Neutrophil Transendothelial Migration Research Articles

Pericytes support neutrophil transmigration via interleukin-8 across a porcine co-culture model of the blood–brain barrier

Transmigration of neutrophils across the blood–brain barrier (BBB) to an inflamed brain tissue is an important process during neuronal inflammation. The process of neutrophil activation as well as their way of rolling along the endothelium and their transmigration is quite well understood. Nevertheless, relatively little is known about the fate of neutrophils after they have transmigrated through the endothelium. The role of the other cells of the neurovascular unit in this process is also poorly understood. Here we studied the potential of pericytes to chemo-attract neutrophils under inflammatory conditions. Quantitative real time PCR, western blot analysis, and a chemotaxis assay showed that pericytes are able to chemo-attract neutrophils by interleukin-8 (IL-8) after stimulation with lipopolysaccharides (LPS), tumor necrosis factor-alpha (TNF-α), or interleukin-1beta (IL-1β). Then, a co-culture model consisting of primary porcine brain capillary endothelial cells (PBCECs) and primary porcine brain capillary pericytes (PBCPs) was used to analyze neutrophil transmigration across the BBB. As a model for inflammation, LPS was used and the effects of the cytokines TNF-α, IL-1β, and interferon-gamma (IFN-γ) were analyzed. In general, all stimulants apart from IFN-γ were able to increase transendothelial neutrophil migration. This effect was significantly reduced by a specific inhibitor of matrix metalloproteinases (MMPs)-2 and -9. MMP-2/-9 secretion is expected to decrease adhesion to pericytes and thus support the transmigration of neutrophils. Additionally, in an adhesion experiment, we showed that MMP-2/-9 inhibition significantly enhances the adhesion of neutrophils to pericytes.

OP0224 Anti-AT1R and Anti-ETAR Autoantibodies from Patients with SSC and their Agonistic Effects

BackgroundAutoantibodies reactive to angiotensin II type 1 receptor (AT1R) and endothelin 1 type A receptor (ETAR), anti-AT1R and anti-ETAR autoantibodies, are found in patients with Systemic sclerosis (SSc) as a...

ICAM1 ligation‐induced transmigratory complex formation regulates leukocyte transendothelial migration

ICAM1 expression is upregulated on activated endothelium and is important in leukocyte adhesion and transendothelial migration (TEM). ICAM1 engagement initiates several signaling events including cortactin phosphorylation and association with the ICAM1 cytoplasmic tail. We hypothesized that interactions between cortactin and a putative SH3 binding domain in the ICAM1 tail regulate leukocyte TEM. To test this we mutated 3 amino acids within the ICAM1 cytoplasmic tail such that the putative SH3 binding domain was no longer present (SH3Mut ICAM1). We used adenoviral infection to express WT ICAM1 or SH3Mut ICAM1 in human umbilical vein endothelial cells (HUVEC). Standard biochemical methods and transmigration assays were used to assess functional relevance of the SH3 mutation. ICAM1 engagement triggers cortactin association with ICAM1. Approximately 50% less cortactin associates with SH3Mut ICAM1 (n=3, p<0.05). Neutrophil TEM is also reduced by 43% in cells expressing SH3Mut ICAM1. Furthermore, ICAM1 ligation induces the formation of a large transmigratory complex which includes ICAM1, VE‐Cad, p120, pSrc, and cortactin. Formation of this complex is, in part, dependent on the SH3 binding domain. Our study identifies an SH3 binding motif in the ICAM‐1 cytoplasmic tail that is necessary for association with cortactin and provides evidence that transmigratory complex formation is important for TEM.

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.

