Neutrophil Arrest Research Articles

Molecular and cellular mechanism of systemic vaso‐occlusion in Sickle Cell Disease (LB544)

SCD is a genetic disease caused by a single point mutation in the β‐globin gene of hemoglobin, which leads to sickling of RBCs under deoxygenated condition. Sickle RBCs (sRBCs) are not only less deformable, but also express enhanced levels of major adhesion molecules compared to normal discoid shaped RBCs of healthy patients. In addition to RBC sickling, SCD is also associated with a hyper‐inflammatory state, which accounts for enhanced adherence of leukocytes to vascular endothelium. Leukocyte‐endothelium adhesion starts with leukocyte rolling mediated by P‐selectin‐glycoprotein‐ligand (PSGL)‐1 on leukocytes binding to P‐selectin on endothelium. Rolling is followed by firm arrest, which is medicated by activated β¬2‐integrins (LFA‐1 and Mac‐1) on the leukocytes binding to inter‐cellular‐adhesion‐molecule (ICAM)‐1 on endothelium. Although neutrophils have been shown to play a role in the onset of vaso‐occlusion by interacting with sRBCs in systemic venules of SCD mice, the cellular and molecular mechanism that enables systemic vaso‐occlusion in human patients is not completely understood. Heparinized blood from SCD patients and race matched control subjects is perfused through a microfluidic flow chamber with a glass bottom coated with either human microvascular endothelial cells or a cocktail of P‐selection, ICAM‐1 and CXCL‐8 at a physiological shear stress (2‐10 dyn cm‐2). Fluorescent Abs against CD16 and glycophorin‐A are added to the blood to stain neutrophils and sRBCs, respectively and cellular interactions recorded using multi‐color Quantitative Dynamic Footprinting or epifluorescence microscopy. Preliminary results reveal that neutrophil arrest is facilitated by the formation of a thick trunk like structure at the rear, which also enables capturing of free flowing sRBCs by arrested neutrophils. Understanding the molecular mechanism behind vaso‐occlusion will be useful in designing prophylactic interventions for SCD patients.Grant Funding Source: This study is supported by 11SDG7340005 from the American Heart Association (P.S.), VMI start‐up funds (P.S.) and CBTP‐T32 fellowship (M.J).

Transendothelial migration of effector T cells across inflamed endothelial barriers does not require heparan sulfate proteoglycans

Leukocyte diapedesis is a chemotactic multistep process that requires optimal chemoattractant presentation by the endothelial barrier. Recent studies have described a critical role for heparan sulfate glycosaminoglycans (HSGAGs) in the presentation and functions of chemokines essential for lymphocyte interactions with the lymph node vasculature. We wished to test whether HS expression by a prototypic endothelial cell type, i.e. human umbilical vein endothelial cells (HUVECs), is critical for their ability to support neutrophil and lymphocyte adhesion and transendothelial migration (TEM) under shear flow. We found that HUVECs deposit HS GAGs mainly at their basolateral compartments in both their resting and inflamed states. We next inactivated the key enzyme involved in HS biosynthesis, exostosin-1 (Ext1). Silencing Ext1 resulted in a complete loss of HS biosynthesis; nonetheless, TNF-α and IL-1β stimulation of key adhesion molecules and inflammatory chemokines necessary for neutrophil or lymphocyte adhesion and TEM remained intact. Ext1 silencing reduced neutrophil arrest and markedly impaired TEM, consistent with a role of basolateral HS GAGs in directing neutrophil crossing of inflamed endothelial barriers. Strikingly, however, the TEM of effector T cells across identically Ext1-silenced HUVECs remained normal. Importantly, the biosynthesis of the main promigratory chemokines for effector T cells and neutrophils, respectively, CCL2 and CXCL1, and their vesicle distributions were also Ext1 independent. These results suggest that transmigrating neutrophils must respond to chemokines transiently presented by apical and basolateral endothelial HS GAGs. In contrast, effector T cells can integrate chemotactic TEM signals directly from intra-endothelial chemokine stores rather than from externally deposited chemokines.

