scholarly journals The LTB4–BLT1 axis regulates actomyosin and β2-integrin dynamics during neutrophil extravasation

2020 ◽  
Vol 219 (10) ◽  
Author(s):  
Bhagawat C. Subramanian ◽  
Nicolas Melis ◽  
Desu Chen ◽  
Weiye Wang ◽  
Devorah Gallardo ◽  
...  
Keyword(s):  
Neutrophil Migration ◽  
Extracellular Vesicle ◽  
Leukotriene B4 ◽  
Cell Polarization ◽  
Sterile Inflammation ◽  
Β2 Integrin ◽  
Neutrophil Extravasation ◽  
Neutrophil Response ◽  
Muscle Myosin

The eicosanoid leukotriene B4 (LTB4) relays chemotactic signals to direct neutrophil migration to inflamed sites through its receptor BLT1. However, the mechanisms by which the LTB4–BLT1 axis relays chemotactic signals during intravascular neutrophil response to inflammation remain unclear. Here, we report that LTB4 produced by neutrophils acts as an autocrine/paracrine signal to direct the vascular recruitment, arrest, and extravasation of neutrophils in a sterile inflammation model in the mouse footpad. Using intravital subcellular microscopy, we reveal that LTB4 elicits sustained cell polarization and adhesion responses during neutrophil arrest in vivo. Specifically, LTB4 signaling coordinates the dynamic redistribution of non-muscle myosin IIA and β2-integrin, which facilitate neutrophil arrest and extravasation. Notably, we also found that neutrophils shed extracellular vesicles in the vascular lumen and that inhibition of extracellular vesicle release blocks LTB4-mediated autocrine/paracrine signaling required for neutrophil arrest and extravasation. Overall, we uncover a novel complementary mechanism by which LTB4 relays extravasation signals in neutrophils during early inflammation response.

scholarly journals The LTB4-BLT1 axis regulates actomyosin and β2 integrin dynamics during neutrophil extravasation

2019 ◽  
Author(s):  
Bhagawat C. Subramanian ◽  
Nicolas Melis ◽  
Desu Chen ◽  
Weiye Wang ◽  
Devorah Gallardo ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Neutrophil Migration ◽  
Cell Polarization ◽  
Sterile Inflammation ◽  
Paracrine Signaling ◽  
Neutrophil Extravasation ◽  
Actomyosin Cytoskeleton ◽  
Neutrophil Response ◽  
Muscle Myosin

ABSTRACTThe eicosanoid Leukotriene B4 (LTB4) relays chemotactic signals to direct neutrophil migration to inflamed sites through its receptor BLT1. However, the mechanisms by which the LTB4-BLT1 axis relays chemotactic signals during intravascular neutrophil response to inflammation remain unclear. Here, we report that LTB4 produced by neutrophils acts as an autocrine/paracrine signal to direct the vascular recruitment, arrest and extravasation of neutrophils in a sterile inflammation model in the mouse footpad. Using Intravital Subcellular Microscopy (ISMic), we reveal that LTB4 elicits sustained cell polarization and adhesion responses during neutrophil arrest in vivo. Specifically, LTB4 signaling coordinates the dynamic redistribution of non-muscle Myosin IIA (NMIIA) and β2-integrin (Itgb2), which facilitate neutrophil arrest and extravasation. Notably, we also found that neutrophils shed extracellular vesicles (EVs) in the vascular lumen, and that inhibition of EV release blocks LTB4-mediated autocrine/paracrine signaling required for neutrophil arrest and extravasation. Overall, we uncover a novel complementary mechanism by which LTB4 relays extravasation signals in neutrophils during early inflammation response.SUMMARYNeutrophils arrest and extravasate from the blood vessels in response to infection and injury. Using intravital subcellular microscopy, Subramanian et al. identify a role for extracellular vesicles-based autocrine/paracrine LTB4-BLT1 signaling in promoting the re-arrangement of actomyosin cytoskeleton and β2-integrin during neutrophil extravasation in live animals.