Anti‐inflammatory effects of tobramycin and a copper–tobramycin complex with superoxide dismutase‐like activity

Airway inflammation in cystic fibrosis (CF) patients is characterized by accumulations of neutrophils in the airway and T cells in bronchial tissue, with activation of platelets in the circulation. CF patients are routinely treated with systemic or inhaled tobramycin for airway infection with Pseudomonas aeruginosa. Clinical trials have indicated an anti-inflammatory effect of tobramycin beyond its bactericidal activity. Here, we investigate the anti-inflammatory properties of tobramycin in vitro and consider if these relate to the ability of tobramycin to bind copper, which is elevated in blood and sputum in CF. A copper-tobramycin complex was synthesized. The effect of tobramycin and copper-tobramycin on neutrophil activation and migration of T cells and neutrophils across human lung microvascular endothelial cells in response to thrombin-activated platelets were investigated in vitro. Tobramycin uptake was detected by immunocytochemistry. Intracellular reactive oxygen species were detected using the fluorescent indicator, 2',7'-dichlorofluorescein diacetate (DCFDA). Neutrophil superoxide, hydrogen peroxide and neutrophil elastase activity were measured using specific substrates. Copper was measured using atomic absorption spectroscopy. Tobramycin and copper-tobramycin were taken up by endothelial cells via a heparan sulphate-dependent mechanism and significantly inhibited T-cell and neutrophil transendothelial migration respectively. Copper-tobramycin has intracellular and extracellular superoxide dismutase-like activity. Neutrophil elastase inhibition by α1-antitrypsin is enhanced in the presence of copper-tobramycin. Tobramycin and copper-tobramycin are equally effective anti-pseudomonal antibiotics. Anti-inflammatory effects of tobramycin in vivo may relate to the spontaneous formation of a copper-tobramycin complex, implying that copper-tobramycin may be more effective therapy.

Thrombin-cleaved Fragments of Osteopontin Are Overexpressed in Malignant Glial Tumors and Provide a Molecular Niche with Survival Advantage

Osteopontin (OPN), which is highly expressed in malignant glioblastoma (GBM), possesses inflammatory activity modulated by proteolytic cleavage by thrombin and plasma carboxypeptidase B2 (CPB2) at a highly conserved cleavage site. Full-length OPN (OPN-FL) was elevated in cerebrospinal fluid (CSF) samples from all cancer patients compared with noncancer patients. However, thrombin-cleaved OPN (OPN-R) and thrombin/CPB2-double-cleaved OPN (OPN-L) levels were markedly increased in GBM and non-GBM gliomas compared with systemic cancer and noncancer patients. Cleaved OPN constituted ∼23 and ∼31% of the total OPN in the GBM and non-GBM CSF samples, respectively. OPN-R was also elevated in GBM tissues. Thrombin-antithrombin levels were highly correlated with cleaved OPN, but not OPN-FL, suggesting that the cleaved OPN fragments resulted from increased thrombin and CPB2 in this extracellular compartment. Levels of VEGF and CCL4 were increased in CSF of GBM and correlated with the levels of cleaved OPN. GBM cell lines were more adherent to OPN-R and OPN-L than OPN-FL. Adhesion to OPN altered gene expression, in particular genes involved with cellular processes, cell cycle regulation, death, and inflammation. OPN and its cleaved forms promoted motility of U-87 MG cells and conferred resistance to apoptosis. Although functional mutation of the RGD motif in OPN largely abolished these functions, OPN(RAA)-R regained significant cell binding and signaling function, suggesting that the SVVYGLR motif in OPN-R may substitute for the RGD motif if the latter becomes inaccessible. OPN cleavage contributes to GBM development by allowing more cells to bind in niches where they acquire anti-apoptotic properties.

Live Brugia malayi Microfilariae Inhibit Transendothelial Migration of Neutrophils and Monocytes

