Molecular Mechanisms of Zinc-Dependent Leukocyte Adhesion Involving the Urokinase Receptor and β2-Integrins

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 2976-2983 ◽  
Author(s):  
Triantafyllos Chavakis ◽  
Andreas E. May ◽  
Klaus T. Preissner ◽  
Sandip M. Kanse
Keyword(s):  
Cell Adhesion ◽  
Molecular Mechanisms ◽  
Leukocyte Adhesion ◽  
Urokinase Receptor ◽  
Cation Binding ◽  
Integrin Activation ◽  
Plasminogen Activator Inhibitor 1 ◽  
Cation Binding Site ◽  
Myelomonocytic Cells ◽  
Β2 Integrins

The trace element Zinc (Zn2+) has been implicated as a mediator in host defense, yet the molecular basis for its extracellular functions remains obscure. Here, we demonstrate that Zn2+can induce the adhesion of myelomonocytic cells to the endothelium, as well as to the provisional matrix proteins vitronectin (VN) and fibrinogen (FBG), which are pivotal steps for the recruitment of leukocytes into inflamed/injured tissue. Physiologic concentrations of Zn2+ increased the urokinase receptor (uPAR)-mediated adhesion of myelomonocytic cells to VN, whereas other divalent cations had smaller effects. Zn2+-induced cell adhesion to VN was abolished by cation chelators such as 1-10-phenanthroline, as well as by plasminogen activator inhibitor-1 (PAI-1) and a monoclonal antibody (MoAb) against uPAR. These characteristics could be recapitulated with a uPAR-transfected cell line emphasizing the specificity of this receptor system for Zn2+-dependent cell adhesion. Like urokinase (uPA), Zn2+ increased the binding of radiolabeled VN to uPAR-expressing cells, as well as the interaction of VN with immobilized uPAR in an isolated system. Moreover, Zn2+ enhanced leukocytic cell adhesion to FBG and endothelial cell monolayers by activating β2-integrins. Instead of the direct β2-integrin activation through the divalent cation binding site, Zn2+-induced integrin activation was mediated via uPAR, a crucial regulator of this system. The present study uncovers for the first time Zn2+-mediated cell adhesion mechanisms that may play a crucial role in modulating leukocyte adhesion to vessel wall components.

Molecular Mechanisms of Zinc-Dependent Leukocyte Adhesion Involving the Urokinase Receptor and β2-Integrins

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 2976-2983 ◽  
Author(s):  
Triantafyllos Chavakis ◽  
Andreas E. May ◽  
Klaus T. Preissner ◽  
Sandip M. Kanse
Keyword(s):  
Cell Adhesion ◽  
Molecular Mechanisms ◽  
Leukocyte Adhesion ◽  
Urokinase Receptor ◽  
Cation Binding ◽  
Integrin Activation ◽  
Plasminogen Activator Inhibitor 1 ◽  
Cation Binding Site ◽  
Myelomonocytic Cells ◽  
Β2 Integrins

Abstract The trace element Zinc (Zn2+) has been implicated as a mediator in host defense, yet the molecular basis for its extracellular functions remains obscure. Here, we demonstrate that Zn2+can induce the adhesion of myelomonocytic cells to the endothelium, as well as to the provisional matrix proteins vitronectin (VN) and fibrinogen (FBG), which are pivotal steps for the recruitment of leukocytes into inflamed/injured tissue. Physiologic concentrations of Zn2+ increased the urokinase receptor (uPAR)-mediated adhesion of myelomonocytic cells to VN, whereas other divalent cations had smaller effects. Zn2+-induced cell adhesion to VN was abolished by cation chelators such as 1-10-phenanthroline, as well as by plasminogen activator inhibitor-1 (PAI-1) and a monoclonal antibody (MoAb) against uPAR. These characteristics could be recapitulated with a uPAR-transfected cell line emphasizing the specificity of this receptor system for Zn2+-dependent cell adhesion. Like urokinase (uPA), Zn2+ increased the binding of radiolabeled VN to uPAR-expressing cells, as well as the interaction of VN with immobilized uPAR in an isolated system. Moreover, Zn2+ enhanced leukocytic cell adhesion to FBG and endothelial cell monolayers by activating β2-integrins. Instead of the direct β2-integrin activation through the divalent cation binding site, Zn2+-induced integrin activation was mediated via uPAR, a crucial regulator of this system. The present study uncovers for the first time Zn2+-mediated cell adhesion mechanisms that may play a crucial role in modulating leukocyte adhesion to vessel wall components.

