Calcium flux in neutrophils synchronizes beta2 integrin adhesive and signaling events that guide inflammatory recruitment.
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ABSTRACT:
SUBMITTER: Schaff UY
PROVIDER: S-EPMC2668576 | biostudies-other | 2008 Apr
REPOSITORIES: biostudies-other
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Project description:On arrested neutrophils a focal adhesive cluster of ~200 high affinity (HA) β2-integrin bonds under tension is sufficient to trigger Ca2+ flux that signals an increase in activation in direct proportion to increments in shear stress. We reasoned that a threshold tension acting on individual β2-integrin bonds provides a mechanical means of transducing the magnitude of fluid drag force into signals that enhance the efficiency of neutrophil recruitment and effector function. Tension gauge tethers (TGT) are a duplex of DNA nucleotides that rupture at a precise shear force, which increases with the extent of nucleotide overlap, ranging from a tolerance of 54pN to 12pN. TGT annealed to a substrate captures neutrophils via allosteric antibodies that stabilize LFA-1 in a high- or low-affinity conformation. Neutrophils sheared on TGT substrates were recorded in real time to form HA β2-integrin bonds and flux cytosolic Ca2+, which elicited shape change and downstream production of reactive oxygen species. A threshold force of 33pN triggered consolidation of HA β2-integrin bonds and triggered membrane influx of Ca2+, whereas an optimum tension of 54pN efficiently transduced activation at a level equivalent to chemotactic stimulation on ICAM-1. We conclude that neutrophils sense the level of fluid drag transduced through individual β2-integrin bonds, providing an intrinsic means to modulate inflammatory response in the microcirculation.
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Project description:ObjectiveWhile the role of antiphospholipid antibodies in activating endothelial cells has been extensively studied, the impact of these antibodies on the adhesive potential of leukocytes has received less attention. This study was undertaken to investigate the extent to which antiphospholipid syndrome (APS) neutrophils adhere to resting endothelial cells under physiologic flow conditions and the surface molecules required for that adhesion.MethodsPatients with primary APS (n = 43), patients with a history of venous thrombosis but negative test results for antiphospholipid antibodies (n = 11), and healthy controls (n = 38) were studied. Cells were introduced into a flow chamber and perfused across resting human umbilical vein endothelial cells (HUVECs). Surface adhesion molecules were quantified by flow cytometry. Neutrophil extracellular trap release (NETosis) was assessed in neutrophil-HUVEC cocultures.ResultsUpon perfusion of anticoagulated blood through the flow chamber, APS neutrophils demonstrated increased adhesion as compared to control neutrophils under conditions representative of either venous (n = 8; P < 0.05) or arterial (n = 15; P < 0.0001) flow. At the same time, APS neutrophils were characterized by up-regulation of CD64, CEACAM1, ?2 -glycoprotein I, and activated Mac-1 on their surface (n = 12-18; P < 0.05 for all markers). Exposing control neutrophils to APS plasma or APS IgG resulted in increased neutrophil adhesion (n = 10-11; P < 0.0001) and surface marker up-regulation as compared to controls. A monoclonal antibody specific for activated Mac-1 reduced the adhesion of APS neutrophils in the flow-chamber assay (P < 0.01). The same monoclonal antibody reduced NETosis in neutrophil-HUVEC cocultures (P < 0.01).ConclusionAPS neutrophils demonstrate increased adhesive potential, which is dependent upon the activated form of Mac-1. In patients, this could lower the threshold for neutrophil-endothelium interactions, NETosis, and possibly thrombotic events.
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Project description:During inflammation, circulating polymorphonuclear neutrophils (PMNs) receive signals to cross the endothelial barrier and migrate through the extracellular matrix (ECM) to reach the injured site. Migration requires complex and poorly understood interactions of chemokines, chemokine receptors, ECM molecules, integrins, and other receptors. Here we show that the ECM protein lumican regulates PMN migration through interactions with specific integrin receptors. Lumican-deficient (Lum(-/-)) mice manifest connective tissue defects, impaired innate immune response, and poor wound healing with reduced PMN infiltration. Lum(-/-) PMNs exhibit poor chemotactic migration that is restored with exogenous recombinant lumican and inhibited by anti-lumican antibody, confirming a role for lumican in PMN migration. Treatment of PMNs with antibodies that block beta(2), beta(1), and alpha(M) integrin subunits inhibits lumican-mediated migration. Furthermore, immunohistochemical and biochemical approaches indicate binding of lumican to beta(2), alpha(M), and alpha(L) integrin subunits. Thus, lumican may regulate PMN migration mediated by MAC-1 (alpha(M)/beta(2)) and LFA-1 (alpha(L)/beta(2)), the two major PMN surface integrins. We detected lumican on the surface of peritoneal PMNs and not bone marrow or peripheral blood PMNs. This suggests that PMNs must acquire lumican during or after crossing the endothelial barrier as they exit circulation. We also found that peritoneal PMNs do not express lumican, whereas endothelial cells do. Taken together these observations suggest a novel endothelial lumican-mediated paracrine regulation of neutrophils early on in their migration path.
