Dynamic shifts in LFA-1 affinity regulate neutrophil rolling, arrest, and transmigration on inflamed endothelium

Polymorphonuclear leukocyte (PMN) recruitment to vascular endothelium during acute inflammation involves cooperation between selectins, G-proteins, and β2-integrins. LFA-1 (CD11a/CD18) affinity correlates with specific adhesion functions because a shift from low to intermediate affinity supports rolling on ICAM-1, whereas high affinity is associated with shear-resistant leukocyte arrest. We imaged PMN adhesion on cytokine-inflamed endothelium in a parallel-plate flow chamber to define the dynamics of β2-integrin function during recruitment and transmigration. After arrest on inflamed endothelium, high-affinity LFA-1 aligned along the uropod-pseudopod major axis, which was essential for efficient neutrophil polarization and subsequent transmigration. An allosteric small molecule inhibitor targeted to the I-domain stabilized LFA-1 in an intermediate-affinity conformation, which supported neutrophil rolling but inhibited cell polarization and abrogated transmigration. We conclude that a shift in LFA-1 from intermediate to high affinity during the transition from rolling to arrest provides the contact-mediated signaling and guidance necessary for PMN transmigration on inflamed endothelium.

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Neutrophil activation via β2 integrins (CD11/CD18): Molecular mechanisms and clinical implications

Thrombosis and Haemostasis ◽

10.1160/th07-02-0156 ◽

2007 ◽

Vol 98 (08) ◽

pp. 262-273 ◽

Cited By ~ 76

Author(s):

Jürgen Schymeinsky ◽

Attila Mócsai ◽

Barbara Walzog

Keyword(s):

Receptor Tyrosine Kinase ◽

Acute Inflammation ◽

Molecular Mechanisms ◽

Inflammatory Diseases ◽

Polymorphonuclear Neutrophils ◽

Downstream Signaling ◽

Β2 Integrin ◽

Pmn Functions ◽

Integrin Family ◽

Β2 Integrins

SummaryPolymorphonuclear neutrophils (PMN) are key components of the innate immunity and their efficient recruitment to the sites of lesion is a prerequisite for acute inflammation. Signaling via adhesion molecules of the β2 integrin family (CD11/CD18) plays an essential role for PMN recruitment and activation during inflammation. In this review, we will focus on the non-receptor tyrosine kinase Syk, an important downstream signaling component of β2 integrins that is required for the control of different PMN functions including adhesion,migration and phagocytosis. The exploration of β2 integrin-mediated Syk activation provided not only novel insights into the control of PMN functions but also led to the identification of Syk as a new molecular target for therapeutic intervention during inflammatory diseases.

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SRC-dependent outside-in signalling is a key step in the process of autoregulation of beta2 integrins in polymorphonuclear cells

Biochemical Journal ◽

10.1042/bj20040151 ◽

2004 ◽

Vol 380 (1) ◽

pp. 57-65 ◽

Cited By ~ 31

Author(s):

Paola PICCARDONI ◽

Stefano MANARINI ◽

Lorenzo FEDERICO ◽

Zsuzsa BAGOLY ◽

Romina PECCE ◽

...

Keyword(s):

