Firm Adhesion Of Neutrophils Research Articles

Autophagy Induced by Palmitic Acid Regulates Neutrophil Adhesion Through the Granule-Dependent Degradation of αMβ2 Integrin in Dairy Cows With Fatty Liver.

β2 integrins are critical for neutrophil firm adhesion, trans-endothelial migration, and the recruitment to the inflamed tissue. Autophagy is implicated in cell migration and tumor metastasis through facilitating the turnover of β1 integrins; however, whether autophagy is able to control neutrophil migration by promoting the degradation of β2 integrins is unexplored. Here, we show that high blood levels of palmitic acid (PA) strongly triggered neutrophil autophagy activation, leading to adhesion deficiency in dairy cows with fatty liver. The three neutrophil granule subtypes, namely, azurophil granules (AGs), specific granules (SGs), and gelatinase granules (GGs), were engulfed by the autophagosomes for degradation, resulting in an increased vacuolation in fatty liver dairy cow neutrophils. Importantly, the adhesion-associated molecules CD11b and CD18 distributed on AGs, SGs, and GGs were degraded with the three granule subtypes by autophagy. Moreover, FGA, Hsc70, and TRIM21 mediated the degradation of cytosolic oxidized–ubiquitinated CD11b and CD18. Collectively, our results demonstrate that high blood PA triggers neutrophil autophagy-dependent vacuolation and granule-dependent adhesion deficiency, decreasing neutrophil mobility, and impairing the innate immune system of dairy cow with fatty liver. This theory extends the category of autophagy in maintaining granule homeostasis and provides a novel strategy to improve the immune of dairy cows with metabolic disease.

Neutrophil-derived heparin binding protein triggers vascular leakage and synergizes with myeloperoxidase at the early stage of severe burns (With video).

BackgroundBurn shock caused by vascular leakage is one of the main causes of high mortality in severe burn injury. However, the pathophysiological mechanism of vascular leakage is still unclear. The purpose of this study was to explore the molecular mechanism of vascular leakage in the early stage of severe burn and provide a new target for the treatment of severe burns.MethodsNeutrophils were isolated from human peripheral blood by magnetic beads sorting. ELISA was used to detect neutrophil-derived granule proteins and glycocalyx injury products in plasma. The vascular leakage and neutrophil movement were assessed by in vivo laser confocal imaging in mice, and high-quality video were provided. Adhesion-related molecules were investigated by qRT-PCR. The damage to glycocalyx of mice vascular endothelial cells was observed by transmission electron microscope and scanning electron microscope. Proteomic analysis, flow cytometry and immunofluorescence were used to further study the relationship between human peripheral blood neutrophil-derived hypochlorite (HOCl) and CD44 of human vascular endothelial cells.ResultsIn this study, we found that rapidly increasing activated neutrophils secrete heparin binding protein (HBP) and myeloperoxidase (MPO) after severe burn injury. Increased HBP triggers vascular leakage with synergy of MPO, results in systemic edema and burn shock. Furthermore, we found that the MPO catalytic product HOCl but not MPO triggers CD44 extracellular domain shedding from vascular endothelial cells to damage the glycocalyx. Damage to the glycocalyx results in firm adhesion of neutrophils and increases vascular leakage. However, MPO inhibitors partially protect the glycocalyx of vascular endothelial cells. The combination of HBP and MPO inhibitors markedly reduces vascular leakage and systemic edema in the early stage of severe burns.ConclusionsTaken together, these data reveal that neutrophil-derived HBP and MPO play an important synergies role in triggering vascular leakage at the early stage of severe burns. Targeted intervention in these two biomolecules may introduce new strategies for helping to reduce large amount of fluid loss and subsequent burn shock.

Small GTPase ARF6 Is a Coincidence-Detection Code for RPH3A Polarization in Neutrophil Polarization.