β2 Integrin–Mediated Crawling on Endothelial ICAM-1 and ICAM-2 Is a Prerequisite for Transcellular Neutrophil Diapedesis across the Inflamed Blood–Brain Barrier

In acute neuroinflammatory states such as meningitis, neutrophils cross the blood-brain barrier (BBB) and contribute to pathological alterations of cerebral function. The mechanisms that govern neutrophil migration across the BBB are ill defined. Using live-cell imaging, we show that LPS-stimulated BBB endothelium supports neutrophil arrest, crawling, and diapedesis under physiological flow in vitro. Investigating the interactions of neutrophils from wild-type, CD11a(-/-), CD11b(-/-), and CD18(null) mice with wild-type, junctional adhesion molecule-A(-/-), ICAM-1(null), ICAM-2(-/-), or ICAM-1(null)/ICAM-2(-/-) primary mouse brain microvascular endothelial cells, we demonstrate that neutrophil arrest, polarization, and crawling required G-protein-coupled receptor-dependent activation of β2 integrins and binding to endothelial ICAM-1. LFA-1 was the prevailing ligand for endothelial ICAM-1 in mediating neutrophil shear resistant arrest, whereas Mac-1 was dominant over LFA-1 in mediating neutrophil polarization on the BBB in vitro. Neutrophil crawling was mediated by endothelial ICAM-1 and ICAM-2 and neutrophil LFA-1 and Mac-1. In the absence of crawling, few neutrophils maintained adhesive interactions with the BBB endothelium by remaining either stationary on endothelial junctions or displaying transient adhesive interactions characterized by a fast displacement on the endothelium along the direction of flow. Diapedesis of stationary neutrophils was unchanged by the lack of endothelial ICAM-1 and ICAM-2 and occurred exclusively via the paracellular pathway. Crawling neutrophils, although preferentially crossing the BBB through the endothelial junctions, could additionally breach the BBB via the transcellular route. Thus, β2 integrin-mediated neutrophil crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed BBB.

Lidocaine Reduces Neutrophil Recruitment by Abolishing Chemokine-Induced Arrest and Transendothelial Migration in Septic Patients

The inappropriate activation, positioning, and recruitment of leukocytes are implicated in the pathogenesis of multiple organ failure in sepsis. Although the local anesthetic lidocaine modulates inflammatory processes, the effects of lidocaine in sepsis are still unknown. This double-blinded, prospective, randomized clinical trial was conducted to investigate the effect of lidocaine on leukocyte recruitment in septic patients. Fourteen septic patients were randomized to receive either a placebo (n = 7) or a lidocaine (n = 7) bolus (1.5 mg/kg), followed by continuous infusion (100 mg/h for patients >70 kg or 70 mg/h for patients <70 kg) over a period of 48 h. Selectin-mediated slow rolling, chemokine-induced arrest, and transmigration were investigated by using flow chamber and transmigration assays. Lidocaine treatment abrogated chemokine-induced neutrophil arrest and significantly impaired neutrophil transmigration through endothelial cells by inhibition of the protein kinase C-θ while not affecting the selectin-mediated slow leukocyte rolling. The observed results were not attributable to changes in surface expression of adhesion molecules or selectin-mediated capturing capacity, indicating a direct effect of lidocaine on signal transduction in neutrophils. These data suggest that lidocaine selectively inhibits chemokine-induced arrest and transmigration of neutrophils by inhibition of protein kinase C-θ while not affecting selectin-mediated slow rolling. These findings may implicate a possible therapeutic role for lidocaine in decreasing the inappropriate activation, positioning, and recruitment of leukocytes during sepsis.