Tyrosine Kinase ◽

Actin Polymerization ◽

Protein Tyrosine Kinase ◽

Polymorphonuclear Cells ◽

High Avidity ◽

Β2 Integrin ◽

High Affinity ◽

Tyrosine Kinase 2 ◽

Oncogenic Protein ◽

Β2 Integrins

In human PMN (polymorphonuclear cells), challenged by P-selectin, the β2-integrin Mac-1 (macrophage antigen-1) promoted the activation of the SRC (cellular homologue of Rous sarcoma virus oncogenic protein) family members HCK (haematopoietic cell kinase) and LYN (an SRC family protein tyrosine kinase) and phosphorylation of a P-110 (110 kDa protein). SRC kinase activity in turn was necessary for macrophage antigen-1-mediated adhesion [Piccardoni, Sideri, Manarini, Piccoli, Martelli, de Gaetano, Cerletti and Evangelista (2001) Blood 98, 108–116]. This suggested that an SRC-dependent outside-in signalling strengthens the β2-integrin interaction with the ligand. To support this hypothesis further, in the present study, we used the monoclonal antibody KIM127 or manganese to lock β2 integrins in a high-affinity state, and homotypic PMN adhesion was analysed to monitor β2-integrin adhesive function. KIM127 or manganese induced PMN homotypic adhesion and P-110 phosphorylation. Both these processes were abolished by blocking antibodies against the common β2 chain, by a combination of antibodies against αL and αM or by inhibitors of SRC activity. Confocal microscopy showed that activation epitopes were expressed by β2 integrins co-localized with patches of F-actin at the adhesion sites. Blockade of SRC kinases or of actin polymerization prevented clustering of activated integrins as well as F-actin accumulation. FACS analysis showed that SRC inhibitors modified neither basal nor manganese-induced KIM127 binding. An SRC-dependent outside-in signalling initiated by β2 integrins was also required for adhesion triggered by interleukin-8. These results confirm the hypothesis that an SRC-dependent outside-in signalling triggered by high affinity and ligand binding is necessary to stabilize β2-integrin-mediated adhesion. Allowing clustering of activated integrins, SRC might link the high-affinity with the high-avidity state. Proline-rich tyrosine kinase-2 appears to be involved in this process.

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Selectin catch-bonds mechanotransduce integrin activation and neutrophil arrest on inflamed endothelium under shear flow

Blood ◽

10.1182/blood-2017-05-783027 ◽

2017 ◽

Vol 130 (19) ◽

pp. 2101-2110 ◽

Cited By ~ 27

Author(s):

Vasilios A. Morikis ◽

Shannon Chase ◽

Ted Wun ◽

Elliot L. Chaikof ◽

John L. Magnani ◽

...

Keyword(s):

Shear Flow ◽

Adhesion Molecule ◽

Bond Formation ◽

Integrin Activation ◽

Β2 Integrin ◽

High Affinity ◽

Intracellular Adhesion Molecule ◽

Key Points ◽

Inflamed Endothelium ◽

Catch Bonds

Key Points Neutrophils rolling on E-selectin form catch-bonds with L-selectin that mechanosignal β2-integrin bond formation with intracellular adhesion molecule 1. Rivipansel blocks E-selectin recognition of sLex on L-selectin, thereby antagonizing outside-in signaling of high-affinity β2-integrin.

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Faculty Opinions recommendation of Neutrophil recruitment limited by high-affinity bent β2 integrin binding ligand in cis.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726701125.793523507 ◽

2016 ◽

Author(s):

M Amin Arnaout

Keyword(s):

Neutrophil Recruitment ◽

Β2 Integrin ◽

High Affinity ◽

In Cis

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VCAM-1 is more effective than MAdCAM-1 in supporting eosinophil rolling under conditions of shear flow

Blood ◽

10.1182/blood.v95.2.592 ◽

2000 ◽

Vol 95 (2) ◽

pp. 592-601 ◽

Cited By ~ 41

Author(s):

P. Sriramarao ◽

Richard G. DiScipio ◽

Ronald R. Cobb ◽

Myron Cybulsky ◽

Greg Stachnick ◽

...

Keyword(s):

Monoclonal Antibody ◽

Parallel Plate ◽

Cell Adhesion Molecule ◽

Flow Chamber ◽

Β1 Integrin ◽

Vascular Cell Adhesion ◽

Parallel Plate Flow Chamber ◽

Vascular Cell

The ability of the 4 integrin counterligands vascular cell adhesion molecule (VCAM)-1 or mucosal addressin (MAd)CAM-1 to support eosinophil rolling or firm adhesion under conditions of physiologic flow has not been delineated. Using a parallel plate flow chamber in vitro and intravital microscopy in vivo, we demonstrate that eosinophil rolling and adhesion on VCAM-1 is mediated by both 4β1 and 4β7 integrins. Eosinophils rolled equally efficiently on both VCAM-1 2 domain and VCAM-1 7 domain, suggesting that the N-terminal 2 domains of VCAM-1 are sufficient to support eosinophil rolling under conditions of flow. Furthermore, activation of the eosinophil β1 integrin with monoclonal antibody (mAb) 8A2 resulted in both resistance to shear stress–induced detachment from VCAM-1 in vitro and in stable arrest of rolling eosinophils on interleukin (IL)-1β–stimulated venules in vivo. Eosinophils rolled less efficiently on MAdCAM-1– than on VCAM-1–coated coverslips under conditions of flow. However, eosinophils firmly adhered as efficiently to MAdCAM-1 as to VCAM-1. Overall, these results demonstrate that both VCAM-1 and MAdCAM-1 can support eosinophil firm adhesion under conditions of flow. In contrast, VCAM-1 is significantly more efficient than MAdCAM-1 in supporting eosinophil rolling under conditions of flow.