VLA-4 (α4β1) engagement defines a novel activation pathway for β2 integrin–dependent leukocyte adhesion involving the urokinase receptor

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 506-513 ◽  
Author(s):  
Andreas E. May ◽  
Franz-Josef Neumann ◽  
Albert Schömig ◽  
Klaus T. Preissner
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Urokinase Receptor ◽  
Β1 Integrin ◽  
Integrin Activation ◽  
Β2 Integrin ◽  
Β1 Integrins ◽  
Dependent Cell ◽  
Cell To Cell Adhesion ◽  
Β2 Integrins

Abstract During acute inflammatory processes, β2 and β1 integrins sequentially mediate leukocyte recruitment into extravascular tissues. We studied the influence of VLA-4 (very late antigen-4) (4β1) engagement on β2 integrin activation-dependent cell-to-cell adhesion. Ligation of VLA-4 by the soluble chimera fusion product vascular cell adhesion molecule-1 (VCAM-1)–Fc or by 2 anti-CD29 (β1 chain) monoclonal antibodies (mAb) rapidly induced adhesion of myelomonocytic cells (HL60, U937) to human umbilical vein endothelial cells (HUVECs). Cell adhesion was mediated via β2 integrin (LFA-1 and Mac-1) activation: induced adhesion to HUVECs was inhibited by blocking mAbs anti-CD18 (70%-90%), anti-CD11a (50%-60%), or anti-CD11b (60%-70%). Adhesion to immobilized ligands of β2 integrins (intercellular adhesion molecule-1 [ICAM-1], fibrinogen, keyhole limpet hemocyanin) as well as to ICAM-1–transfected Chinese hamster ovary cells, but not to ligands of β1 integrins (VCAM-1, fibronectin, laminin, and collagen), was augmented. VCAM-1–Fc binding provoked the expression of the activation-dependent epitope CBRM1/5 of Mac-1 on leukocytes. Clustering of VLA-4 through dimeric VCAM-1–Fc was required for β2 integrin activation and induction of cell adhesion, whereas monovalent VCAM-1 or Fab fragments of anti-β1 integrin mAb were ineffective. Activation of β2 integrins by 4β1 integrin ligation (VCAM-1–Fc or anti-β1 mAb) required the presence of urokinase receptor (uPAR) on leukocytic cells, because the removal of uPAR from the cell surface by phosphatidylinositol-specific phospholipase C reduced cell adhesion to less than 40%. Adhesion was reconstituted when soluble recombinant uPAR was allowed to reassociate with the cells. Finally, VLA-4 engagement by VCAM-1–Fc or anti-β1 integrin mAb induced uPAR-dependent adhesion to immobilized vitronectin as well. These results elucidate a novel activation pathway of β2 integrin–dependent cell-to-cell adhesion that requires 4β1 integrin ligation for initiation and uPAR as activation transducer.

VLA-4 (α4β1) engagement defines a novel activation pathway for β2 integrin–dependent leukocyte adhesion involving the urokinase receptor

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 506-513 ◽  
Author(s):  
Andreas E. May ◽  
Franz-Josef Neumann ◽  
Albert Schömig ◽  
Klaus T. Preissner
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Urokinase Receptor ◽  
Β1 Integrin ◽  
Integrin Activation ◽  
Β2 Integrin ◽  
Β1 Integrins ◽  
Dependent Cell ◽  
Cell To Cell Adhesion ◽  
Β2 Integrins

