Neutrophil Crawling Research Articles

A key role for platelet GPVI in neutrophil recruitment, migration, and NETosis in the early stages of acute lung injury

AbstractAcute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with high morbidity and mortality. Excessive neutrophil infiltration into the pulmonary airspace is the main cause for the acute inflammation and lung injury. Platelets have been implicated in the pathogenesis of ALI/ARDS, but the underlying mechanisms are not fully understood. Here, we show that the immunoreceptor tyrosine-based activation motif–coupled immunoglobulin-like platelet receptor, glycoprotein VI (GPVI), plays a key role in the early phase of pulmonary thrombo-inflammation in a model of lipopolysaccharide (LPS)-induced ALI in mice. In wild-type (WT) control mice, intranasal LPS application triggered severe pulmonary and blood neutrophilia, hypothermia, and increased blood lactate levels. In contrast, GPVI-deficient mice as well as anti-GPVI–treated WT mice were markedly protected from pulmonary and systemic compromises and showed no increased pulmonary bleeding. High-resolution multicolor microscopy of lung sections and intravital confocal microcopy of the ventilated lung revealed that anti-GPVI treatment resulted in less stable platelet interactions with neutrophils and overall reduced platelet–neutrophil complex (PNC) formation. Anti-GPVI treatment also reduced neutrophil crawling and adhesion on endothelial cells, resulting in reduced neutrophil transmigration and alveolar infiltrates. Remarkably, neutrophil activation was also diminished in anti-GPVI–treated animals, associated with strongly reduced formation of PNC clusters and neutrophil extracellular traps (NETs) compared with that in control mice. These results establish GPVI as a key mediator of neutrophil recruitment, PNC formation, and NET formation (ie, NETosis) in experimental ALI. Thus, GPVI inhibition might be a promising strategy to reduce the acute pulmonary inflammation that causes ALI/ARDS.

Endothelial Focal Adhesions Are Functional Obstacles for Leukocytes During Basolateral Crawling.

An inflammatory response requires leukocytes to migrate from the circulation across the vascular lining into the tissue to clear the invading pathogen. Whereas a lot of attention is focused on how leukocytes make their way through the endothelial monolayer, it is less clear how leukocytes migrate underneath the endothelium before they enter the tissue. Upon finalization of the diapedesis step, leukocytes reside in the subendothelial space and encounter endothelial focal adhesions. Using TIRF microscopy, we show that neutrophils navigate around these focal adhesions. Neutrophils recognize focal adhesions as physical obstacles and deform to get around them. Increasing the number of focal adhesions by silencing the small GTPase RhoJ slows down basolateral crawling of neutrophils. However, apical crawling and diapedesis itself are not affected by RhoJ depletion. Increasing the number of focal adhesions drastically by expressing the Rac1 GEF Tiam1 make neutrophils to avoid migrating underneath these Tiam1-expressing endothelial cells. Together, our results show that focal adhesions mark the basolateral migration path of neutrophils.

Priming of microvascular endothelium by microbiota-derived metabolites regulates neutrophil trafficking to the liver during sepsis

Abstract During systemic bacterial infections and sepsis, neutrophils are recruited en mass into the microcirculation of the liver and lungs as part of a coordinated intravascular response to defend the bloodstream against bacterial pathogens. The mechanisms that program this response have not been elucidated. The gut microbiota regulates immune cell development and function. In extra-intestinal organs, the microbiota conducts long-distance communication with immune cells through the release of metabolites into the bloodstream. While gut microbes augment granulopoiesis in the bone marrow, the role the microbiota and commensal-derived metabolites on neutrophil trafficking is poorly understood. Using confocal intravital microscopy, we observed that germ-free (GF) mice had impaired neutrophil recruitment to the liver, but not the lungs, during sepsis. Analysis of microbial metabolites in the gut, portal circulation (between gut and liver), and systemic circulation revealed that commensal-derived D-lactate levels are high in the gut and portal circulation, but nearly absent in the systemic circulation. Reconstitution of gut-to-liver D-lactate signalling by enteral administration restored neutrophil trafficking to the liver. In contrast, D-lactate did not impact neutrophil infiltration to the lungs, systemic levels of pro-inflammatory mediators or granulopoiesis. Instead, commensal D-lactate primed liver endothelium to express adhesion molecules required for neutrophil adhesion and crawling. Taken together, we show that compartmentalized molecular communication between gut microbes and the liver primes the microvascular endothelium for neutrophil recruitment in response to systemic bacterial infection and sepsis.