Lymphatic filariasis is a major tropical disease caused by the parasite Brugia malayi. Microfilariae (Mf) circulate in the peripheral blood for 2–3 hours in synchronisation with maximal feeding of the mosquito vector. When absent from the peripheral blood, Mf sequester in the capillaries of the lungs. Mf are therefore in close contact with vascular endothelial cells (EC) and may induce EC immune function and/or wound repair mechanisms such as angiogenesis. In this study, Mf were co-cultured with human umbilical vein EC (HUVEC) or human lung microvascular EC (HLMVEC) and the transendothelial migration of leukocyte subsets was analysed. In addition, the protein and/or mRNA expression of chemokine, cytokine and angiogenic mediators in endothelial cells in the presence of live microfilariae were measured by a combination of cDNA arrays, protein arrays, ELISA and fluorescence antibody tests.Surprisingly, our findings indicate that Mf presence partially blocked transendothelial migration of monocytes and neutrophils, but not lymphocytes. However, Mf exposure did not result in altered vascular EC expression of key mediators of the tethering stage of extravasation, such as ICAM-1, VCAM-1 and various chemokines. To further analyse the immunological function of vascular EC in the presence of Mf, we measured the mRNA and/or protein expression of a number of pro-inflammatory mediators. We found that expression levels of the mediators tested were predominantly unaltered upon B. malayi Mf exposure. In addition, a comparison of angiogenic mediators induced by intact Mf and Wolbachia-depleted Mf revealed that even intact Mf induce the expression of remarkably few angiogenic mediators in vascular EC. Our study suggests that live microfilariae are remarkably inert in their induction and/or activation of vascular cells in their immediate local environment. Overall, this work presents important insights into the immunological function of the vascular endothelium during an infection with B. malayi.

ADAM15 deficiency attenuates pulmonary hyperpermeability and acute lung injury in lipopolysaccharide-treated mice

ADAM15 is a disintegrin and metalloprotease recently implicated in cancer and chronic immune disorders. We have recently characterized ADAM15 as a mediator of endothelial barrier dysfunction. Whether this molecule contributes to acute inflammation has not been evaluated. The purpose of this study was to investigate the role of ADAM15 in mediating pulmonary microvascular leakage during acute inflammatory injury. Immunofluorescent staining and Western blotting revealed that the endothelium was the main source of ADAM15 in lung tissue. In a mouse model of acute lung injury induced by lipopolysaccharide (LPS), upregulation of ADAM15 was observed in association with pulmonary edema and neutrophil infiltration. The LPS-induced inflammatory injury, as demonstrated by bronchoalveolar lavage neutrophil count, lung wet-to-dry weight ratio, and myeloperoxidase activity, was significantly attenuated in Adam15(-/-) mice. Studies with primary cell culture demonstrated abundant ADAM15 expression in endothelial cells (ECs) of mouse lung but not in neutrophils. Deficiency of ADAM15 in ECs had no obvious effect on basal permeability but significantly attenuated hyperpermeability response to LPS as evidenced by albumin flux assay and measurements of transendothelial electrical resistance, respectively. ADAM15 deficiency also reduced neutrophil chemotactic transmigration across endothelial barriers in the presence or absence of formyl-methionyl-leucyl-phenylalanine (fMLP). Rescue expression of ADAM15 in Adam15(-/-) ECs restored neutrophil transendothelial migration. These data indicate that ADAM15 upregulation contributes to inflammatory lung injury by promoting endothelial hyperpermeability and neutrophil transmigration.

An Inflammation Loop Orchestrated by S100A9 and Calprotectin Is Critical for Development of Arthritis

ObjectiveThe S100A9 and S100A8 proteins are highly expressed by neutrophils and monocytes and are part of a group of damage-associated molecular pattern molecules that trigger inflammatory responses. Sera and synovial fluids of patients with rheumatoid arthritis (RA) contain high concentrations of S100A8/A9 that correlate with disease activity.MethodsIn this study, we investigated the importance of S100A9 in RA by using neutralizing antibodies in a murine lipopolysaccharide-synchronized collagen-induced arthritis model. We also used an in vitro model of stimulation of human immune cells to decipher the role played by S100A9 in leukocyte migration and pro-inflammatory cytokine secretion.ResultsTreatment with anti-S100A9 antibodies improved the clinical score by 50%, diminished immune cell infiltration, reduced inflammatory cytokines, both in serum and in the joints, and preserved bone/collagen integrity. Stimulation of neutrophils with S100A9 protein led to the enhancement of neutrophil transendothelial migration. S100A9 protein also induced the secretion by monocytes of proinflammatory cytokines like TNFα, IL-1β and IL-6, and of chemokines like MIP-1α and MCP-1.ConclusionThe effects of anti-S100A9 treatment are likely direct consequences of inhibiting the S100A9-mediated promotion of neutrophil transmigration and secretion of pro-inflammatory cytokines from monocytes. Collectively, our results show that treatment with anti-S100A9 may inhibit amplification of the immune response and help preserve tissue integrity. Therefore, S100A9 is a promising potential therapeutic target for inflammatory diseases like rheumatoid arthritis for which alternative therapeutic strategies are needed.