Cell polarization is a key step for leukocytes adhesion and transmigration during leukocytes' inflammatory infiltration. Polarized localization of plasma membrane (PM) phosphatidylinositol-4-phosphate (PtdIns4P) directs the polarization of RPH3A, which contains a PtdIns4P binding site. Consequently, RPH3A mediates the RAB21 and PIP5K1C90 polarization, which is important for neutrophil adhesion to endothelia during inflammation. However, the mechanism by which RPH3A is recruited only to PM PtdIns4P rather than Golgi PtdIns4P remains unclear. By using ADP-ribosylation factor 6 (ARF6) small interfering RNA, ARF6 dominant-negative mutant ARF6(T27N), and ARF6 activation inhibitor SecinH3, we demonstrate that ARF6 plays an important role in the polarization of RPH3A, RAB21, and PIP5K1C90 in murine neutrophils. PM ARF6 is polarized and colocalized with RPH3A, RAB21, PIP5K1C90, and PM PtdIns4P in mouse and human neutrophils upon integrin stimulation. Additionally, ARF6 binds to RPH3A and enhances the interaction between the PM PtdIns4P and RPH3A. Consistent with functional roles of polarization of RPH3A, Rab21, and PIP5K1C90, ARF6 is also required for neutrophil adhesion on the inflamed endothelial layer. Our study reveals a previously unknown role of ARF6 in neutrophil polarization as being the coincidence-detection code with PM PtdIns4P. Cooperation of ARF6 and PM PtdIns4P direct RPH3A polarization, which is important for neutrophil firm adhesion to endothelia.

Microvascular Mechanisms of Polyphosphate-Induced Neutrophil-Endothelial Cell Interactions in vivo

Background: Polyphosphates (PolyPs) have been reported to exert pro-inflammatory effects. However, the molecular mechanisms regulating PolyP-provoked tissue accumulation of leukocytes are not known. The aim of the present investigation was to determine the role of specific adhesion molecules in PolyP-mediated leukocyte recruitment. Methods: PolyPs and TNF-α were intrascrotally administered, and anti-P-selectin, anti-E-selectin, anti-P-selectin glycoprotein ligand-1 (PSGL-1), anti-membrane-activated complex-1 (Mac-1), anti-lymphocyte function antigen-1 (LFA-1), and neutrophil depletion antibodies were injected intravenously or intraperitoneally. Intravital microscopy of the mouse cremaster microcirculation was used to examine leukocyte-endothelium interactions and recruitment in vivo. Results: Intrascrotal injection of PolyPs increased leukocyte accumulation. Depletion of neutrophils abolished PolyP-induced leukocyte-endothelium interactions, indicating that neutrophils were the main leukocyte subtype responding to PolyP challenge. Immunoneutralization of P-selectin and PSGL-1 abolished PolyP-provoked neutrophil rolling, adhesion, and emigration. Moreover, immunoneutralization of Mac-1 and LFA-1 had no impact on neutrophil rolling but markedly reduced neutrophil adhesion and emigration evoked by PolyPs. Conclusion: These results suggest that P-selectin and PSGL-1 exert important roles in PolyP-induced inflammatory cell recruitment by mediating neutrophil rolling. In addition, our data show that Mac-1 and LFA-1 are necessary for supporting PolyP-triggered firm adhesion of neutrophils to microvascular endothelium. These novel findings define specific molecules as potential targets for pharmacological intervention in PolyP-dependent inflammatory diseases.

Endothelial cell SHP-2 negatively regulates neutrophil adhesion and promotes transmigration by enhancing ICAM-1-VE-cadherin interaction.

Intercellular adhesion molecule-1 (ICAM-1) mediates the firm adhesion of leukocytes to endothelial cells and initiates subsequent signaling that promotes their transendothelial migration (TEM). Vascular endothelial (VE)-cadherin plays a critical role in endothelial cell-cell adhesion, thereby controlling endothelial permeability and leukocyte transmigration. This study aimed to determine the molecular signaling events that originate from the ICAM-1-mediated firm adhesion of neutrophils that regulate VE-cadherin's role as a negative regulator of leukocyte transmigration. We observed that ICAM-1 interacts with Src homology domain 2-containing phosphatase-2 (SHP-2), and SHP-2 down-regulation via silencing of small interfering RNA in endothelial cells enhanced neutrophil adhesion to endothelial cells but inhibited neutrophil transmigration. We also found that VE-cadherin associated with the ICAM-1-SHP-2 complex. Moreover, whereas the activation of ICAM-1 leads to VE-cadherin dissociation from ICAM-1 and VE-cadherin association with actin, SHP-2 down-regulation prevented ICAM-1-VE-cadherin association and promoted VE-cadherin-actin association. Furthermore, SHP-2 down-regulation in vivo promoted LPS-induced neutrophil recruitment in mouse lung but delayed neutrophil extravasation. These results suggest that SHP-2-via association with ICAM-1-mediates ICAM-1-induced Src activation and modulates VE-cadherin switching association with ICAM-1 or actin, thereby negatively regulating neutrophil adhesion to endothelial cells and enhancing their TEM.-Yan, M., Zhang, X., Chen, A., Gu, W., Liu, J., Ren, X., Zhang, J., Wu, X., Place, A. T., Minshall, R. D., Liu, G. Endothelial cell SHP-2 negatively regulates neutrophil adhesion and promotes transmigration by enhancing ICAM-1-VE-cadherin interaction.