Immune Imbalance in Nasal Polyps of Caucasian Chronic Rhinosinusitis Patients Is Associated with a Downregulation of E-Selectin

Chronic rhinosinusitis with nasal polyps (CRSwNP) in Caucasians is a chronic Th2 inflammatory disease of the nasal and paranasal mucosa and the recruitment of leukocytes to the site of inflammation is poorly understood. We studied mRNA and protein expression profiles of adhesion molecules in nasal polyp and associated inferior turbinate tissues using molecular, biochemical, and immunohistological methods. Analysis showed a strongly decreased E-selectin expression in nasal polyps with a significant difference between eosinophil and neutrophil counts in nasal polyps and balanced counts in inferior turbinates. E-selectin expression is known to be downregulated in a Th2 milieu and has an essential role in immunosurveillance by locally activating neutrophil arrest and migratory function. A downregulation of E-selectin may come along with an immune imbalance in Caucasian nasal polyps due to a significant inhibition of neutrophil recruitment. Therefore, we suggest that an upregulation of E-selectin and the associated influx of neutrophils may play a significant role in the resolution of inflammation as well as for the pathophysiology of nasal polyps of Caucasian chronic rhinosinusitis patients.

Neutrophil Rolling on Patches of Selectin

For a neutrophil to reach an infection site, it circulates through the blood, adheres to the blood vessel wall, initially rolling, then adhering firmly; finally, the neutrophil actively passes through the wall. The initial rolling of a neutrophil is primarily mediated by adhesion of receptors on the neutrophil to selectins on the blood vessel wall. A neutrophil has hundreds of surface protrusions called microvilli, little fingers; most of the adhesion receptors involved in initial rolling are situated on the tips of microvilli. Here, I present theoretical analysis showing that the dynamics of a rolling neutrophil are qualitatively different depending on whether its microvilli are typically bound with single or multiple receptors. The role of multiple binding on a single microvillus increases with the surface concentration of selectin, up to a limit. P-selectin is ordinarily stored in granules in an endothelial cell, and released in patches on the cell membrane during inflammation. There is some experimental evidence suggesting additionally that patches of P-selectin are concentrated near the boundaries of endothelial cells. Here, a numerical model of neutrophil rolling which accounts for discrete receptor-ligand interactions is used to show how patchy distribution of P-selectin can promote neutrophil arrest and direct neutrophils to boundaries of endothelial cells, facilitating extravasation.

Elevated CXCL1 expression in gp130-deficient endothelial cells impairs neutrophil migration in mice

AbstractNeutrophils emigrate from venules to sites of infection or injury in response to chemotactic gradients. How these gradients form is not well understood. Some IL-6 family cytokines stimulate endothelial cells to express adhesion molecules and chemokines that recruit leukocytes. Receptors for these cytokines share the signaling subunit gp130. We studied knockout mice lacking gp130 in endothelial cells. Unexpectedly, gp130-deficient endothelial cells constitutively expressed more CXCL1 in vivo and in vitro, and even more upon stimulation with tumor necrosis factor-α. Mobilization of this increased CXCL1 from intracellular stores to the venular surface triggered β2 integrin–dependent arrest of neutrophils rolling on selectins but impaired intraluminal crawling and transendothelial migration. Superfusing CXCL1 over venules promoted neutrophil migration only after intravenously injecting mAb to CXCL1 to diminish its intravascular function or heparinase to release CXCL1 from endothelial proteoglycans. Remarkably, mice lacking gp130 in endothelial cells had impaired histamine-induced venular permeability, which was restored by injecting anti–P-selectin mAb to prevent neutrophil rolling and arrest. Thus, excessive CXCL1 expression in gp130-deficient endothelial cells augments neutrophil adhesion but hinders migration, most likely by disrupting chemotactic gradients. Our data define a role for endothelial cell gp130 in regulating integrin-dependent adhesion and de-adhesion of neutrophils during inflammation.Key Points

Kupffer cells and activation of endothelial TLR4 coordinate neutrophil adhesion within liver sinusoids during endotoxemia