Antibody Opsonization Enhances Early Interactions between Yersinia pestis and Neutrophils in the Skin and Draining Lymph Node in a Mouse Model of Bubonic Plague.

Bubonic plague results when Yersinia pestis is deposited in the skin via the bite of an infected flea. Bacteria then traffic to the draining lymph node (dLN) where they replicate to large numbers. Without treatment, this infection can result in highly fatal septicemia. Several plague vaccine candidates are currently at various stages of development, but no licensed vaccine is available in the United States. Though polyclonal and monoclonal antibodies (Ab) can provide complete protection against bubonic plague in animal models, the mechanisms responsible for this antibody-mediated immunity (AMI) to Y. pestis remain poorly understood. Here, we examine the effects of Ab opsonization on Y. pestis interactions with phagocytes in vitro and in vivo Opsonization of Y. pestis with polyclonal antiserum modestly increased phagocytosis/killing by an oxidative burst of murine neutrophils in vitro Intravital microscopy (IVM) showed increased association of Ab-opsonized Y. pestis with neutrophils in the dermis in a mouse model of bubonic plague. IVM of popliteal LNs after intradermal (i.d.) injection of bacteria in the footpad revealed increased Y. pestis-neutrophil interactions and increased neutrophil crawling and extravasation in response to Ab-opsonized bacteria. Thus, despite only having a modest effect in in vitro assays, opsonizing Ab had a dramatic effect in vivo on Y. pestis-neutrophil interactions in the dermis and dLN very early after infection. These data shed new light on the importance of neutrophils in AMI to Y. pestis and may provide a new correlate of protection for evaluation of plague vaccine candidates.

Igniting the flame in arthritis: C5aR2 controls endothelial transcytosis of C5a.

C5aR2 transports C5a generated in the arthritic joint to the blood vessel endothelium as the first step in C5aR1-driven neutrophil arrest and crawling.

Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis

SummaryNeutrophils require directional cues to navigate through the complex structure of venular walls and into inflamed tissues. Here we applied confocal intravital microscopy to analyze neutrophil emigration in cytokine-stimulated mouse cremaster muscles. We identified differential and non-redundant roles for the chemokines CXCL1 and CXCL2, governed by their distinct cellular sources. CXCL1 was produced mainly by TNF-stimulated endothelial cells (ECs) and pericytes and supported luminal and sub-EC neutrophil crawling. Conversely, neutrophils were the main producers of CXCL2, and this chemokine was critical for correct breaching of endothelial junctions. This pro-migratory activity of CXCL2 depended on the atypical chemokine receptor 1 (ACKR1), which is enriched within endothelial junctions. Transmigrating neutrophils promoted a self-guided migration response through EC junctions, creating a junctional chemokine “depot” in the form of ACKR1-presented CXCL2 that enabled efficient unidirectional luminal-to-abluminal migration. Thus, CXCL1 and CXCL2 act in a sequential manner to guide neutrophils through venular walls as governed by their distinct cellular sources.