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Project description:Stromal-derived factor-1 (SDF-1) and its receptor, CXCR4, are essential for normal hematopoietic progenitor cell movement and adherence within the bone marrow microenvironment. In leukemia, the BCR-ABL1 oncoprotein inhibits SDF-1-dependent cell trafficking within the bone marrow through a mechanism that is not fully understood. Here, we report that BCR-ABL1 in malignant cells constitutively increases expression of activation-dependent epitopes of the beta(2) integrin LFA-1. This is associated with the complete loss of responsiveness of LFA-1 to SDF-1-induced "inside-out" signaling involving CXCR4 and Lyn, leading to aberrant adhesive responses. These data provide a novel, LFA-1-mediated mechanism whereby BCR-ABL1 inhibits SDF-1 action in malignant progenitors.
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Project description:Acute inflammation triggers the innate immune response of neutrophils that efficiently traffic from the bloodstream to concentrate at high numbers at the site of tissue infection or wounding. A gatekeeper in this process is activation of ?(2) integrins, which form bond clusters with ICAM-1 on the endothelial surface. These bond clusters serve dual functions of providing adhesive strength to anchor neutrophils under the shear forces of blood flow and directional guidance for cell polarization and subsequent transmigration on inflamed endothelium. We hypothesized that shear forces transmitted through high-affinity LFA-1 facilitates the cooperation with the calcium release-activated channel Orai1 in directing localized cytoskeletal activation and directed migration. By using vascular mimetic microfluidic channels, we observed neutrophil arrest on a substrate of either ICAM-1 or allosteric Abs that stabilize a high- or low-affinity conformation of LFA-1. Neutrophils captured via low-affinity LFA-1 did not exhibit intracellular calcium flux, F-actin polymerization, cell polarization, or directional migration under shear flow. In contrast, high-affinity LFA-1 provided orientation along a uropod-pseudopod axis that required calcium flux through Orai1. We demonstrate how the shear stress of blood flow can transduce distinct outside-in signals at focal sites of high-affinity LFA-1 that provide contact-mediated guidance for neutrophil emigration.
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Project description:Ly6G is a glycosylphosphatidylinositol (GPI)-anchored protein of unknown function that is commonly targeted to induce experimental neutrophil depletion in mice. In the present study, we found that doses of anti-Ly6G Abs too low to produce sustained neutropenia remained capable of inhibiting experimental arthritis, leaving joint tissues free of infiltrating neutrophils. Thioglycollate-stimulated peritonitis was also attenuated. No alteration in neutrophil apoptosis was observed, implicating impaired recruitment. Indeed, Ly6G ligation abrogated neutrophil migration toward LTB(4) and other chemoattractants in a transwell system. Exploring the basis for this blockade, we identified colocalization of Ly6G and ?2-integrins by confocal microscopy and confirmed close association by both coimmunoprecipitation and fluorescence lifetime imaging microscopy. Anti-Ly6G Ab impaired surface expression of ?2-integrins in LTB(4)-stimulated neutrophils and mimicked CD11a blockade in inhibiting both ICAM-1 binding and firm adhesion to activated endothelium under flow conditions. Correspondingly, migration of ?2-integrin-deficient neutrophils was no longer inhibited by anti-Ly6G. These results demonstrate that experimental targeting of Ly6G has functional effects on the neutrophil population and identify a previously unappreciated role for Ly6G as a modulator of neutrophil migration to sites of inflammation via a ?2-integrin-dependent mechanism.
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Project description:Calcium signaling has long been associated with key events of immunity, including chemotaxis, phagocytosis, and activation. However, imaging and manipulation of calcium flux in motile immune cells in live animals remain challenging. Using light-sheet microscopy for in vivo calcium imaging in zebrafish, we observe characteristic patterns of calcium flux triggered by distinct events, including phagocytosis of pathogenic bacteria and migration of neutrophils toward inflammatory stimuli. In contrast to findings from ex vivo studies, we observe enriched calcium influx at the leading edge of migrating neutrophils. To directly manipulate calcium dynamics in vivo, we have developed transgenic lines with cell-specific expression of the mammalian TRPV1 channel, enabling ligand-gated, reversible, and spatiotemporal control of calcium influx. We find that controlled calcium influx can function to help define the neutrophil's leading edge. Cell-specific TRPV1 expression may have broad utility for precise control of calcium dynamics in other immune cell types and organisms.