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

2019 ◽  
Vol 3 (3) ◽  
pp. 256-267 ◽  
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.

scholarly journals The Macrophage-derived Lectin, MNCF, Activates Neutrophil Migration through a Pertussis Toxin-sensitive Pathway

2005 ◽  
Vol 53 (6) ◽  
pp. 715-723 ◽  
Author(s):  
Andréa N. Moreno ◽  
Gabriela Pereira-da-Silva ◽  
Constance Oliver ◽  
Maria Célia Jamur ◽  
Ademilson Panunto-Castelo ◽  
...  
Keyword(s):  
Cell Surface ◽  
Pertussis Toxin ◽  
Neutrophil Migration ◽  
Cell Polarization ◽  
Glucocorticoid Treatment ◽  
Neutrophil Chemotactic Factor ◽  
G Protein Coupled ◽  
Neutrophil Surface

The macrophage-derived neutrophil chemotactic factor (MNCF) is a d-galactose-binding lectin that induces neutrophil migration in vitro and in vivo. Neutrophil recruitment induced by MNCF is resistant to glucocorticoid treatment and is inhibited by the lectin-specific sugar, d-galactose. In the present study, we characterized the binding of MNCF to neutrophils and the responses triggered by this binding. Exposure to MNCF resulted in cell polarization, formation of a lamellipodium, and deep ruffles on the cell surface. By confocal microscopy, we observed that MNCF was evenly distributed on the cell surface after 30 min of incubation. The labeling intensity progressively diminished with longer incubations. Internalization kinetics showed that MNCF/ligand complexes were rapidly internalized, reaching maximum intracellular concentrations at 120 min and then decreased thereafter. The binding and internalization of MNCF were selectively inhibited by d-galactose. MNCF-induced neutrophil chemotaxis was inhibited by pertussis toxin. This fact strongly suggests that the MNCF-ligand on the neutrophil surface is a G-protein-coupled receptor (GPCR), similar to receptors for well-established neutrophil attractants. Our observations on the ability of MNCF to activate neutrophils are consistent with the increasing evidence for the participation of animal lectins in the innate immune response.

Leukotriene B4-induced neutrophil-mediated endothelial leakage in vitro and in vivo

1991 ◽  
Vol 71 (4) ◽  
pp. 1322-1330 ◽  
Author(s):  
S. Rosengren ◽  
A. M. Olofsson ◽  
U. H. von Andrian ◽  
E. Lundgren-Akerlund ◽  
K. E. Arfors
Keyword(s):  
Umbilical Vein ◽  
Neutrophil Migration ◽  
Fluorescein Isothiocyanate ◽  
Leukotriene B4 ◽  
Vascular Leakage ◽  
Neutrophil Granulocytes ◽  
Vitro Assay ◽  
Umbilical Vein Endothelial Cells

The vascular leakage of macromolecules seen in several models after application of leukotriene B4 (LTB4) is mediated by neutrophil granulocytes. We describe here an in vitro assay for this event. Human umbilical vein endothelial cells were grown on polycarbonate filters separating luminal and abluminal compartments of fluid. Both clearance rate of fluorescein isothiocyanate albumin and neutrophil migration through the endothelial monolayer were increased when LTB4 (10–100 nM) was added to the abluminal compartment. However, if LTB4 was instead added to the luminal compartments together with the neutrophils, no migration or change in clearance could be detected. These findings were confirmed in vivo in the cheek pouches of anesthetized hamsters, where extravascular application of LTB4 induced intravascular adhesion of neutrophils, accompanied by neutrophil-dependent vascular leakage. On the other hand, intravascular deposition of LTB4 with micropipettes induced adhesion of leukocytes but no leakage. In conclusion, the presence of neutrophils adhering to endothelium does not necessarily imply the development of neutrophil-mediated vascular leakage. Instead, the leakage appears connected to the process of neutrophil chemotaxis.