A key pathological feature of the systemic inflammatory response of sepsis/endotoxemia is the accumulation of neutrophils within the microvasculature of organs such as the liver, where they cause tissue damage and vascular dysfunction. There is emerging evidence that the vascular endothelium is critical to the orchestration of inflammatory responses to blood-borne microbes and microbial products in sepsis/endotoxemia. In this study, we aimed to understand the role of endothelium, and specifically endothelial TLR4 activation, in the regulation of neutrophil recruitment to the liver during endotoxemia. Intravital microscopy of bone marrow chimeric mice revealed that TLR4 expression by non-bone marrow-derived cells was required for neutrophil recruitment to the liver during endotoxemia. Furthermore, LPS-induced neutrophil adhesion in liver sinusoids was equivalent between wild-type mice and transgenic mice that express TLR4 only on endothelium (tlr4(-/-)Tie2(tlr4)), revealing that activation of endothelial TLR4 alone was sufficient to initiate neutrophil adhesion. Neutrophil arrest within sinusoids of endotoxemic mice requires adhesive interactions between neutrophil CD44 and endothelial hyaluronan. Intravital immunofluorescence imaging demonstrated that stimulation of endothelial TLR4 alone was sufficient to induce the deposition of serum-derived hyaluronan-associated protein (SHAP) within sinusoids, which was required for CD44/hyaluronan-dependent neutrophil adhesion. In addition to endothelial TLR4 activation, Kupffer cells contribute to neutrophil recruitment via a distinct CD44/HA/SHAP-independent mechanism. This study sheds new light on the control of innate immune activation within the liver vasculature during endotoxemia, revealing a key role for endothelial cells as sentinels in the detection of intravascular infections and coordination of neutrophil recruitment to the liver.

Kinetic effects of TiO2 fine particles and nanoparticles aggregates on the nanomechanical properties of human neutrophils assessed by force spectroscopy

BackgroundIncreasing applications of titanium dioxide (TiO2) fine particles (FPs) and nanoparticles (NPs) require coupled knowledge improvement concerning their biokinetic effects. Neutrophils are quickly recruited to titanium implantation areas. Neutrophils mechanical properties display a crucial role on cell physiology and immune responsive functions. Then, micro and nanomechanical characterization assessed by force spectroscopy (FS) technique has been largely applied in this field.ResultsScanning electron microscopy (SEM) images highlighted neutrophils morphological changes along TiO2 FPs and NPs aggregates exposure time (1, 5, and 30 min) compared to controls. FS approaches showed an increasing on attraction forces to TiO2 FPs and NPs treated neutrophils. This group depicted stronger stiffness features than controls just at 1 min of exposure. Treated neutrophils showed a tendency to increase adhesive properties after 1 and 5 min of exposure. These cells maintained comparatively higher elasticity behavior for a longer time possibly due to intense phagocytosis and cell stiffness opposing to the tip indentation. Neutrophils activation caused by FPs and NPs uptake could be related to increasing dissipated energy results.ConclusionsMechanical modifications resulted from TiO2 FPs and NPs aggregates interaction with neutrophils showed increasing stiffness and also cell morphology alteration. Cells treatment by this metal FPs and NPs caused an increase in attractive forces. This event was mainly observed on the initial exposure times probably regarding to the interaction of neutrophils membrane and phagocytosis. Similar results were found to adhesion forces and dissipated energy outcomes. Treated cells presented comparatively higher elasticity behavior for a longer time. SEM images clearly suggested cell morphology alteration along time course probably related to activation, cytoskeleton rearrangement and phagocytosis. This scenario with increase in stiffness strongly suggests a direct relationship over neutrophil rolling, arrest, and transmigration. Scrutinizing these interactions represents an essential step to clarify the mechanisms involved on treatments containing micro and nanomaterials and their fates on the organisms.

Leukocyte Function Antigen-1, Kindlin-3, and Calcium Flux Orchestrate Neutrophil Recruitment during Inflammation

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.