INHIBITION OF PROTEIN ARGININE DEIMINASES IMPROVES COGNITION AND REDUCES NEUROPATHOLOGICAL CHANGES IN MOUSE MODELS OF ALZHEIMER’S DISEASE

Inflammation has been correlated to Alzheimer's disease (AD) and a better understanding of its mechanisms may help to develop new therapies for AD. We have recently demonstrated that neutrophils infiltrate the brain and produce neutrophil extracellular traps (NETs) in subjects with AD and mice with AD-like disease. Furthermore, inhibition of neutrophil trafficking has therapeutic effect in animal models of AD. Recent studies have shown that protein arginine deiminases (PADs) mediate NET formation and have a role in innate immunity responses. PAD inhibitors have demonstrated remarkable efficacy in numerous inflammatory diseases including rheumatoid arthritis, lupus, ulcerative colitis, multiple sclerosis and atherosclerosis. The aim of our study was to test the potential therapeutic effect of PAD inhibition in AD models. BB-Cl-amidine, a pan-PAD inhibitor, and GSK199, a PAD4 specific inhibitor were used to study the role of PADs in vitro and in vivo. The role of PADs in rapid adhesion in vitro was assessed in adhesion assays on integrin ligands. Epifluorescence intravital microscopy and two-photon microscopy were performed to study the role of PADs in neutrophil adhesion in vivo. 5xFAD mice, which develop amyloid pathology, and 3xTg-AD mice, which develop both amyloid and tau pathology, were used to test the effect of PAD inhibition on disease. Treatment of neutrophils with PAD inhibitors reduced integrin-dependent rapid adhesion triggered by chemoattractants and TNF. PAD inhibition also reduced rolling interactions and drastically inhibited firm adhesion in inflamed brain microcirculation. Moreover, two-photon microscopy performed in the brain of AD mice showed a drastic reduction of adhesion and intravascular crawling of neutrophils treated with BB-Cl-amidine compared to untreated cells, suggesting that PADs control neutrophil trafficking in the AD brain. Notably, treatment with BB-Cl-amidine of 3xTg-AD and 5xFAD mice led to a significant restoration of memory in Y maze and contextual fear conditioning behavioral tests when compared to control animals. Neuropathological studies showed that treatment with BB-Cl-amidine reduced microglial activation and amyloid deposition compared to control animals. PAD inhibition interferes with neutrophil trafficking and ameliorates cognitive impairment and neuropathological changes in animal models of AD, suggesting PADs represent new therapeutic targets in AD.

Endothelial CD2AP Binds the Receptor ICAM-1 To Control Mechanosignaling, Leukocyte Adhesion, and the Route of Leukocyte Diapedesis In Vitro.

Inflammation is driven by excessive transmigration (diapedesis) of leukocytes from the blood to the tissue across the endothelial cell monolayer that lines blood vessels. Leukocyte adhesion, crawling, and transmigration are regulated by clustering of the endothelial mechanosensitive receptor intercellular adhesion molecule-1 (ICAM-1). Whereas several proteins are known to promote ICAM-1 function, the molecular mechanisms that limit ICAM-1-mediated adhesion to prevent excessive leukocyte transmigration remain unknown. We identify the endothelial actin-binding protein CD2-associated protein (CD2AP) as a novel interaction partner of ICAM-1. Loss of CD2AP stimulates the dynamics of ICAM-1 clustering, which facilitates the formation of ICAM-1 complexes on the endothelial cell surface. Consequently, neutrophil adhesion is increased, but crawling is decreased. In turn, this promotes the neutrophil preference for the transcellular over the paracellular transmigration route. Mechanistically, CD2AP is required for mechanosensitive ICAM-1 downstream signaling toward activation of the PI3K, and recruitment of F-actin and of the actin-branching protein cortactin. Moreover, CD2AP is necessary for ICAM-1-induced Rac1 recruitment and activation. Mechanical force applied on ICAM-1 impairs CD2AP binding to ICAM-1, suggesting that a tension-induced negative feedback loop promotes ICAM-1-mediated neutrophil crawling and paracellular transmigration. To our knowledge, these data show for the first time that the mechanoreceptor ICAM-1 is negatively regulated by an actin-binding adaptor protein, i.e., CD2AP, to allow a balanced and spatiotemporal control of its adhesive function. CD2AP is important in kidney dysfunction that is accompanied by inflammation. Our findings provide a mechanistic basis for the role of CD2AP in inflamed vessels, identifying this adaptor protein as a potential therapeutic target.