During acute inflammatory processes, β2 and β1 integrins sequentially mediate leukocyte recruitment into extravascular tissues. We studied the influence of VLA-4 (very late antigen-4) (4β1) engagement on β2 integrin activation-dependent cell-to-cell adhesion. Ligation of VLA-4 by the soluble chimera fusion product vascular cell adhesion molecule-1 (VCAM-1)–Fc or by 2 anti-CD29 (β1 chain) monoclonal antibodies (mAb) rapidly induced adhesion of myelomonocytic cells (HL60, U937) to human umbilical vein endothelial cells (HUVECs). Cell adhesion was mediated via β2 integrin (LFA-1 and Mac-1) activation: induced adhesion to HUVECs was inhibited by blocking mAbs anti-CD18 (70%-90%), anti-CD11a (50%-60%), or anti-CD11b (60%-70%). Adhesion to immobilized ligands of β2 integrins (intercellular adhesion molecule-1 [ICAM-1], fibrinogen, keyhole limpet hemocyanin) as well as to ICAM-1–transfected Chinese hamster ovary cells, but not to ligands of β1 integrins (VCAM-1, fibronectin, laminin, and collagen), was augmented. VCAM-1–Fc binding provoked the expression of the activation-dependent epitope CBRM1/5 of Mac-1 on leukocytes. Clustering of VLA-4 through dimeric VCAM-1–Fc was required for β2 integrin activation and induction of cell adhesion, whereas monovalent VCAM-1 or Fab fragments of anti-β1 integrin mAb were ineffective. Activation of β2 integrins by 4β1 integrin ligation (VCAM-1–Fc or anti-β1 mAb) required the presence of urokinase receptor (uPAR) on leukocytic cells, because the removal of uPAR from the cell surface by phosphatidylinositol-specific phospholipase C reduced cell adhesion to less than 40%. Adhesion was reconstituted when soluble recombinant uPAR was allowed to reassociate with the cells. Finally, VLA-4 engagement by VCAM-1–Fc or anti-β1 integrin mAb induced uPAR-dependent adhesion to immobilized vitronectin as well. These results elucidate a novel activation pathway of β2 integrin–dependent cell-to-cell adhesion that requires 4β1 integrin ligation for initiation and uPAR as activation transducer.

scholarly journals Protein Kinase Cδ-Mediated Phosphorylation of Phospholipase D Controls Integrin-Mediated Cell Spreading

2010 ◽  
Vol 30 (21) ◽  
pp. 5086-5098 ◽  
Author(s):  
Young Chan Chae ◽  
Kyung Lock Kim ◽  
Sang Hoon Ha ◽  
Jaeyoon Kim ◽  
Pann-Ghill Suh ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Molecular Mechanisms ◽  
Cell Spreading ◽  
Integrin Signaling ◽  
Integrin Activation ◽  
Actin Remodeling ◽  
And Migration ◽  
Protein Kinase Cδ

ABSTRACT Integrin signaling plays critical roles in cell adhesion, spreading, and migration, and it is generally accepted that to regulate these integrin functions accurately, localized actin remodeling is required. However, the molecular mechanisms that control the targeting of actin regulation molecules to the proper sites are unknown. We previously demonstrated that integrin-mediated cell spreading and migration on fibronectin are dependent on the localized activation of phospholipase D (PLD). However, the mechanism underlying PLD activation by integrin is largely unknown. Here we demonstrate that protein kinase Cδ (PKCδ) is required for integrin-mediated PLD signaling. After integrin stimulation, PKCδ is activated and translocated to the edges of lamellipodia, where it colocalizes with PLD2. The abrogation of PKCδ activity inhibited integrin-induced PLD activation and cell spreading. Finally, we show that Thr566 of PLD2 is directly phosphorylated by PKCδ and that PLD2 mutation in this region prevents PLD2 activation, PLD2 translocation to the edge of lamellipodia, Rac translocation, and cell spreading after integrin activation. Together, these results suggest that PKCδ is a primary regulator of integrin-mediated PLD activation via the direct phosphorylation of PLD, which is essential for directing integrin-induced cell spreading.

scholarly journals LDL-receptor–related protein regulates β2-integrin–mediated leukocyte adhesion

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 170-177 ◽  
Author(s):  
Patricia P. E. M. Spijkers ◽  
Paula da Costa Martins ◽  
Erik Westein ◽  
Carl G. Gahmberg ◽  
Jaap J. Zwaginga ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Leukocyte Adhesion ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
U937 Cells ◽  
Related Protein ◽  
Β2 Integrin ◽  
Adhesive Surface ◽  
Β2 Integrins