Distinct roles for talin-1 and kindlin-3 in LFA-1 extension and affinity regulation

AbstractIn inflammation, neutrophils and other leukocytes roll along the microvascular endothelium before arresting and transmigrating into inflamed tissues. Arrest requires conformational activation of the integrin lymphocyte function-associated antigen-1 (LFA-1). Mutations of the FERMT3 gene encoding kindlin-3 underlie the human immune deficiency known as leukocyte adhesion deficiency-III. Both kindlin-3 and talin-1, another FERM domain-containing cytoskeletal protein, are required for integrin activation, but their individual roles in the induction of specific integrin conformers are unclear. Here, we induce differential LFA-1 activation in neutrophils through engagement of the selectin ligand P-selectin glycoprotein ligand-1 or the chemokine receptor CXCR2. We find that talin-1 is required for inducing LFA-1 extension, which corresponds to intermediate affinity and induces neutrophil slow rolling, whereas both talin-1 and kindlin-3 are required for induction of the high-affinity conformation of LFA-1 with an open headpiece, which results in neutrophil arrest. In vivo, both slow rolling and arrest are defective in talin-1–deficient neutrophils, whereas only arrest is defective in kindlin-3–deficient neutrophils. We conclude that talin-1 and kindlin-3 serve distinct functions in LFA-1 activation.

Distinct roles for talin‐1 and kindlin‐3 in LFA‐1‐dependent neutrophil rolling and arrest

β2 integrins, including LFA‐1 (αLβ2), play critical roles in leukocyte recruitment during inflammation by binding to ICAM‐1 expressed on the endothelium. The affinity of integrin for ligand is determined by its global conformation, regulated by the binding of proteins to the integrin cytoplasmic tails. At least three different integrin affinity states have been observed: low affinity with bent ectodomain, intermediate affinity with extended ectodomain, and high affinity with extended ectodomain and open headpiece. Intermediate affinity LFA‐1 contributes to neutrophil rolling interactions with the endothelium, whereas high affinity LFA‐1 mediates neutrophil arrest. In the current study, we determined the roles for two integrin activating proteins, talin‐1 and kindlin‐3, in neutrophil rolling and arrest. Using established in vivo (intravital microscopy of post‐capillary venules) and ex vivo (microfluidic flow chamber) models, we found that talin‐1 is necessary for both LFA‐1‐dependent neutrophil rolling and arrest. In contrast, kindlin‐3 was required only for neutrophil arrest and was dispensable for rolling mediated by intermediate affinity LFA‐1. We further corroborate our results with imaging and in vitro studies using antibodies that directly report β2 integrin conformation. These data indicate that talin‐1 is sufficient to induce LFA‐1 extension to an intermediate affinity state whereas kindlin‐3 is critical for the transition of LFA‐1 to a high affinity state.

Neutrophil motion in straight and tapering P‐selectin‐coated in vitro models of lung capillaries

Neutrophil sequestration in lung capillaries is a key step in the inflammatory response to lung infection. Neutrophils were aspirated into capillary‐sized glass microvessels of varying diameters and taper rates coated with soluble P‐selectin or BSA. Cell velocity distributions were measured for pressure differences thought to be present across single pulmonary capillary segments. A creeping motion was observed with velocity significantly lower on P‐selectin than BSA, and that decreased with increasing P‐selectin concentration. The gap width between neutrophil surface and microvessel wall decreased with increasing concentration of P‐selectin while the force exerted by the bonds on the cell increased. It was also shown that high concentrations of anti‐P‐selectin F(ab&amp;#900;)2 were effective to inhibit the P‐selectin mediated adhesion. These results demonstrate that even low densities of P‐selectin mediate neutrophil adhesion in the pulmonary capillary geometry. No mechanical arrest of neutrophils was observed in tapering vessels with taper angles of up to 9°, however, there is a transition point where cells slow down drastically, that does not depend on the P‐selectin concentrations, taper rate, or aspiration pressure. These results suggest that neutrophil arrest is primarily influenced by adhesion rather than mechanical trapping. This work was supported by grant BES‐0547165 from the NSF.