Neutrophils mediate blood-spinal cord barrier disruption in demyelinating neuroinflammatory diseases.

Disruption of the blood-brain and blood-spinal cord barriers (BBB and BSCB, respectively) and immune cell infiltration are early pathophysiological hallmarks of multiple sclerosis (MS), its animal model experimental autoimmune encephalomyelitis (EAE), and neuromyelitis optica (NMO). However, their contribution to disease initiation and development remains unclear. In this study, we induced EAE in lys-eGFP-ki mice and performed single, nonterminal intravital imaging to investigate BSCB permeability simultaneously with the kinetics of GFP(+) myeloid cell infiltration. We observed a loss in BSCB integrity within a day of disease onset, which paralleled the infiltration of GFP(+) cells into the CNS and lasted for ∼4 d. Neutrophils accounted for a significant proportion of the circulating and CNS-infiltrating myeloid cells during the preclinical phase of EAE, and their depletion delayed the onset and reduced the severity of EAE while maintaining BSCB integrity. We also show that neutrophils collected from the blood or bone marrow of EAE mice transmigrate more efficiently than do neutrophils of naive animals in a BBB cell culture model. Moreover, using intravital videomicroscopy, we demonstrate that the IL-1R type 1 governs the firm adhesion of neutrophils to the inflamed spinal cord vasculature. Finally, immunostaining of postmortem CNS material obtained from an acutely ill multiple sclerosis patient and two neuromyelitis optica patients revealed instances of infiltrated neutrophils associated with regions of BBB or BSCB leakage. Taken together, our data provide evidence that neutrophils are involved in the initial events that take place during EAE and that they are intimately linked with the status of the BBB/BSCB.

Human neutrophil flow chamber adhesion assay.

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the β2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both β2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the β2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both β2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

Phosphodiesterase type 4 blockade prevents platelet-mediated neutrophil recruitment at the site of vascular injury.

Platelet-neutrophil interactions play a key role in cardiovascular disease and inflammatory processes. Src family kinases mediate P-selectin glycoprotein ligand-1-Mac-1 cross talk necessary for firm platelet-neutrophil adhesion. Because Src family kinase activity can be regulated by cAMP-dependent pathways, in this work, we evaluated the role of phosphodiesterases in the signaling events that are required to sustain platelet-neutrophil interactions and neutrophil recruitment at the site of vascular injury. In neutrophils exposed to P-selectin, selective phosphodiesterase 4 (PDE4) inhibition prevented Src family kinase-mediated phosphorylation of the proline-rich tyrosine kinase 2 on Tyr579/Tyr580. The effects of PDE4 inhibition required protein kinase A, likely through protein kinase A-mediated activation of COOH-terminal Src kinase, a major negative regulator of Src family kinases. PDE4, but not other phosphodiesterase inhibitors, reduced platelet-neutrophil conjugates as well as neutrophil firm adhesion on spread platelets under flow conditions. The effect of PDE4 inhibition on neutrophil adhesion was primarily mediated by downregulation of P-selectin-induced activation of Mac-1. In a murine model of endovascular injury, selective inhibition of PDE4 significantly reduced neutrophil recruitment at the site of vascular damage. This study identifies PDE4 as a central node in the signaling network that mediates platelet-neutrophil adhesion and suggests that pharmacological inhibition of PDE4 may represent a novel therapeutic avenue for the treatment of cardiovascular disease.