GEF-H1 is necessary for neutrophil shear stress-induced migration during inflammation.

Leukocyte crawling and transendothelial migration (TEM) are potentiated by shear stress caused by blood flow. The mechanism that couples shear stress to migration has not been fully elucidated. We found that mice lacking GEF-H1 (GEF-H1-/-), a RhoA-specific guanine nucleotide exchange factor (GEF), displayed limited migration and recruitment of neutrophils into inflamed tissues. GEF-H1-/- leukocytes were deficient in in vivo crawling and TEM in the postcapillary venules. We demonstrated that although GEF-H1 deficiency had little impact on the migratory properties of neutrophils under static conditions, shear stress triggered GEF-H1-dependent spreading and crawling of neutrophils and relocalization of GEF-H1 to flotillin-2-rich uropods. Our results identify GEF-H1 as a component of the shear stress response machinery in neutrophils required for a fully competent immune response to bacterial infection.

Identification and characterization of VEGF-A–responsive neutrophils expressing CD49d, VEGFR1, and CXCR4 in mice and humans

AbstractVascular endothelial growth factor A (VEGF-A) is upregulated during hypoxia and is the major regulator of angiogenesis. VEGF-A expression has also been found to recruit myeloid cells to ischemic tissues where they contribute to angiogenesis. This study investigates the mechanisms underlying neutrophil recruitment to VEGF-A as well as the characteristics of these neutrophils. A previously undefined circulating subset of neutrophils shown to be CD49d+VEGFR1highCXCR4high was identified in mice and humans. By using chimeric mice with impaired VEGF receptor 1 (VEGFR1) or VEGFR2 signaling (Flt-1tk−/−, tsad−/−), we found that parallel activation of VEGFR1 on neutrophils and VEGFR2 on endothelial cells was required for VEGF-A-induced recruitment of circulating neutrophils to tissue. Intravital microscopy of mouse microcirculation revealed that neutrophil recruitment by VEGF-A versus by the chemokine macrophage inflammatory protein 2 (MIP-2 [CXCL2]) involved the same steps of the recruitment cascade but that an additional neutrophil integrin (eg, VLA-4 [CD49d/CD29]) played a crucial role in neutrophil crawling and emigration to VEGF-A. Isolated CD49d+ neutrophils featured increased chemokinesis but not chemotaxis compared with CD49d– neutrophils in the presence of VEGF-A. Finally, by targeting the integrin α4 subunit (CD49d) in a transplantation-based angiogenesis model that used avascular pancreatic islets transplanted to striated muscle, we demonstrated that inhibiting the recruitment of circulating proangiogenic neutrophils to hypoxic tissue impairs vessel neoformation. Thus, angiogenesis can be modulated by targeting cell-surface receptors specifically involved in VEGF-A-dependent recruitment of proangiogenic neutrophils without compromising recruitment of the neutrophil population involved in the immune response to pathogens.

Cross-Talk between Shp1 and PIPKIγ Controls Leukocyte Recruitment.

Neutrophil recruitment to the site of inflammation plays a pivotal role in host defense. However, overwhelming activation and accumulation of neutrophils in the tissue may cause tissue damage and autoimmunity due to the release of cytokines, oxidants, and proteases. Neutrophil adhesion in acute inflammation is initiated by activation of αLβ2 (LFA-1), which can be induced by rolling on E-selectin (slowly) or by exposure to the chemokine CXCL1 (rapidly). Despite the clinical importance, cell-intrinsic molecular mechanisms of negative regulation of integrin adhesiveness and neutrophil recruitment are poorly understood. Mice deficient in the tyrosine phosphatase Src homology 2 domain-containing protein tyrosine phosphatase 1 (Shp1) show increased leukocyte adhesion, but the interpretation of these data is limited by the severe global phenotype of these mice. In this study, we used mice with global and myeloid-restricted deletion of Shp1 to study neutrophil arrest, adhesion, crawling, and transendothelial migration in vitro and in vivo. Shp1 deficiency results in increased neutrophil adhesion in vivo; however, neutrophil crawling, transmigration, and chemotaxis were reduced in these mice. Mechanistically, Shp1 binds and controls PIPKIγ activity and, thereby, modulates phosphatidylinositol (4,5)-bisphosphate levels and adhesion. Thus, Shp1 is involved in the deactivation of integrins and regulation of neutrophil recruitment into inflamed tissue.