Distinct Roles for Talin-1 and Kindlin-3 in Regulating LFA-1 Conformation during Neutrophil Rolling and Arrest

β2 integrins, including LFA-1 (αLβ2), play critical roles in leukocyte trafficking. LFA-1 mediates the recruitment of neutrophils during inflammation through interaction with ICAM-1 expressed on the endothelium. The affinity of integrin for ligand is determined by its global conformation, regulated by the binding of proteins to the integrin cytoplasmic tails. At least three different integrin affinity states have been observed: low affinity with bent ectodomain, intermediate affinity with extended ectodomain, and high affinity with extended ectodomain and open headpiece. Intermediate affinity LFA-1 contributes to neutrophil rolling interactions with the endothelium, whereas high affinity LFA-1 mediates neutrophil arrest. In the current study, we determined the roles for two integrin tail-binding activating proteins, talin-1 and kindlin-3, in neutrophil rolling and arrest. Using established in vivo (intravital microscopy of post-capillary venules) and ex vivo (microfluidic flow chamber) models, we found that talin-1 is necessary for both LFA-1-dependent neutrophil rolling and arrest. In contrast, kindlin-3 is required only for neutrophil arrest and is dispensable for rolling mediated by intermediate affinity LFA-1. These data indicate that talin-1 is sufficient to induce LFA-1 extension to an intermediate affinity state whereas kindlin-3 is critical for the transition of LFA-1 to a high affinity state. We further corroborated our results with imaging and in vitro studies using antibodies that directly report β2 integrin conformation. Our results are the first to demonstrate that members of the talin and kindlin protein families play distinct roles in regulating integrin conformation.

Neutrophil arrest by LFA-1 activation

Lymphocyte function-associated antigen-1 (LFA-1) is a heterodimeric integrin consisting of αL (gene name, Itgal) and β2 (gene name, Itgb2) subunits expressed in all leukocytes. LFA-1 is essential for neutrophil recruitment to inflamed tissue. Activation of LFA-1 by chemokines allows neutrophils and other leukocytes to undergo arrest, resulting in firm adhesion on endothelia expressing intercellular adhesion molecules (ICAMs). In mice, CXCR2 is the primary chemokine receptor involved in triggering neutrophil arrest, and it does so through “inside-out” activation of LFA-1. CXCR2 signaling induces changes in LFA-1 conformation that are coupled to affinity upregulation of the ligand-binding headpiece (extended with open I domain). Unlike naïve lymphocytes, engagement of P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils stimulates a slow rolling behavior that is mediated by LFA-1 in a distinct activation state (extended with closed I domain). How inside-out signaling cascades regulate the structure and function of LFA-1 is being studied using flow chambers, intravital microscopy, and flow cytometry for ligand and reporter antibody binding. Here, we review how LFA-1 activation is regulated by cellular signaling and ligand binding. Two FERM domain-containing proteins, talin-1 and Kindlin-3, are critical integrin co-activators and have distinct roles in the induction of LFA-1 conformational rearrangements. This review integrates these new results into existing models of LFA-1 activation.

Leukocyte rolling and adhesion via ICAM-1 signals to endothelial permeability. Focus on “Leukocyte rolling and adhesion both contribute to regulation of microvascular permeability to albumin via ligation of ICAM-1”