Exacerbated tissue homing of neutrophils during sepsis and TLR2-induced cytokine production are regulated by integrin α3β1. (CAM5P.230)

Abstract Neutrophil migration to sites of infection is vital for pathogen clearance, but massive neutrophil infiltration during systemic inflammation leads to tissue injury and host mortality. While β2 integrins are well known to mediate neutrophil firm adhesion and crawling, the role of β1 integrins in vascular extravasation has been less explored. In this study, we evaluated the surface expression kinetics of β1 and β3 integrin heterodimers on neutrophils during sepsis using mouse models (CLP and endotoxemia) and human samples. We show that only integrin α3β1 becomes significantly upregulated. Moreover, human subjects with sepsis, but not SIRS (systemic inflammation w/o infection) upregulated α3β1. The increased expression of α3β1 on neutrophils correlated with their activation state. Administration of synthetic peptide (LXY2), which binds at the ligand binding site on α3β1, and conditional gene deletion of the integrin in granulocytes significantly reduced the number of extravasated neutrophils and improved mouse survival. Host bacterial clearance remained unaffected. Interestingly, LXY2 binding to α3β1 on neutrophils was associated with a reduction in TLR2-, but not TLR4-induced IL-10 secretion in vitro. Previous studies suggest a role for IL-10 as a regulator of the transition from mild sepsis to irreversible septic shock. Thus, integrin α3β1 upregulation promotes enhanced neutrophil migration and, in turn, could regulate sepsis progression by modulating neutrophil IL-10 release.

Multiple Lupus‐Associated ITGAM Variants Alter Mac‐1 Functions on Neutrophils

Multiple studies have demonstrated that single-nucleotide polymorphisms (SNPs) in the ITGAM locus (including the nonsynonymous SNPs rs1143679, rs1143678, and rs1143683) are associated with systemic lupus erythematosus (SLE). ITGAM encodes the protein CD11b, a subunit of the β2 integrin Mac-1. The purpose of this study was to determine the effects of ITGAM genetic variation on the biologic functions of neutrophil Mac-1. Neutrophils from ITGAM-genotyped and -sequenced healthy donors were isolated for functional studies. The phagocytic capacity of neutrophil ITGAM variants was probed with complement-coated erythrocytes, serum-treated zymosan, heat-treated zymosan, and IgG-coated erythrocytes. The adhesion capacity of ITGAM variants, in adhering to either purified intercellular adhesion molecule 1 or tumor necrosis factor α-stimulated endothelial cells, was assessed in a flow chamber. Expression levels of total CD11b and activation of CD11b were assessed by flow cytometry. Mac-1-mediated neutrophil phagocytosis, determined in cultures with 2 different complement-coated particles, was significantly reduced in individuals with nonsynonymous variant alleles of ITGAM. This reduction in phagocytosis was related to variation at either rs1143679 (in the β-propeller region) or rs1143678/rs1143683 (highly linked SNPs in the cytoplasmic/calf-1 regions). Phagocytosis mediated by Fcγ receptors was also significantly reduced in donors with variant ITGAM alleles. Similarly, firm adhesion of neutrophils was significantly reduced in individuals with variant ITGAM alleles. These functional alterations were not attributable to differences in total receptor expression or activation. The nonsynonymous ITGAM variants rs1143679 and rs1143678/rs113683 contribute to altered Mac-1 function on neutrophils. These results underscore the need to consider multiple nonsynonymous SNPs when assessing the functional consequences of ITGAM variation on immune cell processes and the risk of SLE.

Soluble CD40 Ligand Stimulates CD40-Dependent Activation of the β2 Integrin Mac-1 and Protein Kinase C Zeda (PKCζ) in Neutrophils: Implications for Neutrophil-Platelet Interactions and Neutrophil Oxidative Burst

Recent work has revealed an essential involvement of soluble CD40L (sCD40L) in inflammation and vascular disease. Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined. We aimed to determine the impact and the mechanisms of sCD40L on neutrophils. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte recruitment to the sites of injury. Our data show that CD40L contributes to neutrophil firm adhesion to and transmigration across activated surface-adherent platelets, possibly through two potential mechanisms. One involves the direct interaction of ligand-receptor (CD40L-CD40), i.e., platelet surface CD40L interaction with neutrophil CD40; another involves an indirect mechanism, i.e. soluble CD40L stimulates activation of the leukocyte-specific β2 integrin Mac-1 in neutrophils and thereby further promotes neutrophil adhesion and migration. Activation of the integrin Mac-1 is known to be critical for mediating neutrophil adhesion and migration. sCD40L activated Mac-1 in neutrophils and enhanced neutrophil-platelet interactions in wild-type neutrophils, but failed to elicit such responses in CD40-deficient neutrophils. Furthermore, our data show that the protein kinase C zeta (PKCζ) is critically required for sCD40L-induced Mac-1 activation and neutrophil adhesive function. sCD40L strongly stimulated the focal clustering of Mac-1 (CD11b) and the colocalization of Mac-1 with PKCζ in wild-type neutrophils, but had minimal effect in CD40-deficient neutrophils. Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion. Moreover, sCD40L strongly stimulates neutrophil oxidative burst via CD40-dependent activation of PI3K/NF-KB, but independent of Mac-1 and PKCζ. These findings may contribute to a better understanding of the underlying mechanisms by which sCD40L/CD40 pathway contributes to inflammation and vascular diseases.