scholarly journals Novel protective role for MAP kinase phosphatase 2 in inflammatory arthritis

RMD Open ◽  
2019 ◽  
Vol 5 (1) ◽  
pp. e000711
Author(s):  
Juliane Schroeder ◽  
Kirsty Ross ◽  
Kathryn McIntosh ◽  
Shilan Jabber ◽  
Stuart Woods ◽  
...  
Keyword(s):  
Inflammatory Arthritis ◽  
Neutrophil Migration ◽  
Protective Role ◽  
Leukotriene B4 ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Map Kinase Phosphatase ◽  
Tnf Α ◽  
The Impact

ObjectivesWe have previously shown mitogen-activated protein kinase phosphatase 2 (MKP-2) to be a key regulator of proinflammatory cytokines in macrophages. In the study presented here, we investigated the role of MKP-2 in inflammatory arthritis with a particular focus on neutrophils.MethodsTo achieve this, we subjected MKP-2 deficient and wild type mice to collagen antibody induced arthritis, an innate model of arthritis, and determined disease pathology. To further our investigation, we depleted neutrophils in a prophylactic and therapeutic fashion. Last, we used chemotaxis assays to analyse the impact of MKP-2 deletion on neutrophil migration.ResultsMKP-2-/- mice showed a significant increase in disease pathology linked to elevated levels of proarthritic cytokines and chemokines TNF-α, IL-6 and MCP-1 in comparison to wild type controls. This phenotype is prevented or abolished after administration of neutrophil depleting antibody prior or after onset of disease, respectively. While MCP-1 levels were not affected, neutrophil depletion diminished TNF-α and reduced IL-6, thus linking these cytokines to neutrophils. In vivo imaging showed that MKP-2-/- mice had an increased influx of neutrophils into affected joints, which was higher and potentially prolonged than in wild type animals. Furthermore, using chemotaxis assays we revealed that MKP-2 deficient neutrophils migrate faster towards a Leukotriene B4 gradient. This process correlated with a reduced phosphorylation of ERK in MKP-2-/- neutrophils.ConclusionsThis is the first study to show a protective role for MKP-2 in inflammatory arthritis.

scholarly journals Differential attenuation of β2 integrin-dependent and -independent neutrophil migration by Ly6G ligation

2018 ◽  
Author(s):  
Pierre Cunin ◽  
Pui Y. Lee ◽  
Edy Kim ◽  
Angela B. Schmider ◽  
Nathalie Cloutier ◽  
...  
Keyword(s):  
Neutrophil Migration ◽  
Surface Protein ◽  
Joint Inflammation ◽  
Selective Inhibition ◽  
List Type ◽  
E Coli ◽  
Β2 Integrin ◽  
Β2 Integrins ◽  
Neutrophil Surface

AbstractAntibody 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 E. coli peritonitis but contributory in 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.KEY POINTSThe contribution of β2 integrins to neutrophil migration into inflamed tissues varies with site and stimulus.Ligation of Ly6G, a GPI-linked neutrophil surface protein, selectively attenuates β2 integrin-dependent neutrophil migration in vivo.Blockade correlates with disrupted interaction between Ly6G and β2 integrins and impaired integrin-mediated postadhesion strengthening.

scholarly journals Molecular Insights Into Neutrophil Biology From the Zebrafish Perspective: Lessons From CD18 Deficiency

2021 ◽  
Vol 12 ◽  
Author(s):  
Almke Bader ◽  
Jincheng Gao ◽  
Thibaud Rivière ◽  
Bettina Schmid ◽  
Barbara Walzog ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Neutrophil Migration ◽  
Amino Acid Sequences ◽  
Sterile Inflammation ◽  
Type I ◽  
Hematopoietic Tissue ◽  
Sequence Alignments ◽  
Zebrafish Larvae ◽  
Inflamed Tissue