THE VASCULAR ENDOTHELIUM plays a vital role in the inflammatory response by induction and surface expression of adhesion molecules and chemokines. At sites of acute inflammation, blood flow is increased and postcapillary venules exhibit increased permeability (leakiness) and support the influx of blood leukocytes, primarily neutrophils. The relationship between neutrophil influx and increased vessel permeability has been a topic of investigation for decades. Many investigations have reported a direct neutrophil-dependent increase in vessel permeability during inflammation using in vivo and in vitro models (reviewed in Ref. 4). One aspect that has received increased attention is whether endothelial cell adhesion molecules contribute to the regulation of vessel permeability, in addition to their well-characterized role in neutrophil rolling and adhesion. Intercellular adhesion molecule-1 (ICAM-1, also known as CD54) is an immunoglobulin superfamily member that is abundantly expressed on the endothelial cell surface and is enriched at endothelial cell borders in vivo and in vitro (2, 12, 14, 17). ICAM-1 surface expression is upregulated in endothelial cells by proinflammatory cytokines like TNF-, IL-1, IFN-, or bacterial endotoxins, and ICAM-1 serves as a receptor for leukocyte 2-integrins (LFA-1 and Mac-1) (reviewed in Ref. 15). In an experiment of nature, patients with leukocyte adhesion deficiency-1 (LAD-1) have a severe primary immune deficiency in which blood neutrophils fail to localize to sites of inflammation or injury. This defect occurs because patients’ neutrophils either lack 2-integrins or contain mutations in these molecules. Studies using neutrophils from LAD-1 patients or 2-integrin-deficient mice clearly demonstrate that these cells have normal selectin-dependent rolling on activated endothelium but fail to stably arrest and transmigrate. Additional studies have demonstrated that endothelial-expressed ICAM-1 contributes to both kinetics of leukocyte rolling and arrest in vivo as determined by intravital microscopy of the cremaster muscle microcirculation in ICAM-1 / mice (16). In this issue of American Journal of Physiology-Cell Physiology, Sumagin and colleagues (18) use elegant in vivo studies to demonstrate that microvascular permeability in the cremaster muscle model is regulated by leukocyte engagement of ICAM-1. The authors monitored permeability (Ps) in cremaster microcirculation by efflux of luminal fluorescent-tagged albumin by fluorescence confocal intravital microscopy. Prior studies by Sarelius and colleagues (19) in the same model demonstrated that ICAM-1 engagement induced a localized increase in vessel permeability in unstimulated arterioles and in TNF-stimulated venules. In agreement with this finding ,4ho f TNF- stimulation of arterioles and venules in ICAM-1 knockout (KO) animals did not result in increased vessel permeability as compared with wild-type (WT) mice under identical conditions. Similar results were seen with 2-integrin KO mice. In the current study, the authors provide further evidence linking ICAM-1 expression to regulation of endothelial permeability. Comparisons between unstimulated venules and TNF--treated venules reveal that neutrophil rolling regulates venule permeability in unstimulated venules while leukocyte adhesion regulates venule permeability in TNF--stimulated venules. The 2-integrin-blocking antibodies, which inhibit neutrophil arrest on endothelium, reduced the vessel permeability, and this was further reduced by treatment with a combination of 2-integrin and P-selectin blocking antibodies that inhibits both rolling and arrest. Interestingly, the reduction in venule permeability observed by blockade of P-selectin and 2-integrin function were suggested to be due to insufficient ICAM-1 engagement due to the absence of rolling leukocytes. To examine the role that neutrophils play, circulating neutrophils were depleted by antineutrophil GR-1 antibody treatment. Notably, depletion of neutrophils caused a marked decrease in

Migrational Guidance of Neutrophils Is Mechanotransduced via High-Affinity LFA-1 and Calcium Flux

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 β(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.

Stressed microvilli and long tethers in rolling, tight adhesion zones and aft trunks in arresting neutrophils revealed using Total Internal Reflection Fluorescence Microscopy (TIRFM)