Relocalization of Junctional Adhesion Molecule A during Inflammatory Stimulation of Brain Endothelial Cells

Junctional adhesion molecule A (JAM-A) is a unique tight junction (TJ) transmembrane protein that under basal conditions maintains endothelial cell-cell interactions but under inflammatory conditions acts as a leukocyte adhesion molecule. This study investigates the fate of JAM-A during inflammatory TJ complex remodeling and paracellular route formation in brain endothelial cells. The chemokine (C-C motif) ligand 2 (CCL2) induced JAM-A redistribution from the interendothelial cell area to the apical surface, where JAM-A played a role as a leukocyte adhesion molecule participating in transendothelial cell migration of neutrophils and monocytes. JAM-A redistribution was associated with internalization via macropinocytosis during paracellular route opening. A tracer study with dextran-Texas Red indicated that internalization occurred within a short time period (~10 min) by dextran-positive vesicles and then became sorted to dextran-positive/Rab34-positive/Rab5-positive vesicles and then Rab4-positive endosomes. By ~20 min, most internalized JAM-A moved to the brain endothelial cell apical membrane. Treatment with a macropinocytosis inhibitor, 5-(N-ethyl-N-isopropyl)amiloride, or Rab5/Rab4 depletion with small interfering RNA oligonucleotides prevented JAM-A relocalization, suggesting that macropinocytosis and recycling to the membrane surface occur during JAM-A redistribution. Analysis of the signaling pathways indicated involvement of RhoA and Rho kinase in JAM-A relocalization. These data provide new insights into the molecular and cellular mechanisms involved in blood-brain barrier remodeling during inflammation.

Dying Cells and Extracellular Histones in AKI

Dying Cells and Extracellular Histones in AKI

Comparative evaluation of the role of the adhesion molecule CD177 in neutrophil interactions with platelets and endothelium

Neutrophil-specific glycoprotein CD177 is expressed on a subset of human neutrophils and has been shown to be a counter-receptor for platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31). Previous studies have demonstrated that the interaction of CD177 with endothelial PECAM-1 supports neutrophil transendothelial migration resulting in preferential transmigration of the CD177-expressing neutrophil subset. As PECAM-1 is also abundantly expressed on platelets, we addressed a follow-up suggestion that CD177/PECAM-1 adhesive interaction may mediate platelet-neutrophil interactions and CD177-positive neutrophils may have a competitive advantage over CD177-negative neutrophils in binding platelets. Here, we report that CD177-positive and CD177-negative neutrophils do not differ significantly in their capacity to form platelet-neutrophil conjugates as assayed in whole blood and in mixed preparations of isolated platelets and neutrophils. Under flow conditions, neither platelet nor neutrophil activation resulted in preferential binding of platelets to CD177-expressing neutrophils. Furthermore, no significant difference was found in the ability of both neutrophil subsets to adhere to and migrate across surface-adherent activated platelets, whereas predominantly CD177-positive neutrophils migrated across HUVEC monolayers. In addition, we demonstrated that S(536) N dimorphism of PECAM-1, which affects CD177/PECAM-1 interaction, did not influence the equal capacity of the two neutrophil subsets to interact with platelets but influenced significantly the transendothelial migration of CD177-expressing neutrophils. Thus, CD177/PECAM-1 adhesive interaction, while contributing to neutrophil-endothelial cell interaction in neutrophil transendothelial migration, does not contribute to or is redundant in platelet-neutrophil interactions.