Mutation in the CD45 inhibitory wedge modulates integrin activation and leukocyte recruitment during inflammation.

Neutrophil recruitment to the site of inflammation plays a pivotal role in host defense. Src family kinases (SFKs) activation is required for integrin and chemokine signaling as well as immune cell function. The receptor-like protein tyrosine phosphatase CD45 positively regulates chemoattractant signaling acting on SFK activity. To further investigate the role of CD45 in neutrophil recruitment and function, we analyzed transgenic mice carrying a single point mutation (CD45E613R), which constitutively activates CD45. By using intravital microscopy experiments, we demonstrated that different steps of the leukocyte recruitment cascade were affected in CD45E613R mutant mice. The rolling velocity of CD45E613R mutant neutrophils was decreased compared with wild-type neutrophils that subsequently resulted in an increased number of adherent cells. The analysis of β2 integrins LFA-1 and macrophage-1 Ag (Mac-1) showed that in CD45E613R mutant neutrophils LFA-1 adhesiveness was impaired, and avidity was enhanced, whereas Mac-1 adhesiveness was increased. Because of the increased Mac-1 adhesiveness, neutrophil crawling was impaired in CD45E613R mutant compared with wild-type neutrophils. In an Escherichia coli lung infection model, CD45E613R mice displayed a decreased neutrophil recruitment into the alveolar compartment, which resulted in an increased number of CFUs in the lung. Our data demonstrate that the CD45E613R mutation modulates integrin activation and leukocyte recruitment during inflammation.

The physiological roles of ICAM-1 and ICAM-2 in neutrophil migration into tissues.

Neutrophil extravasation from the blood into tissues is initiated by tethering and rolling of neutrophils on endothelial cells, followed by neutrophil integrin activation and shear resistant arrest, crawling, diapedesis and breaching the endothelial basement membrane harbouring pericytes. Endothelial intercellular cell adhesion molecule (ICAM)-1 and ICAM-2, in conjunction with ICAM-1 on pericytes, critically contribute to each step. In addition, epithelial ICAM-1 is involved in neutrophil migration to peri-epithelial sites. The most recent findings on the role of ICAM-1 and ICAM-2 for neutrophil migration into tissues will be reviewed here. Signalling via endothelial ICAM-1 and ICAM-2 contributes to stiffness of the endothelial cells at sites of chronic inflammation and junctional maturation, respectively. Endothelial ICAM-2 contributes to neutrophil crawling and initiation of paracellular diapedesis, which then proceeds independent of ICAM-2. Substantial transcellular neutrophil diapedesis across the blood-brain barrier is strictly dependent on endothelial ICAM-1 and ICAM-2. Endothelial ICAM-1 or ICAM-2 is involved in neutrophil-mediated plasma leakage. ICAM-1 on pericytes assists the final step of neutrophil extravasation. Epithelial ICAM-1 rather indirectly promotes neutrophil migration to peri-epithelial sites. ICAM-1 and ICAM-2 are involved in each step of neutrophil extravasation, and have redundant but also distinct functions. Analysis of the role of endothelial ICAM-1 requires simultaneous consideration of ICAM-2.

