scholarly journals A novel pathway of rapid TLR-triggered activation of integrin-dependent leukocyte adhesion that requires Rap1 GTPase

2014 ◽  
Vol 25 (19) ◽  
pp. 2948-2955 ◽  
Author(s):  
Kyoung-Jin Chung ◽  
Ioannis Mitroulis ◽  
Johannes R. Wiessner ◽  
Ying Yi Zheng ◽  
Gabriele Siegert ◽  
...  
Keyword(s):  
Leukocyte Adhesion ◽  
Integrin Activation ◽  
Pathogen Invasion ◽  
Β2 Integrin ◽  
Tlr2 Ligand ◽  
Species Production ◽  
Rapid Activation ◽  
Slow Rolling ◽  
Inside Out

Rapid β2-integrin activation is indispensable for leukocyte adhesion and recruitment to sites of infection and is mediated by chemokine- or P-selectin glycoprotein ligand-1–induced inside-out signaling. Here we uncovered a novel pathway for rapid activation of integrin-dependent leukocyte adhesion, triggered by toll-like receptor (TLR)–mediated signaling. TLR2 or TLR5 ligation rapidly activated integrin-dependent leukocyte adhesion to immobilized ICAM-1 and fibronectin. Consistently, in vivo administration of the TLR2-ligand Pam3CSK4 increased integrin-dependent slow rolling and adhesion to endothelium within minutes, as identified by intravital microscopy in the cremaster model. TLR2 and TLR5 ligation increased β2-integrin affinity, as assessed by the detection of activation-dependent neoepitopes. TLR2- and TLR5-triggered integrin activation in leukocytes required enhanced Rap1 GTPase activity, which was mediated by Rac1 activation and NADPH oxidase-2–dependent reactive oxygen species production. This novel direct pathway linking initial pathogen recognition by TLRs to rapid β2-integrin activation may critically regulate acute leukocyte infiltration to sites of pathogen invasion.

scholarly journals Direct Rap1/Talin1 interaction regulates platelet and neutrophil integrin activity in mice

Blood ◽  
2018 ◽  
Vol 132 (26) ◽  
pp. 2754-2762 ◽  
Author(s):  
Thomas Bromberger ◽  
Sarah Klapproth ◽  
Ina Rohwedder ◽  
Liang Zhu ◽  
Laura Mittmann ◽  
...  
Keyword(s):  
Leukocyte Adhesion ◽  
Thrombus Formation ◽  
Alternative Mechanism ◽  
Integrin Activation ◽  
Vessel Occlusion ◽  
Crucial Step ◽  
Β2 Integrin ◽  
Biological Studies ◽  
Integrin Ligands

Abstract Targeting Talin1 to the plasma membrane is a crucial step in integrin activation, which in leukocytes is mediated by a Rap1/RIAM/Talin1 pathway, whereas in platelets, it is RIAM independent. Recent structural, biochemical, and cell biological studies have suggested direct Rap1/Talin1 interaction as an alternative mechanism to recruit Talin1 to the membrane and induce integrin activation. To test whether this pathway is of relevance in vivo, we generated Rap1 binding–deficient Talin1 knockin (Tln13mut) mice. Although Tln13mut mice showed no obvious abnormalities, their platelets exhibited reduced integrin activation, aggregation, adhesion, and spreading, resulting in prolonged tail-bleeding times and delayed thrombus formation and vessel occlusion in vivo. Surprisingly, neutrophil adhesion to different integrin ligands and β2 integrin–dependent phagocytosis were also significantly impaired, which caused profound leukocyte adhesion and extravasation defects in Tln13mut mice. In contrast, macrophages exhibited no defect in adhesion or spreading despite reduced integrin activation. Taken together, our findings suggest that direct Rap1/Talin1 interaction is of particular importance in regulating the activity of different integrin classes expressed on platelets and neutrophils, which both depend on fast and dynamic integrin-mediated responses.

scholarly journals Skap2 is required for β2 integrin–mediated neutrophil recruitment and functions

2017 ◽  
Vol 214 (3) ◽  
pp. 851-874 ◽  
Author(s):  
Mark Boras ◽  
Stephanie Volmering ◽  
Arne Bokemeyer ◽  
Jan Rossaint ◽  
Helena Block ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Leukocyte Adhesion ◽  
Direct Interaction ◽  
Neutrophil Recruitment ◽  
Integrin Activation ◽  
Leukocyte Adhesion Deficiency ◽  
Β2 Integrin ◽  
Syndrome Protein