Abstract β2-Integrin clustering on activation is a key event in leukocyte adhesion to the endothelium during the inflammatory response. In the search for molecular mechanisms leading to this clustering, we have identified low-density lipoprotein (LDL) receptor–related protein (LRP) as a new partner for β2-integrins at the leukocyte surface. Immobilized recombinant LRP fragments served as an adhesive surface for blood-derived leukocytes and the U937 cell line. This adhesion was decreased up to 95% in the presence of antibodies against β2-integrins, pointing to these integrins as potential partners for LRP. Using purified proteins, LRP indeed associated with the αMβ2 complex and the αM and αL I-domains (Kd, app ≈ 0.5 μM). Immunoprecipitation experiments and confocal microscopy revealed that endogenously expressed LRP and αLβ2 colocalized in monocytes and U937 cells. Furthermore, activation of U937 cells resulted in clustering of αLβ2 and LRP to similar regions at the cell surface, indicating potential cooperation between both proteins. This was confirmed by the lack of αLβ2 clustering in U937 cells treated by antisense oligonucleotides to down-regulate LRP. In addition, the absence of LRP resulted in complete abrogation of β2-integrin–dependent adhesion to endothelial cells in a perfusion system, demonstrating the presence of a previously unrecognized link between LRP and leukocyte function.

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

1998 ◽  
Vol 188 (6) ◽  
pp. 1029-1037 ◽  
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.

Leukocyte Integrin Activation and Alterations in Parathyroid Hormone Levels Following G-CSF Administration

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2164-2164
Author(s):  
Robert E. Donahue ◽  
Laura Tuschong ◽  
Thomas R. Bauer ◽  
Dennis D. Hickstein ◽  
Yu Ying Yau ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Neutrophil Count ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Cation Binding ◽  
Integrin Activation ◽  
Healthy Human ◽  
Hematopoietic Stem ◽  
Α Subunit ◽  
Cation Binding Site

Abstract Abstract 2164 Following administration of granulocyte colony-stimulating factor (G-CSF) there is a marked but transient drop in circulating neutrophils. To determine the contribution of integrin activation to these events, a monoclonal antibody (mAb 24) that recognizes the activated α-subunit cation binding domain of the integrin family of receptors was used to evaluate human leukocytes following G-CSF administration. A single 480 mcg dose of human recombinant G-CSF (Neupogen, Amgen) was administered in the upper arm of six healthy human subjects (3 male, 3 female). EDTA and heparinized blood samples were collected prior to and at 15, 30, 60, 90, 120 and 240 minutes after injection. Mean absolute neutrophil count decreased from 2,557 ± 654/μl at baseline to 227 ± 193 (p<0.001) at 30 and 45 minutes. Flow cytometric analysis revealed a dramatic increase in binding of mAb24, which recognizes the occupied divalent cation binding site of the activated integrin α subunit, to neutrophils but not other circulating nucleated cells within 15 to 30 minutes after G-CSF administration, concurrent with a dramatic decrease in circulating neutrophil numbers. This activation, like the drop in neutrophil count, was transient, with a return to baseline status by 60 minutes. Intact parathyroid hormone (iPTH) levels were also evaluated. Within 30–90 minutes following G-CSF injection there was a significant drop in iPTH from a baseline of 52.5 ± 31.1 to a nadir value of 30.6 ± 20.9 pg/ml (p=0.01). A rebound in iPTH levels to 63.4 ± 27.0 pg/ml (p<0.001) occurred at 90 to 240 minutes. There were no significant changes in ionized calcium or magnesium levels. These results demonstrate that the integrin on circulating neutrophils is transiently activated following G-CSF administration and that this activation may mediate the transient neutropenia seen in this setting. In addition, alterations in PTH levels occur following G-CSF administration. As it is known that iPTH influences the hematopoietic stem cell (HSC) niche, fluctuations in iPTH levels following G-CSF administration may play a role in HSC expansion. Disclosures: No relevant conflicts of interest to declare.