Neutrophil crawling in capillaries; a novel immune response to Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA), particularly the USA300 strain, is a highly virulent pathogen responsible for an increasing number of skin and soft tissue infections globally. Furthermore, MRSA-induced soft tissue infections can rapidly progress into life-threatening conditions, such as sepsis and necrotizing fasciitis. The importance of neutrophils in these devastating soft tissue infections remains ambiguous, partly because of our incomplete understanding of their behaviour. Spinning disk confocal microscopy was used to visualize the behaviour of GR1-labelled neutrophils in subcutaneous tissue in response to GFP-expressing MRSA attached to a foreign particle (agarose bead). We observed significant directional neutrophil recruitment towards the S. aureus agarose bead but not a control agarose bead. A significant increase in neutrophil crawling within the capillaries surrounding the infectious nidus was noted, with impaired capillary perfusion in these vessels and increased parenchymal cell death. No neutrophils were able to emigrate from capillaries. The crawling within these capillaries was mediated by the β2 and α4 integrins and blocking these integrins 2 hours post infection eliminated neutrophil crawling, improved capillary perfusion, reduced cell death and reduced lesion size. Blocking prior to infection increased pathology. Neutrophil crawling within capillaries during MRSA soft tissue infections, while potentially contributing to walling off or preventing early dissemination of the pathogen, resulted in impaired perfusion and increased tissue injury with time.

Ultra structure analysis of cell–cell interactions between pericytes and neutrophils in vitro

Neutrophils’ adhesion to the endothelium during inflammatory is a well-known processes. In contrast the interaction of neutrophils with cells of the neurovascular unit after they have been transmigrated into the brain is less clear. Recently, lymphocyte function-associated antigen-1 (LFA-1) dependent subendothelial crawling of neutrophils has been observed in vivo. This is mediated by intracellular adhesion molecule-1 (ICAM-1), which is expressed on the cell surface of pericytes. In our work we demonstrated in vitro a cell–cell interaction between porcine brain capillary pericytes (PBCPs) and neutrophils, with further characterization of the initial contact between these cells. PBCPs increase ICAM-1 protein expression in response to the cytokine tumor necrosis factor-alpha (TNF-α). Furthermore, an increase in neutrophil adhesion to PBCPs was determined by immunofluorescence staining. By means of scanning force microscopy (SFM), we could additionally show that pericytes as well as neutrophils form cell extensions towards the neighboring cell. Interestingly, these extensions differ for different cell types.

Pharmacological inhibition of p38 mitogen‐activated protein kinases affects KC/CXCL1‐induced intraluminal crawling, transendothelial migration, and chemotaxis of neutrophils in vivo

Leukocyte recruitment to the inflammation site is a multi‐step process governed by specific signalling cascades. After adhesion in the lumen, leukocytes crawl to optimal sites at endothelial junctions and transmigrate to extravascular tissue in a Mac‐1‐dependent manner. The signalling mechanisms that regulate post‐adhesion steps of intraluminal crawling, transmigration and chemotaxis in tissue remain incompletely understood. The present study explored the effect of p38 MAPK inhibitor SB203580 on various steps of neutrophil recruitment triggered by chemokine KC (CXCL1) gradient in microvasculature of murine cremaster muscle using intravital microscopy and time‐lapsed video analysis. SB203580 (100 nM) did not change leukocyte rolling but significantly attenuated neutrophil adhesion, emigration, transmigration time and detachment time, and impaired the initiation of crawling and transmigration. In response to KC chemotactic gradient, SB203580 significantly reduced the velocity of migration and chemotaxis index of neutrophils in tissue. The upregulation of Mac‐1 expression in neutrophils stimulated by KC was significantly blunted by SB203580 in vitro. Collectively, our findings demonstrate that pharmacological suppression of p38 MAPK significantly impairs multiple steps of neutrophil recruitment in vivo.