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Project description:In recent years, several studies suggested that the ability of hyperbaric oxygen therapy (HBOT) to promote healing in patients with diabetic ulcers and chronic wounds is due to the reduction of inflammatory cytokines and to a significant decrease in neutrophils recruitment to the damaged area. α4 and β2 integrins are receptors mediating the neutrophil adhesion to the endothelium and the comprehension of the effects of hyperbaric oxygenation on their expression and functions in neutrophils could be of great importance for the design of novel therapeutic protocols focused on anti-inflammatory agents. In this study, the α4 and β2 integrins' expression and functions have been evaluated in human primary neutrophils obtained from patients with chronic non-healing wounds and undergoing a prolonged HBOT (150 kPa per 90 minutes). The effect of a peptidomimetic α4β1 integrin antagonist has been also analyzed under these conditions. A statistically significant decrease (68%) in β2 integrin expression on neutrophils was observed during the treatment with HBO and maintained one month after the last treatment, while α4 integrin levels remained unchanged. However, cell adhesion function of both neutrophilic integrins α4β1 and β2 was significantly reduced 70 and 67%, respectively), but α4β1 integrin was still sensitive to antagonist inhibition in the presence of fibronectin, suggesting that a combined therapy between HBOT and integrin antagonists could have greater antinflammatory efficacy.
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Project description:Neutrophil arrest and migration on inflamed endothelium involves a conformational shift in CD11a/CD18 (leukocyte function antigen-1; LFA-1) to a high-affinity and clustered state that determines the strength and lifetime of bond formation with ICAM-1. Cytoskeletal adapter proteins Kindlin-3 and Talin-1 anchor clustered LFA-1 to the cytoskeleton and facilitate the transition from neutrophil rolling to arrest. We recently reported that tensile force acts on LFA-1 bonds inducing their colocalization with Orai1, the predominant membrane store operated Ca(2+) channel that cooperates with the endoplasmic reticulum to elicit cytosolic flux. Because Kindlin-3 was recently reported to initiate LFA-1 clustering in lymphocytes, we hypothesized that it cooperates with Orai1 and LFA-1 in signaling local Ca(2+) flux necessary for shear-resistant neutrophil arrest. Using microfluidic flow channels combined with total internal reflection fluorescence microscopy, we applied defined shear stress to low- or high-affinity LFA-1 and imaged the spatiotemporal regulation of bond formation with Kindlin-3 recruitment and Ca(2+) influx. Orai1 and Kindlin-3 genes were silenced in neutrophil-like HL-60 cells to assess their respective roles in this process. Kindlin-3 was enriched within focal clusters of high-affinity LFA-1, which promoted physical linkage with Orai1. This macromolecular complex functioned to amplify inside-out Ca(2+) signaling in response to IL-8 stimulation by catalyzing an increased density of Talin-1 and consolidating LFA-1 clusters within sites of contact with ICAM-1. In this manner, neutrophils use focal adhesions as mechanosensors that convert shear stress-mediated tensile force into local bursts of Ca(2+) influx that catalyze cytoskeletal engagement and an adhesion-strengthened migratory phenotype.
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Project description:CD47 plays an important but incompletely understood role in the innate and adaptive immune responses. CD47, also called integrin-associated protein, has been demonstrated to associate in cis with ?1 and ?3 integrins. Here we test the hypothesis that CD47 regulates adhesive functions of T-cell ?4?1 (VLA-4) and ?L?2 (LFA-1) in in vivo and in vitro models of inflammation. Intravital microscopy studies reveal that CD47(-/-) Th1 cells exhibit reduced interactions with wild-type (WT) inflamed cremaster muscle microvessels. Similarly, murine CD47(-/-) Th1 cells, as compared with WT, showed defects in adhesion and transmigration across tumor necrosis factor-? (TNF-?)-activated murine endothelium and in adhesion to immobilized intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion protein 1 (VCAM-1) under flow conditions. Human Jurkat T-cells lacking CD47 also showed reduced adhesion to TNF-?-activated endothelium and ICAM-1 and VCAM-1. In cis interactions between Jurkat T-cell ?2 integrins and CD47 were detected by fluorescence lifetime imaging microscopy. Unexpectedly, Jurkat CD47 null cells exhibited a striking defect in ?1 and ?2 integrin activation in response to Mn(2+) or Mg(2+)/ethylene glycol tetraacetic acid treatment. Our results demonstrate that CD47 associates with ?2 integrins and is necessary to induce high-affinity conformations of LFA-1 and VLA-4 that recognize their endothelial cell ligands and support leukocyte adhesion and transendothelial migration.
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