Integrin activation is required for neutrophil functions. Impaired integrin activation on neutrophils is the hallmark of leukocyte adhesion deficiency (LAD) syndrome in humans, characterized by impaired leukocyte recruitment and recurrent infections. The Src kinase–associated phosphoprotein 2 (Skap2) is involved in integrin functions in different leukocyte subtypes. However, the role of Skap2 in β2 integrin activation and neutrophil recruitment is unknown. In this study, we demonstrate the crucial role of Skap2 in regulating actin polymerization and binding of talin-1 and kindlin-3 to the β2 integrin cytoplasmic domain, thereby being indispensable for β2 integrin activation and neutrophil recruitment. The direct interaction of Skap2 with the Wiskott–Aldrich syndrome protein via its SH3 domain is critical for integrin activation and neutrophil recruitment in vivo. Furthermore, Skap2 regulates integrin-mediated outside-in signaling events and neutrophil functions. Thus, Skap2 is essential to activate the β2 integrins, and loss of Skap2 function is sufficient to cause a LAD-like phenotype in mice.

scholarly journals Blocking neutrophil integrin activation prevents ischemia–reperfusion injury

2015 ◽  
Vol 212 (8) ◽  
pp. 1267-1281 ◽  
Author(s):  
Tadayuki Yago ◽  
Brian G. Petrich ◽  
Nan Zhang ◽  
Zhenghui Liu ◽  
Bojing Shao ◽  
...  
Keyword(s):  
Reperfusion Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Renal Ischemia ◽  
Integrin Activation ◽  
Β2 Integrin ◽  
Renal Ischemia Reperfusion Injury ◽  
Slow Rolling

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.

scholarly journals A LAD-III syndrome is associated with defective expression of the Rap-1 activator CalDAG-GEFI in lymphocytes, neutrophils, and platelets

2007 ◽  
Vol 204 (7) ◽  
pp. 1571-1582 ◽  
Author(s):  
Ronit Pasvolsky ◽  
Sara W. Feigelson ◽  
Sara Sebnem Kilic ◽  
Amos J. Simon ◽  
Guy Tal-Lapidot ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Leukocyte Adhesion ◽  
Splice Junction ◽  
Integrin Activation ◽  
Leukocyte Adhesion Deficiency ◽  
Protein Levels ◽  
Guanine Exchange Factor ◽  
Β3 Integrin ◽  
Inside Out

Leukocyte and platelet integrins rapidly alter their affinity and adhesiveness in response to various activation (inside-out) signals. A rare leukocyte adhesion deficiency (LAD), LAD-III, is associated with severe defects in leukocyte and platelet integrin activation. We report two new LAD cases in which lymphocytes, neutrophils, and platelets share severe defects in β1, β2, and β3 integrin activation. Patients were both homozygous for a splice junction mutation in their CalDAG-GEFI gene, which is a key Rap-1/2 guanine exchange factor (GEF). Both mRNA and protein levels of the GEF were diminished in LAD lymphocytes, neutrophils, and platelets. Consequently, LAD-III platelets failed to aggregate because of an impaired αIIbβ3 activation by key agonists. β2 integrins on LAD-III neutrophils were unable to mediate leukocyte arrest on TNFα-stimulated endothelium, despite normal selectin-mediated rolling. In situ subsecond activation of neutrophil β2 integrin adhesiveness by surface-bound chemoattractants and of primary T lymphocyte LFA-1 by the CXCL12 chemokine was abolished. Chemokine inside-out signals also failed to stimulate lymphocyte LFA-1 extension and high affinity epitopes. Chemokine-triggered VLA-4 adhesiveness in T lymphocytes was partially defective as well. These studies identify CalDAG-GEFI as a critical regulator of inside-out integrin activation in human T lymphocytes, neutrophils, and platelets.

Antimyeloperoxidase antibodies rapidly induce α4-integrin–dependent glomerular neutrophil adhesion

Blood ◽  
2009 ◽  
Vol 113 (25) ◽  
pp. 6485-6494 ◽  
Author(s):  
Michael P. Kuligowski ◽  
Rain Y. Q. Kwan ◽  
Cecilia Lo ◽  
Cyndi Wong ◽  
Will G. James ◽  
...  
Keyword(s):  
Intravenous Administration ◽  
Intravital Microscopy ◽  
Leukocyte Adhesion ◽  
Antineutrophil Cytoplasmic Antibodies ◽  
Neutrophil Adhesion ◽  
Inflammatory Stimulus ◽  
Β2 Integrin ◽  
Higher Doses

Abstract Patients with antineutrophil cytoplasmic antibodies (ANCAs) frequently develop severe vasculitis and glomerulonephritis. Although ANCAs, particularly antimyeloperoxidase (anti-MPO), have been shown to promote leukocyte adhesion in postcapillary venules, their ability to promote adhesion in the glomerular vasculature is less clear. We used intravital microscopy to examine glomerular leukocyte adhesion induced by anti-MPO. In mice pretreated with LPS, 50 μg anti-MPO induced LFA-1–dependent adhesion in glomeruli. In concert with this finding, in mice pretreated with LPS, more than 80% of circulating neutrophils bound anti-MPO within 5 minutes of intravenous administration. However, even in the absence of LPS, more than 40% of circulating neutrophils bound anti-MPO in vivo, a response not seen in MPO−/− mice. In addition, a higher dose of anti-MPO (200 μg) induced robust glomerular leukocyte adhesion in the absence of LPS. The latter response was β2-integrin independent, instead requiring the α4-integrin, which was up-regulated on neutrophils in response to anti-MPO. These data indicate that anti-MPO antibodies bind to circulating neutrophils, and can induce glomerular leukocyte adhesion via multiple pathways. Lower doses induce adhesion only after an infection-related stimulus, whereas higher doses are capable of inducing responses in the absence of an additional inflammatory stimulus, via alternative adhesion mechanisms.