ICAM-2 facilitates luminal neutrophil-endothelial cell interactionsin vivo

ABSTRACTIntercellular adhesion molecule 2 (ICAM-2) is expressed on endothelial cells (ECs) and supports neutrophil extravasation. However, the full details of its role remain unknown, and the present study investigates the functional mechanisms of ICAM-2 in neutrophil–endothelial-cell interactions. Our initial studies showed expression of ICAM-2 at both EC junctions and on the EC body. In line with the observed expression profile analysis of neutrophil–vessel-wall interactions using real-time in vivo confocal microscopy identified numerous functional roles for ICAM-2 within the vascular lumen and at the stage of neutrophil extravasation. Functional or genetic blockade of ICAM-2 significantly reduced neutrophil crawling velocity, increased frequency of crawling with a disrupted stop-start profile, and prolonged interaction of neutrophils with EC junctions prior to transendothelial cell migration (TEM), collectively resulting in significantly reduced extravasation. Pharmacological blockade of the leukocyte integrin MAC-1 indicated that some ICAM-2-dependent functions might be mediated through ligation of this integrin. These findings highlight novel roles for ICAM-2 in mediating luminal neutrophil crawling and the effect on subsequent levels of extravasation.

Pharmacological Inhibition of p38 Mitogen-Activated Protein Kinases Affects KC/CXCL1-Induced Intraluminal Crawling, Transendothelial Migration, and Chemotaxis of NeutrophilsIn Vivo

p38 mitogen-activated protein kinase (MAPK) signalling is critical in the pathophysiology of a variety of inflammatory processes. Leukocyte recruitment to the site of inflammation is a multistep process governed by specific signalling cascades. After adhesion in the lumen, many leukocytes crawl to optimal sites at endothelial junctions and transmigrate to extravascular tissue in a Mac-1-dependent manner. The signalling mechanisms that regulate postadhesion steps of intraluminal crawling, transmigration, and chemotaxis in tissue remain incompletely understood. The present study explored the effect of p38 MAPK inhibitor SB203580 on various parameters of neutrophil recruitment triggered by chemokine KC (CXCL1) gradient. Neutrophil-endothelial interactions in microvasculature of murine cremaster muscle were determined using intravital microscopy and time-lapsed video analysis. SB203580 (100 nM) did not change leukocyte rolling but significantly attenuated neutrophil adhesion, emigration, and transmigration and impaired the initiation of neutrophil crawling and transmigration. In response to KC chemotactic gradient, SB203580 significantly reduced the velocity of migration and chemotaxis index of neutrophils in tissue. The upregulation of Mac-1 expression in neutrophils stimulated by KC was significantly blunted by SB203580 in vitro. Collectively, our findings demonstrate that pharmacological suppression of p38 MAPK significantly impairs multiple steps of neutrophil recruitment in vivo.

Neutrophil Interactions With ICAM-1 at the Luminal Surface of the Intestinal Epithelium Enhance Neutrophil Survival and Alter Epithelial Barrier Function

