain, leading to activation of LFA-1. Consistently, disruption of the 14-3-3/β2-integrin interaction abrogated the enhanced ICAM-1 adhesion of Cbl-b−/− BMDMs. In conclusion, Cbl-b deficiency activates LFA-1 and LFA-1–mediated inflammatory cell recruitment by stimulating the interaction between the LFA-1 β-chain and 14-3-3 proteins.

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

Journal of Experimental Medicine ◽

10.1084/jem.20070846 ◽

2008 ◽

Vol 205 (6) ◽

pp. 1435-1446 ◽

Cited By ~ 52

Author(s):

David Frommhold ◽

Andreas Ludwig ◽

M. Gabriele Bixel ◽

Alexander Zarbock ◽

Inna Babushkina ◽

...

Keyword(s):

Leukocyte Adhesion ◽

Hek293 Cells ◽

In Vitro Assays ◽

Human Neutrophils ◽

Neutrophil Degranulation ◽

Maackia Amurensis Lectin ◽

Deficient Mice ◽

Neutrophil Surface

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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Endogenous developmental endothelial locus-1 limits ischaemia- related angiogenesis by blocking inflammation

Thrombosis and Haemostasis ◽

10.1160/th16-05-0354 ◽

2017 ◽

Vol 117 (06) ◽

pp. 1150-1163 ◽

Cited By ~ 10

Author(s):

Anne Klotzsche - von Ameln ◽

Sebastian Cremer ◽

Jedrzej Hoffmann ◽

Peggy Schuster ◽

Sherif Khedr ◽

...

Keyword(s):

Hind Limb ◽

Immune Cell ◽

Ex Vivo ◽

Aortic Ring ◽

Limb Ischaemia ◽

Β2 Integrin ◽

Haematopoietic Cells ◽

Deficient Mice

SummaryWe have recently identified endothelial cell-secreted developmental endothelial locus-1 (Del-1) as an endogenous inhibitor of β2-integrin–dependent leukocyte infiltration. Del-1 was previously also implicated in angiogenesis. Here, we addressed the role of endogenously produced Del-1 in ischaemia-related angiogenesis. Intriguingly, Del-1–deficient mice displayed increased neovascularisation in two independent ischaemic models (retinopathy of prematurity and hind-limb ischaemia), as compared to Del-1–proficient mice. On the contrary, angiogenic sprouting in vitro or ex vivo (aortic ring assay) and physiological developmental retina angiogenesis were not affected by Del-1 deficiency. Mechanistically, the enhanced ischaemic neovascularisation in Del-1-deficiency was linked to higher infiltration of the ischaemic tissue by CD45+ haematopoietic and immune cells. Moreover, Del-1-deficiency promoted β2-integrin–dependent adhesion of haematopoietic cells to endothelial cells in vitro, and the homing of hematopoietic progenitor cells and of immune cell populations to ischaemic muscles in vivo. Consistently, the increased hind limb ischaemia-related angiogenesis in Del-1 deficiency was completely reversed in mice lacking both Del-1 and the β2-integrin LFA-1. Additionally, enhanced retinopathy-associated neovascularisation in Del-1-deficient mice was reversed by LFA-1 blockade. Our data reveal a hitherto unrecognised function of endogenous Del-1 as a local inhibitor of ischaemia-induced angiogenesis by restraining LFA-1–dependent homing of pro-angiogenic haematopoietic cells to ischaemic tissues. Our findings are relevant for the optimisation of therapeutic approaches in the context of ischaemic diseases.Supplementary Material to this article is available online at www.thrombosis-online.com.

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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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