P120-Catenin prevents neutrophil transmigration independently of RhoA inhibition by impairing Src dependent VE-cadherin phosphorylation

Leukocyte transendothelial migration (TEM) is regulated by several signaling pathways including Src family kinases (SFK) and the small RhoGTPases. Previous studies have shown that vascular endothelial-cadherin (VE-cad) forms a complex with β-,γ-, and p120-catenins and this complex disassociates to form a transient gap during leukocyte TEM. Additionally, p120-catenin (p120-1A) overexpression in human umbilical vein endothelial cells (HUVEC) stabilizes VE-cad surface expression, prevents tyrosine phosphorylation of VE-cad, and inhibits leukocyte TEM. Based on reports showing that p120 overexpression in fibroblasts or epithelial cells inhibits RhoA and activates Rac and Cdc42 GTPases, and on other reports showing that RhoA activation in endothelial cells is necessary for leukocyte TEM, we reasoned that p120 overexpression inhibited TEM through inhibition of RhoA. To test this idea, we overexpressed a mutant p120 isoform, p120-4A, which does not interact with RhoA. p120-4A colocalized with VE-cad in HUVEC junctions and enhanced VE-cad surface expression, similar to overexpression of p120-1A. Interestingly, overexpression of either p120-4A or p120-1A dramatically blocked TEM, and overexpression of p120-1A in HUVEC did not affect RhoA basal activity or activation of RhoA and Rac induced by thrombin or ICAM-1 crosslinking. In contrast, biochemical studies revealed that overexpression of p120-1A reduced activated pY416-Src association with VE-cad. In summary, p120 overexpression inhibits neutrophil TEM independently of an effect on RhoA or Rac and instead blocks TEM by preventing VE-cad tyrosine phosphorylation and association of active Src with the VE-cad complex.

Distinctive roles of neutrophil LSP1 and endothelial cell LSP1 during neutrophil recruitment (173.29)

Abstract Leukocyte-specific protein 1 (LSP1) is an intracellular, F-actin- and Ca2+-binding, phosphoprotein expressed in leukocytes and endothelial cells (ECs). Using intravital microscopy and time-lapse video photography, we studied the role of neutrophil LSP1 (N-LSP1) and endothelial cell LSP1 (EC-LSP1) during neutrophil recruitment in the post-capillary venules in mice. The wild-type control, LSP1-deficient (Lsp1−/−) and their chimeric mice were used in this study. Neutrophil recruitment was induced by addition of chemokine MIP-2-containing gel to the observed cremasteric venule. Neutrophil intraluminal crawling, transendothelial migration, and chemotaxis in tissue were determined. We found that both N-LSP1 and EC-LSP1 were involved in neutrophil intraluminal crawling and neutrophil transendothelial migration. After neutrophil transmigration, only N-LSP1 was found to be important for neutrophil migration in the tissue, whereas EC-LSP1, but not N-LSP1, was essential for the directional neutrophil chemotaxis. Although N-LSP1 did not play an important role in neutrophil directional chemotaxis, both neutrophil migration and directional chemotaxis were dependent on the function of p38 mitogen-activated protein kinase, an upstream signaling molecule for N-LSP1, in the emigrated neutrophils in the tissue. Our results suggest that N-LSP1 and EC-LSP1 are both important for neutrophil recruitment but play distinctive roles in different steps of neutrophil recruitment in vivo.

Specific Role of Neutrophil Inducible Nitric Oxide Synthase in Murine Sepsis-Induced Lung Injury In Vivo