Neutrophils are key players in innate immunity and originate from the bone marrow of the adult mammalian organism. In mammals, mature neutrophils are released from the bone marrow into the peripheral blood where they circulate until their recruitment to sites of inflammation in a multistep adhesion cascade. Here, adhesion molecules of the β2 integrin family (CD11/CD18) are critically required for the initial neutrophil adhesion to the inflamed endothelium and several post-adhesion steps allowing their extravasation into the inflamed tissue. Within the mammalian tissue, interstitial neutrophil migration can occur widely independent of β2 integrins. This is in sharp contrast to neutrophil recruitment in zebrafish larvae (Danio rerio) where neutrophils originate from the caudal hematopoietic tissue and mainly migrate interstitially to sites of lesion upon the early onset of inflammation. However, neutrophils extravasate from the circulation to the inflamed tissue in zebrafish larvae at later-time points. Although zebrafish larvae are a widely accepted model system to analyze neutrophil trafficking in vivo, the functional impact of β2 integrins for neutrophil trafficking during acute inflammation is completely unknown in this model. In this study, we generated zebrafish with a genetic deletion of CD18, the β subunit of β2 integrins, using CRISPR/Cas9 technology. Sequence alignments demonstrated a high similarity of the amino acid sequences between zebrafish and human CD18 especially in the functionally relevant I-like domain. In addition, the cytoplasmic domain of CD18 harbors two highly conserved NXXF motifs suggesting that zebrafish CD18 may share functional properties of human CD18. Accordingly, CD18 knock-out (KO) zebrafish larvae displayed the key symptoms of patients suffering from leukocyte adhesion deficiency (LAD) type I due to defects in ITGB2, the gene for CD18. Importantly, CD18 KO zebrafish larvae showed reduced neutrophil trafficking to sites of sterile inflammation despite the fact that an increased number of neutrophils was detectable in the circulation. By demonstrating the functional importance of CD18 for neutrophil trafficking in zebrafish larvae, our findings shed new light on neutrophil biology in vertebrates and introduce a new model organism for studying LAD type I.

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.

Short-chain fatty acids stimulate the migration of neutrophils to inflammatory sites

2009 ◽  
Vol 117 (9) ◽  
pp. 331-338 ◽  
Author(s):  
Marco A. R. Vinolo ◽  
Hosana G. Rodrigues ◽  
Elaine Hatanaka ◽  
Cristina B. Hebeda ◽  
Sandra H. P. Farsky ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Neutrophil Migration ◽  
Short Chain Fatty Acids ◽  
Short Chain ◽  
Mrna Levels ◽  
Air Pouch ◽  
Β2 Integrin ◽  
Chain Fatty Acids

SCFAs (short-chain fatty acids) are produced by anaerobic bacterial fermentation. Increased concentrations of these fatty acids are observed in inflammatory conditions, such as periodontal disease, and at sites of anaerobic infection. In the present study, the effect of the SCFAs acetate, propionate and butyrate on neutrophil chemotaxis and migration was investigated. Experiments were carried out in rats and in vitro. The following parameters were measured: rolling, adherence, expression of adhesion molecules in neutrophils (L-selectin and β2 integrin), transmigration, air pouch influx of neutrophils and production of cytokines [CINC-2αβ (cytokine-induced neutrophil chemoattractant-2αβ), IL-1β (interleukin-1β), MIP-1α (macrophage inflammatory protein-1α) and TNF-α (tumour necrosis factor-α)]. SCFAs induced in vivo neutrophil migration and increased the release of CINC-2αβ into the air pouch. These fatty acids increased the number of rolling and adhered cells as evaluated by intravital microscopy. SCFA treatment increased L-selectin expression on the neutrophil surface and L-selectin mRNA levels, but had no effect on the expression of β2 integrin. Propionate and butyrate also increased in vitro transmigration of neutrophils. These results indicate that SCFAs produced by anaerobic bacteria raise neutrophil migration through increased L-selectin expression on neutrophils and CINC-2αβ release.

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