α-Chain phosphorylation of the human leukocyte CD11b/CD18 (Mac-1) integrin is pivotal for integrin activation to bind ICAMs and leukocyte extravasation

Blood ◽  
2006 ◽  
Vol 108 (10) ◽  
pp. 3379-3386 ◽  
Author(s):  
Susanna C. Fagerholm ◽  
Minna Varis ◽  
Michael Stefanidakis ◽  
Tiina J. Hilden ◽  
Carl G. Gahmberg
Keyword(s):  
Leukocyte Adhesion ◽  
Phosphorylation Site ◽  
Human Leukocyte ◽  
Serine Phosphorylation ◽  
Integrin Activation ◽  
Endothelial Ligands ◽  
Mutant Cells ◽  
Inside Out ◽  
Selective Mechanism

Abstract The promiscuous CD11b/CD18 (Mac-1) integrin has important roles in regulating many immunologic functions such as leukocyte adhesion and emigration from the bloodstream via interactions with the endothelial ligands ICAM-1 and ICAM-2, iC3b-mediated phagocytosis, and apoptosis. However, the mechanisms for Mac-1 inside-out activation have remained poorly understood. Phosphorylation of integrin cytoplasmic domains is emerging as an important mechanism of regulating integrin functions. Here, we have studied phosphorylation of human CD11b, which takes place on the cytoplasmic Ser1126 in neutrophils. We show that mutation of the serine phosphorylation site leads to inability of Mac-1 to become activated to bind the cellular ligands ICAM-1 and ICAM-2. However, CD11b-mutant cells are fully capable of binding other studied CD11b ligands (ie, iC3b and denatured BSA). Activation epitopes expressed in the extracellular domain of the integrin and affinity for soluble ICAM ligands were decreased for the mutated integrin. Additionally, the mutation resulted in inhibition of chemokine-induced migration in a transendothelial assay in vitro and significantly reduced the accumulation of intravenously administered cells in the spleen and lungs of Balb/c mice. These results characterize a novel selective mechanism of Mac-1–integrin activation, which mediates leukocyte emigration from the bloodstream to the tissues.

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 ◽  
2006 ◽  
Vol 108 (12) ◽  
pp. 3919-3927 ◽  
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.

scholarly journals Endothelial CD99 supports arrest of mouse neutrophils in venules and binds to neutrophil PILRs

Blood ◽  
2017 ◽  
Vol 129 (13) ◽  
pp. 1811-1822 ◽  
Author(s):  
Debashree Goswami ◽  
Sigrid März ◽  
Yu-Tung Li ◽  
Annette Artz ◽  
Kerstin Schäfer ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Integrin Activation ◽  
Β2 Integrin ◽  
Neutrophil Extravasation ◽  
Key Points

Key Points Only CD99 on endothelial cells, not on neutrophils, participates in neutrophil extravasation in vivo. A new function was found for CD99: support of chemokine-induced β2-integrin activation and neutrophil arrest by binding to PILR.

Ser756 of β2 integrin controls Rap1 activity during inside-out activation of αMβ2

2011 ◽  
Vol 437 (3) ◽  
pp. 461-467 ◽  
Author(s):  
Jenson Lim ◽  
Neil A. Hotchin ◽  
Emmanuelle Caron
Keyword(s):  
Phorbol Ester ◽  
Physiological Role ◽  
Small Gtpase ◽  
Integrin Activation ◽  
Calmodulin Antagonist ◽  
Β2 Integrin ◽  
Inside Out

During αMβ2-mediated phagocytosis, the small GTPase Rap1 activates the β2 integrin by binding to a region between residues 732 and 761. Using COS-7 cells transfected with αMβ2, we show that αMβ2 activation by the phorbol ester PMA involves Ser756 of β2. This residue is critical for the local positioning of talin and biochemically interacts with Rap1. Using the CaM (calmodulin) antagonist W7, we found Rap1 recruitment and the inside-out activation of αMβ2 to be affected. We also report a role for CaMKII (calcium/CaM-dependent kinase II) in the activation of Rap1 during integrin activation. These results demonstrate a distinct physiological role for Ser756 of β2 integrin, in conjunction with the actions of talin and Rap1, during αMβ2 activation in macrophages.

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.

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