Nitric oxide produced by inducible nitric oxide synthase (iNOS) contributes importantly to acute lung injury (ALI), but the specific contribution of neutrophil iNOS has not been defined. Thus, we defined the role of neutrophils and specifically neutrophil iNOS in a murine model of septic ALI. Four hours after cecal ligation/perforation, ALI was characterized by increases in pulmonary neutrophil infiltration (tissue myeloperoxidase activity, bronchoalveolar lavage neutrophils), microvascular leak of Evans blue (EB) dye-labeled albumin, and oxidant stress (8-isoprostane levels). Septic ALI was neutrophil dependent, as pretreatment with anti-CD18 before cecal ligation/perforation significantly (P < 0.05) attenuated septic increases in pulmonary myeloperoxidase (39 ± 11 vs. 85 ± 14 mU/mg protein), bronchoalveolar lavage neutrophils (0.5% ± 0.2% vs. 2.1% ± 0.6%), microvascular EB-albumin leak (1.3 ± 0.3 vs. 2.6 ± 0.7 μg EB/g per minute), and 8-isoprostane content (74 ± 15 vs. 115 ± 16 pg/mg protein). The role of neutrophil iNOS was assessed by creation of neutrophil-iNOS chimeric mice: iNOS(+/+) versus iNOS(-/-) mice were bone marrow depleted by irradiation and selectively reconstituted with iNOS(+/+) versus iNOS(-/-) neutrophils. Cecal ligation/perforation resulted in significant septic ALI in + to - neutrophil-iNOS chimeric mice (iNOS(+/+) neutrophils in iNOS(-/-) mice), but not in - to + neutrophil depleted-reconstituted mice (iNOS(-/-) neutrophils in iNOS(+/+) mice). There were no significant differences between iNOS(+/+) and iNOS(-/-) neutrophils in phagocytosis, respiratory burst, or CD11a/b/CD18 surface expression, although septic shedding of CD62L was blunted in iNOS(-/-) neutrophils. Neutrophil iNOS contributes importantly to murine septic ALI in vivo, but not simply through a change in neutrophil phenotype. We speculate that neutrophil iNOS may modulate neutrophil-endothelial interactions in complex fashion, including regulation of transendothelial neutrophil migration and pulmonary neutrophil infiltration.

P‐Rex1 is critical for vascular hyper‐permeability and edema in the lungs

The primary objective of this study was to investigate a role for P‐Rex1 (PIP3 dependent Rac exchanger 1) a guanine nucleotide exchange factor for Rac, previously known for G protein‐coupled receptor signaling, in mediating TNF‐α‐induced vascular hyper‐permeability. Using gene deletion and knockdown approaches, we investigated the potential role of P‐Rex1 in TNF‐α‐induced lung vascular injury. P‐Rex1, previously found in neutrophils and neurons, is also expressed in endothelial cells. In cultured human lung microvascular endothelial cells (HLMVECs), small interference (si) RNA‐mediated knockdown of P‐Rex1 prevented TNF‐α‐induced intercellular gap formation, Rac activation and reactive oxygen species (ROS) production. TNF‐α stimulated P‐Rex1 membrane translocation and the resulting Rac activation in a PI3K dependent manner. P‐Rex1 knockout mice were also refractory to TNF‐α‐induced lung vascular hyper‐permeability and edema. Both, in vivo and in vitro, absence of P‐Rex1 resulted in significantly less ICAM‐1 induction and neutrophil transendothelial migration. These results demonstrate for the first time that P‐Rex1 is activated downstream of TNF‐α, which is not a GPCR. We conclude that P‐Rex1 is a novel mediator of endothelial barrier disruption and therefore could be a potential therapeutic target in the control of vascular permeability and neutrophil infiltration to inflammatory tissues.

Proteinase 3 Contributes to Transendothelial Migration of NB1-Positive Neutrophils

Neutrophil transmigration requires the localization of neutrophils to endothelial cell junctions, in which receptor-ligand interactions and the action of serine proteases promote leukocyte diapedesis. NB1 (CD177) is a neutrophil-expressed surface molecule that has been reported to bind proteinase 3 (PR3), a serine protease released from activated neutrophils. PR3 has demonstrated proteolytic activity on a number of substrates, including extracellular matrix proteins, although its role in neutrophil transmigration is unknown. Recently, NB1 has been shown to be a heterophilic binding partner for the endothelial cell junctional protein, PECAM-1. Disrupting the interaction between NB1 and PECAM-1 significantly inhibits neutrophil transendothelial cell migration on endothelial cell monolayers. Because NB1 interacts with endothelial cell PECAM-1 at cell junctions where transmigration occurs, we considered that NB1-PR3 interactions may play a role in aiding neutrophil diapedesis. Blocking Abs targeting the heterophilic binding domain of PECAM-1 significantly inhibited transmigration of NB1-positive neutrophils through IL-1β-stimulated endothelial cell monolayers. PR3 expression and activity were significantly increased on NB1-positive neutrophils following transmigration, whereas neutrophils lacking NB1 demonstrated no increase in PR3. Finally, using selective serine protease inhibitors, we determined that PR3 activity facilitated transmigration of NB1-positive neutrophils under both static and flow conditions. These data demonstrate that PR3 contributes in the selective recruitment of the NB1-positive neutrophil population.