Neutrophils are covered with microvilli. P‐selectin‐PSGL‐1 bonds are predicted to form when the microvillus tip approaches the P‐selectin substrate to within 70 nm. A microfluidic device with a substrate coated with P‐selectin/ICAM‐1 was perfused with blood from mice expressing GFP in neutrophils. Rolling interactions were studied at realistic wall shear stresses (τw = 6–8 dynes/cm2) using TIRFM. Following bond formation, microvilli undergo compression approaching the substrate to within 25 nm near the cell center. At the trailing edge, the P‐selectin‐PSGL‐1 bonds stretch to 125–150 nm before they dissociate. Rolling is stabilized by the formation of 3–4 long (up to 16 μm) tethers (life time~1–10s). Adding the chemokine CXCL1 to the substrate induces neutrophil arrest and formation of a trunk‐like structure that stretches up to 10 μm behind the cell. The closest contact (area~1–3 μm2) between the arrested neutrophil and the substrate is always found near the cell center. Its distance from the substrate (44 nm) corresponds to the length of the ICAM‐1‐LFA‐1 bond. These results identify the molecular and cellular dimensions of neutrophils during rolling and transition to arrest, thus providing a framework for the biomechanical analysis of this fundamental process. This work was supported by 09POST2230093 from American Heart Association (P.S) and NIH EB 02185 (K.L).

Stressed and Compressed Molecular Bonds Revealed in Footprints of Rolling Neutrophils using Total Internal Reflection Fluorescence Microscopy

Neutrophil recruitment to the sites of inflammation involves selectin-mediated rolling followed by chemokine-induced activation and beta2 integrin-mediated arrest. PSGL-1, a ligand for endothelial P-selectin is presented on the tips of neutrophil microvilli. It has been predicted that P-selectin-PSGL-1 bonds form when the microvillus tip approaches the P-selectin expressing substrate to within 70 nm, but this prediction has not been tested experimentally. A PDMS based microfluidic device with a glass substrate coated with P-selectin/ICAM-1 was perfused with blood from an anesthetized mouse expressing green fluorescent protein (GFP) in neutrophils. Rolling interactions were studied at wall shear stress of 6-8 dynes/cm2 using TIRF microscopy which provides high resolution in z-direction. The contact zones of rolling neutrophils were revealed as footprints which were 3-6 μm in diameter, about twice as large as what would be expected for spherical cells. Following bond formation, microvilli in the footprint undergo compression, approaching the substrate to within 25 nm near the center of the cell. At the trailing edge, the P-selectin-PSGL-1 bonds stretch to a length of 125-150 nm before they dissociate. Adding the chemokine CXCL1 to the substrate induced neutrophil arrest and formation of single, long, branched tethers that stretch for up to 10 μm behind the arrested cells. The closest contact between the arrested neutrophil and the substrate is always found in front of the cell center and covers 1-3 μm2. Its distance from the substrate (44 nm) corresponds to the length of the ICAM-1-LFA-1 bond. These results identify the molecular and cellular dimensions of rolling neutrophils and provide a framework for the biomechanical analysis of this fundamental process. This work was supported by a postdoctoral fellowship 09POST2230093 from American Heart Association (P.S) and NIH EB 02185 (K.L).

Calpain inhibition impairs TNF-α-mediated neutrophil adhesion, arrest and oxidative burst

Proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), are increased in many chronic inflammatory disorders, including rheumatoid arthritis, and contribute to recruitment of neutrophils into areas of inflammation. TNF-α induces a stop signal that promotes neutrophil firm adhesion and inhibits neutrophil polarization and chemotaxis. Calpain is a calcium-dependent protease that mediates cytoskeletal reorganization during cell migration. Here, we show that calpain inhibition impairs TNF-α-induced neutrophil firm adhesion to fibrinogen-coated surfaces and the formation of vinculin-containing focal complexes. Calpain inhibition induces random migration in TNF-α-stimulated cells and prevents the generation of reactive oxygen species, but does not alter TNF-α-mediated activation of p38 MAPK and ERK MAPK. These findings suggest that the TNF-α-induced neutrophil arrest requires the activity of calpain independent of p38 MAPK and ERK signaling seen after TNF-α stimulation. Together, our data suggest that therapeutic inhibition of calpain may be beneficial for limiting TNF-α-induced inflammatory responses.