A novel genetic leukocyte adhesion deficiency in subsecond triggering of integrin avidity by endothelial chemokines results in impaired leukocyte arrest on vascular endothelium under shear flow

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4437-4445 ◽  
Author(s):  
Ronen Alon ◽  
Memet Aker ◽  
Sara Feigelson ◽  
Maya Sokolovsky-Eisenberg ◽  
Donald E. Staunton ◽  
...  
Keyword(s):  
Shear Flow ◽  
Vascular Endothelium ◽  
Leukocyte Adhesion ◽  
Genetic Disorder ◽  
High Avidity ◽  
Bleeding Tendency ◽  
Integrin Activation ◽  
Multistep Process ◽  
Β2 Integrins

Abstract Leukocyte arrest on vascular endothelium under disruptive shear flow is a multistep process that requires in situ integrin activation on the leukocyte surface by endothelium-displayed chemoattractants, primarily chemokines. A genetic deficiency of leukocyte adhesion to endothelium associated with defective β2 integrin expression or function (LAD-1) has been described. We now report a novel severe genetic disorder in this multistep process associated with functional defects in multiple leukocyte integrins, reflected in recurrent infections, profound leukocytosis, and a bleeding tendency. This syndrome is associated with an impaired ability of neutrophil and lymphocyte β1 and β2 integrins to generate high avidity to their endothelial ligands and arrest cells on vascular endothelium in response to endothelial chemoattractant signals. Patient leukocytes roll normally on endothelial selectins, express intact integrins and G protein–coupled chemokine receptors (GPCR), spread on integrin ligands, and migrate normally along a chemotactic gradient. Activation of β2 integrins in response to GPCR signals and intrinsic soluble ligand binding properties of the very late activation antigen-4 (VLA-4) integrin are also retained in patient leukocytes. Nevertheless, all integrins fail to generate firm adhesion to immobilized ligands in response to in situ GPCR-mediated activation by chemokines or chemoattractants, a result of a primary defect in integrin rearrangement at ligand-bearing contacts. This syndrome is the first example of a human integrin-activation deficiency associated with defective GPCR stimulation of integrin avidity at subsecond contacts, a key step in leukocyte arrest on vascular endothelium under shear flow.

scholarly journals Leukocyte integrin activation mediates transient neutropenia after G-CSF administration

Blood ◽  
2011 ◽  
Vol 118 (15) ◽  
pp. 4209-4214 ◽  
Author(s):  
Robert E. Donahue ◽  
Laura Tuschong ◽  
Thomas R. Bauer ◽  
Yu Ying Yau ◽  
Susan F. Leitman ◽  
...  
Keyword(s):  
Leukocyte Adhesion ◽  
Cation Binding ◽  
Gene Correction ◽  
Integrin Activation ◽  
Α Subunit ◽  
Leukocyte Integrins ◽  
Specific Increase ◽  
Stimulating Factor ◽  
Activation Status

Abstract After administration of granulocyte colony-stimulating factor (G-CSF), there is a marked, albeit transient, drop in circulating neutrophils. To determine the role of leukocyte integrins in this disappearance, a dog having canine leukocyte adhesion deficiency (CLAD) or CLAD dogs who had undergone gene correction either by matched littermate allogeneic transplant or autologous gene therapy were evaluated. Shortly after G-CSF administration, a dramatic, yet transient, neutropenia was observed in the control littermates. This neutropenia was not as marked in the CLAD dogs. In all instances, it was CD18+ neutrophils that preferentially egressed from the circulation. The association of CD18 with this rapid loss suggested leukocyte integrin activation after G-CSF administration. To determine the activation status of the integrin, a monoclonal antibody recognizing the activated α-subunit cation binding domain (mAb24) was used to evaluate human leukocytes after G-CSF administration. Mirroring the dramatic decrease in circulating neutrophil numbers, there was a dramatic and specific increase in the activation of the α-subunit after G-CSF expression on polymorphonuclear leukocytes. This activation, like the drop in neutrophil count, was transient. These results demonstrate that the leukocyte integrin on circulating neutrophils is transiently activated after G-CSF administration and mediates the transient neutropenia observed after G-CSF administration.

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