Identification of VLDLR as a novel endothelial cell receptor for fibrin that modulates fibrin-dependent transendothelial migration of leukocytes

While testing the effect of the (β15-66)(2) fragment, which mimics a pair of fibrin βN-domains, on the morphology of endothelial cells, we found that this fragment induces redistribution of vascular endothelial-cadherin in a process that is inhibited by the receptor-associated protein (RAP). Based on this finding, we hypothesized that fibrin may interact with members of RAP-dependent low-density lipoprotein (LDL) receptor family. To test this hypothesis, we examined the interaction of (β15-66)(2), fibrin, and several fibrin-derived fragments with 2 members of this family by ELISA and surface plasmon resonance. The experiments showed that very LDL (VLDL) receptor (VLDLR) interacts with high affinity with fibrin through its βN-domains, and this interaction is inhibited by RAP and (β15-66)(2). Furthermore, RAP inhibited transendothelial migration of neutrophils induced by fibrin-derived NDSK-II fragment containing βN-domains, suggesting the involvement of VLDLR in fibrin-dependent leukocyte transmigration. Our experiments with VLDLR-deficient mice confirmed this suggestion by showing that, in contrast to wild-type mice, fibrin-dependent leukocyte transmigration does not occur in such mice. Altogether, the present study identified VLDLR as a novel endothelial cell receptor for fibrin that promotes fibrin-dependent leukocyte transmigration and thereby inflammation. Establishing the molecular mechanism underlying this interaction may result in the development of novel inhibitors of fibrin-dependent inflammation.

A versatile assay for monitoring in vivo-like transendothelial migration of neutrophils

Spatiotemporal analysis of the inflammatory response has been limited by the difficulties of in vivo imaging and reconstitution of inflammation in vitro. Here, we present a novel method for establishing in vivo-like inflammatory models in a microfluidic device and quantitatively measuring the three-dimensional transmigration of neutrophils during the inflammatory process. This enabled us to concurrently characterize transendothelial migration behaviors of neutrophils under the influence of various inflammatory stimuli.

Anti Inflammatory Effects of Statins in Cardiac Surgery Patients

Objectives: Cardiac surgery with cardiopulmonary bypass provokes systemic inflammatory response, which may cause organ dysfunction. Studies have suggested that pre-operative statin therapy can reduce morbidity and mortality associated with cardiac surgery; the mechanism for this remains unclear. We hypothesise that underlying mechanism of action for these effects of statins is through inhibition of neutrophil transendothelial migration. Methods: We recruited 151 patients undergoing cardiac surgery. Of these 41 patients were on no-statin; 48 patients on low-dose (10 - 30 mg) and 62 patients were on high-dose statin (40 - 80 mg). Ex vivo neutrophil migration was performed on pre-operative blood samples of 90 patients. Of these 90 patients we used 36 patients to assess the levels of TNF-α and sICAM-1. Clinical parameters of total 151 patients were assessed to analyse outcome. Results: Ex vivo neutrophil migration was decreased in high-dose statin group when compared to neutrophils isolated from no-statin or low-dose statin groups (p < 0.001). Serum TNF-α levels were lower in the high-dose statin group (p = 0.002) and sICAM-1 levels were decreased in both low (p = 0.02) and high-dose statin (p = 0.01) groups. Conclusion: Our study demonstrates that high-dose statins reduce ex-vivo transendothelial neutrophil migration, TNF-α and sICAM-1 serum levels suggesting that statins may help in decreasing the post-cardiac surgery morbidity.