Neutrophil transepithelial migration (TEM) is a hallmark of inflammatory bowel disease (IBD), and is associated with epithelial injury and impaired barrier function. While TEM has been extensively modeled, little is known about neutrophil interactions with ligands expressed on the luminal surface of the intestinal epithelium. ICAM-1 is a well-recognized signaling receptor and endothelial ligand for migrating neutrophils. Its expression is also induced on the luminal (apical) membrane of intestinal epithelial cells (IEC) exposed to interferon γ (IFNγ), and in the colonic mucosa of individuals with active IBD. However, the role of ICAM-1 in mediating functional effects of neutrophil interactions with intestinal epithelial cells is undefined. ICAM-1 expression in cultured IECs and mouse intestinal epithelium was induced by exposure to IFNγ (100U/mL, 24h) and IFNγ+TNFα (500ng each, 24h, ip) respectively, and analyzed by flow cytometry and immunofluorescence. The effects of neutrophil-IEC interactions, and specifically of ICAM-1 engagement by antibody crosslinking on the regulation of epithelial barrier function was examined in T84 IECs by measuring changes in transepithelial resistance (TER) and permeability to 4kDa FITC-Dextran, and by using an intestinal loop model from anesthetized mice, measuring absorption of FITC-dextran from the intestinal lumen into the circulation. We confirmed upregulation of ICAM-1 expression in IFNγ treated T84 cells, in the intestinal epithelium of patients with IBD, and further showed induction in ICAM-1 expression in-vivo in murine small intestine pre-treated with IFNγ and TNFa. In cultured IECs, expression of ICAM-1 enhanced neutrophil adhesion to the apical epithelial membrane. The majority (˜60%) of apically adhered neutrophil that completed TEM were found to exhibit luminal crawling behavior, which was significantly enhanced (>33%) when T84 cells were pre-exposed to IFNγ. Neutrophil interactions with apical epithelial membrane of IFNγ pretreated IECs also resulted in enhanced neutrophil survival as evident by decreased apoptosis. The effects on neutrophil crawling and apoptosis were ICAM-1-dependent and were reversed by treatment with an anti-ICAM-1 function-blocking antibody. Furthermore, neutrophil interactions with the apical epithelial membrane resulted in time-dependent decrease in TER. This decrease was prevented by inhibition of neutrophil adhesion and crawling using an anti-CD11b/CD18 function-blocking antibody, suggesting that neutrophil induced effects on barrier function were due to direct engagement of ligands on epithelial surface. Experiments modeling neutrophil apical engagement using antibody-mediated crosslinking of cell surface ICAM-1 in cultured IECs and in-vivo, using murine intestinal loop model confirmed a direct role for ICAM-1 engagement in regulating epithelial barrier function. Antibody crosslinking of ICAM-1 resulted in time-dependent decrease in TER (up to 30%) and increased flux of FITC-dextran (˜2.5-fold at 4 hours) in-vitro and increased flux of FITC-dextran (˜1.6-fold) in-vivo. ICAM-1 mediated changes in barrier function were accompanied by actin rearrangement and dependent on MLCK. These studies suggest that neutrophil engagement of apically expressed ICAM-1during IBD results in signaling events that can inhibit neutrophil clearance and alter barrier. Such interactions may contribute to further recruitment of inflammatory cells and impede resolution of inflammation. Thus, targeting ICAM-1 in the intestinal epithelium may provide new/improved therapeutic approaches for treating IBD.

The Mammalian Actin-Binding Protein 1 Is Critical for Spreading and Intraluminal Crawling of Neutrophils under Flow Conditions

Recently, the mammalian actin-binding protein 1 (mAbp1; Hip-55, SH3P7, debrin-like protein) was identified as a novel component of the β(2) integrin-mediated signaling cascade during complement-mediated phagocytosis and firm adhesion of polymorphonuclear neutrophils (PMN) under physiological shear stress conditions. In this study, we found that the genetic ablation of mAbp1 severely compromised not only the induction of adhesion, but also subsequent spreading of leukocytes to the endothelium as assessed by intravital microscopy of inflamed vessels of the cremaster muscle of mice. In vitro studies using murine PMN confirmed that mAbp1 was required for β(2) integrin-mediated spreading under shear stress conditions, whereas mAbp1 was dispensable for spreading under static conditions. Upon β(2) integrin-mediated adhesion and chemotactic migration of human neutrophil-like differentiated HL-60 cells, mAbp1 was enriched at the leading edge of the polarized cell. Total internal reflection fluorescence microscopy revealed that mAbp1 formed propagating waves toward the front of the lamellipodium, which are characteristic for dynamic reorganization of the cytoskeleton. Accordingly, binding of mAbp1 to actin was increased upon β(2) integrin-mediated adhesion, as shown by coimmunoprecipitation experiments. However, chemotactic migration under static conditions was unaffected in the absence of mAbp1. In contrast, the downregulation of mAbp1 by RNA interference technique in neutrophil-like differentiated HL-60 cells or the genetic ablation of mAbp1 in leukocytes led to defective migration under flow conditions in vitro and in inflamed cremaster muscle venules in the situation in vivo. In conclusion, mAbp1 is of fundamental importance for spreading and migration under shear stress conditions, which are critical prerequisites for efficient PMN extravasation during inflammation.