Acute-phase concentrations of soluble fibrinogen inhibit neutrophil adhesion under flow conditions in vitro through interactions with ICAM-1 and MAC-1 (CD11b/CD18)

Immobilized fibrinogen and fibrin facilitate leukocyte adhesion, as they are potent ligands for leukocyte MAC-1 (CD11b/CD18). However, fibrinogen in its soluble form also binds to MAC-1, albeit with low affinity. The level of soluble fibrinogen is increased during chronic and acute inflammation, but the function of this increase is unknown. To study the effect of soluble fibrinogen in concentrations found in severe acute inflammation on leukocyte adhesion. Isolated leukocytes and soluble fibrinogen were studied in various in vitro settings under static and under flow conditions. Soluble fibrinogen functioned as a natural antagonist of neutrophil functions that are dependent on MAC-1, such as the respiratory burst induced by unopsonized zymosan and adhesion to ICAM-1 and heparin. In addition, soluble fibrinogen inhibited lymphocyte function-associated antigen 1-dependent lymphocyte binding to ICAM-1 through a direct interaction with ICAM-1. Soluble fibrinogen reduced MAC-1-dependent binding of interleukin-8-activated neutrophils to ICAM-1-expressing cells under flow conditions. Importantly soluble fibrinogen in acute-phase concentrations (4-10 mg mL(-1) ) dose-dependently reduced neutrophil firm adhesion to tumor necrosis factor-α-activated endothelium to 40% under flow conditions. We propose a model in which the increased circulating concentrations of soluble fibrinogen found during the acute-phase response can act as a natural antagonist of leukocyte recruitment, and therefore might contribute to the resolution of inflammation.

Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion

BackgroundChronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated proline–glycine–proline (N-ac-PGP). In the current study, we investigate whether N-ac-PGP influences β2-integrin activation and function in neutrophilic firm adhesion to endothelium. MethodsHuman polymorphonuclear leukocytes (PMNs) were isolated from fresh human blood. Subsequently, a transmigration assay was performed to evaluate the active migration of PMNs towards N-ac-PGP. Furthermore, the effect of the tripeptide on β2-integrin activation was assessed by performing the adhesion assay using fibrinogen as a ligand. To determine whether this effect was due to conformational change of β2-integrins, antibodies against CD11b and CD18 were used in the adhesion assay and the expression pattern of CD11b was determined. ResultsHuman neutrophils transmigrated through an endothelial cell layer in response to basolateral N-ac-PGP. N-ac-PGP induced also a neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that CD11b/CD18 (Mac-1) was responsible for the N-ac-PGP-induced firm adhesion of neutrophils to fibrinogen. Pertussis toxin decreased the Mac-1 activation indicating the involvement of G-proteins. N-ac-PGP most likely activated Mac-1 by initiating a conformational change, since the expression pattern of Mac-1 on the cell surface did not change significantly. ConclusionsChemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion. General significanceThis is the first study to describe that the chemo-attractant N-ac-PGP also activates Mac-1 on the surface of neutrophils, which can additionally contribute to neutrophilic transmigration into the lung tissue during lung inflammation.