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Wall Shear Stress Determination in a Small-Scale Parallel Plate Flow Chamber Using Laser Doppler Velocimetry Under Laminar, Pulsatile and Low-Reynolds Number Turbulent Flows

Journal of Fluids Engineering ◽

10.1115/1.4039158 ◽

2018 ◽

Vol 140 (6) ◽

Cited By ~ 1

Author(s):

Hamed Avari ◽

Kem A. Rogers ◽

Eric Savory

Keyword(s):

Shear Stress ◽

Wall Shear Stress ◽

Laser Doppler Velocimetry ◽

Parallel Plate ◽

Flow Chamber ◽

Laser Doppler ◽

Small Scale ◽

Doppler Velocimetry ◽

Flow Conditions ◽

Parallel Plate Flow Chamber

The parallel plate flow chamber (PPFC) has gained popularity due to its applications in fields such as biological tissue engineering. However, most of the studies using PPFC refer to theoretical relations for estimating the wall shear stress (WSS) and, hence, the accuracy of such quantifications remains elusive for anything other than steady laminar flow. In the current study, a laser Doppler velocimetry (LDV) method was used to quantify the flow in a PPFC (H = 1.8 mm × W = 17.5 mm, Dh = 3.26 mm, aspect ratio = 9.72) under steady Re = 990, laminar pulsatile (carotid Re0-mean = 282 as well as a non-zero-mean sinusoidal Re0-mean = 45 pulse) and low-Re turbulent Re = 2750 flow conditions. A mini-LDV probe was applied, and the absolute location of the LDV measuring volume with the respect to the wall was determined using a signal monitoring technique with uncertainties being around ±27 μm. The uniformity of the flow across the span of the channel, as well as the WSS assessment for all the flow conditions, was measured with the uncertainties all being less than 16%. At least two points within the viscous sublayer of the low-Re turbulent flow were measured (with the y+ for the first point < 3) and the WSS was determined using two methods with the differences between the two methods being within 5%. This paper for the first time presents the experimental determination of WSS using LDV in a small-scale PPFC under various flow conditions, the challenges associated with each condition, and a comparison between the cases. The present data will be useful for those conducting biological or numerical modeling studies using such devices.

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Differential attenuation of β2 integrin–dependent and –independent neutrophil migration by Ly6G ligation

Blood Advances ◽

10.1182/bloodadvances.2018026732 ◽

2019 ◽

Vol 3 (3) ◽

pp. 256-267 ◽

Cited By ~ 2

Author(s):

Pierre Cunin ◽

Pui Y. Lee ◽

Edy Kim ◽

Angela B. Schmider ◽

Nathalie Cloutier ◽

...

Keyword(s):

Interleukin 1 ◽

Neutrophil Migration ◽

Surface Protein ◽

Joint Inflammation ◽

Selective Inhibition ◽

Β2 Integrin ◽

Β2 Integrins ◽

Neutrophil Surface

Abstract Antibody ligation of the murine neutrophil surface protein Ly6G disrupts neutrophil migration in some contexts but not others. We tested whether this variability reflected divergent dependence of neutrophil migration on β2 integrins, adhesion molecules that interact with Ly6G at the neutrophil surface. In integrin-dependent murine arthritis, Ly6G ligation attenuated joint inflammation, even though mice lacking Ly6G altogether developed arthritis normally. By contrast, Ly6G ligation had no impact on integrin-independent neutrophil migration into inflamed lung. In peritoneum, the role of β2 integrins varied with stimulus, proving dispensable for neutrophil entry in Escherichia coli peritonitis but contributory in interleukin 1 (IL-1)–mediated sterile peritonitis. Correspondingly, Ly6G ligation attenuated only IL-1 peritonitis, disrupting the molecular association between integrins and Ly6G and inducing cell-intrinsic blockade restricted to integrin-dependent migration. Consistent with this observation, Ly6G ligation impaired integrin-mediated postadhesion strengthening for neutrophils arresting on activated cremaster endothelium in vivo. Together, these findings identify selective inhibition of integrin-mediated neutrophil emigration through Ly6G ligation, highlighting the marked site and stimulus specificity of β2 integrin dependence in neutrophil migration.

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