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes. This method focuses on neutrophil transmigration across human endothelial cells. One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC). Neutrophil isolation has been described visually elsewhere; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-α results in increased E-selectin and IL-8 expression. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature. The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass. Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi μslide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

Cleavage of the CD11b extracellular domain by the leukocyte serprocidins is critical for neutrophil detachment during chemotaxis (173.7)

Abstract The β(2)-integrin CD11b/CD18 mediates the firm adhesion of neutrophils (PMNs) to epithelial monolayers, a key step in PMN transepithelial migration. To complete the transmigration process, adherent PMNs must detach from epithelial surfaces to move forward. The mechanism that governs the detachment of adherent PMNs, however, is not clear. Here, we present evidence that cleavage of the CD11b extracellular domain containing the ligand-binding I-domain by 3 structural and functional related serine proteases (elastase, proteinase-3 and cathepsin G) serves as a novel mechanism for PMN detachment after the initial cell adhesion. Kinetic studies showed that the cleavage of CD11b is positively correlated with PMN detachment and subsequent transmigration. Moreover, the results demonstrated that elastase, proteinase-3 and cathepsin G all cleaved the purified, functionally active form of CD11b in a pattern similar to the CD11b shedding that occurs during PMN transmigration. Their cleavage sites on purified CD11b were located at (761)Thr-Ala(762) (elastase/proteinase-3) and (760)Phe-Thr(761) (cathepsin G), respectively. CD11b cleavage and PMN detachment and chemotaxis, were impaired in elastase/cathepsin G-deficient Beige mice; this defect could be restored by the addition of extracellular elastase. By illustrating CD11b shedding by elastase, proteinase-3 and cathepsin G as a novel mechanism for PMN detachment, our study provides novel therapeutic targets for controlling inflammation.

A better biomarker for cerebral malaria

A better biomarker for cerebral malaria

Cigarette smoke induces β2-integrin-dependent neutrophil migration across human endothelium

BackgroundCigarette smoking induces peripheral inflammatory responses in all smokers and is the major risk factor for neutrophilic lung disease such as chronic obstructive pulmonary disease. The aim of this study was to investigate the effect of cigarette smoke on neutrophil migration and on β2-integrin activation and function in neutrophilic transmigration through endothelium.Methods and resultsUtilizing freshly isolated human PMNs, the effect of cigarette smoke on migration and β2-integrin activation and function in neutrophilic transmigration was studied. In this report, we demonstrated that cigarette smoke extract (CSE) dose dependently induced migration of neutrophils in vitro. Moreover, CSE promoted neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that Mac-1 (CD11b/CD18) is responsible for the cigarette smoke-induced firm adhesion of neutrophils to fibrinogen. Furthermore, neutrophils transmigrated through endothelium by cigarette smoke due to the activation of β2-integrins, since pre-incubation of neutrophils with functional blocking antibodies against CD11b and CD18 attenuated this transmigration.ConclusionThis is the first study to describe that cigarette smoke extract induces a direct migratory effect on neutrophils and that CSE is an activator of β2-integrins on the cell surface. Blocking this activation of β2-integrins might be an important target in cigarette smoke induced neutrophilic diseases.

Cleavage of the CD11b extracellular domain by the leukocyte serprocidins is critical for neutrophil detachment during chemotaxis

AbstractThe β2-integrin CD11b/CD18 mediates the firm adhesion of neutrophils (PMNs) to epithelial monolayers, a key step in PMN transepithelial migration. To complete the transmigration process, adherent PMNs must detach from epithelial monolayer surfaces to move forward. The mechanism that governs the detachment of adherent PMNs, however, is not clear. Here, we present evidence that cleavage of the CD11b extracellular domain containing the ligand-binding I-domain by 3 structural and functional related serine proteases (elastase, proteinase-3 and cathepsin G) serves as a novel mechanism for PMN detachment after the initial cell adhesion. Kinetic studies showed that the cleavage of CD11b is positively correlated with PMN detachment and subsequent transmigration. Moreover, the results demonstrated that elastase, proteinase-3 and cathepsin G all cleaved the purified, functionally active form of CD11b in a pattern similar to the CD11b shedding that occurs during PMN transmigration. Their cleavage sites on purified CD11b were located at 761Thr-Ala762 (elastase/proteinase-3) and 760Phe-Thr761 (cathepsin G), respectively. CD11b cleavage and PMN detachment and chemotaxis, were impaired in elastase/cathepsin G–deficient Beige mice; this defect could be restored by the addition of extracellular elastase. By illustrating CD11b shedding by elastase, proteinase-3 and cathepsin G as a novel mechanism for PMN detachment, our study provides novel therapeutic targets for controlling inflammation.

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.