Neutrophil-endothelial Adhesion Research Articles

DPP-4 exacerbates LPS-induced endothelial cells inflammation via integrin-α5β1/FAK/AKT signaling

Endothelial dysfunction plays a pivotal role in the pathogenesis of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Dipeptidyl peptidase IV (DPP-4), a cell surface glycoprotein, has been implicated in endothelial inflammation and barrier dysfunction. In this study, the role of DPP-4 on lipopolysaccharide (LPS)-induced pulmonary microvascular endothelial cells (HPMECs) dysfunction and the underlying mechanism were investigated by siRNA-mediated knockdown of DPP-4. Our results indicated that LPS (1 μg/ml) challenge resulted in either the production and releasing of DPP-4, as well as the secretion of IL-6 and IL-8 in HPMECs. DPP-4 knockdown inhibited chemokine releasing and monolayer hyper-permeability in LPS challenged HPMECs. When cocultured with human polymorphonuclear neutrophils (PMNs), DPP4 knockdown suppressed LPS-induced neutrophil-endothelial adhesion, PMN chemotaxis and trans-endothelial migration. Western blotting showed that DPP-4 knockdown attenuated LPS-induced activation of TLR4/NF-κB pathway. Immunoprecipitation and liquid chromatography-tandem mass spectrometry revealed that DPP-4 mediated LPS-induced endothelial inflammation by interacting with integrin-α5β1. Moreover, exogenous soluble DPP-4 treatment sufficiently activated integrin-α5β1 downstream FAK/AKT/NF-κB signaling, thereafter inducing ICAM-1 upregulation in HPMECs. Collectively, our results suggest that endothelia synthesis and release DPP-4 under the stress of endotoxin, which interact with integrin-α5β1 complex in an autocrine or paracrine manner to exacerbate endothelial inflammation and enhance endothelial cell permeability. Therefore, blocking DDP-4 could be a potential therapeutic strategy to prevent endothelial dysfunction in ALI/ARDS.

Neutrophil-Membrane-Coated Biomineralized Metal-Organic Framework Nanoparticles for Atherosclerosis Treatment by Targeting Gene Silencing.

Antisense oligonucleotides (ASOs) are promising tools for gene silencing and have been exploited as therapeutics for human disease. However, delivery of therapeutic ASOs to diseased tissues or cells and subsequent escape from the endosomes and release of ASO in the cytosol remain a challenge. Here, we reported a neutrophil-membrane-coated zeolitic imidazolate framework-8 (ZIF-8) nanodelivery platform (AM@ZIF@NM) for the targeted transportation of ASOs against microRNA-155 (anti-miRNA-155) to the endothelial cells in atherosclerotic lesions. Neutrophil membrane could improve plaque endothelial cells targeting through the interaction between neutrophil membrane protein CD18 and endothelial cell membrane protein intercellular adhesion molecule-1 (ICAM-1). The ZIF-8 "core" provided high loading capacity and efficient endolysosomal escaping ability. Delivery of anti-miR-155 effectively downregulated miR-155 expression and also saved the expression of its target gene BCL6. Moreover, RELA expression and the expression of its downstream target genes CCL2 and ICAM-1 were correspondingly reduced. Consequently, this anti-miR-155 nanotherapy can inhibit the inflammation of atherosclerotic lesions and alleviate atherosclerosis. Our study shows that the designed biomimetic nanodelivery system has great application prospects in the treatment of other chronic diseases.

The effect of tranexamic acid dosing regimen on trauma/hemorrhagic shock-related glycocalyx degradation and endothelial barrier permeability: An in vitro model.

Improved outcomes with early tranexamic acid (TXA) following trauma hemorrhagic shock (T/HS) may be related to its antifibrinolytic, as well as anti-inflammatory properties. Previous in vitro studies have shown that early TXA administration protects against T/HS endothelial barrier dysfunction and associated glycocalyx degradation. An intact endothelial glycocalyx may protect against subsequent neutrophil mediated tissue injury. We postulated that early TXA administration would mitigate against glycocalyx damage and resultant neutrophil adherence and transmigration through the endothelial barrier. This was studied in vitro using a microfluidic flow platform. Human umbilical vein endothelial cell monolayers were subjected to control or shock conditions (hypoxia + epinephrine) followed by administration of TXA 90 minutes or 180 minutes later. "Early" TXA administration protected against glycocalyx degradation, biomarkers of increased permeability and the development of a fibrinolytic phenotype. This was associated with decreased neutrophil endothelial adherence and transmigration. There were no differences in low versus high TXA concentrations. The protective effects were only significant with "early" TXA administration. There was a concentration and temporal effect of TXA administration on endothelial glycocalyx degradation. This was associated with "vascular leakiness" as indexed by the relative ratio of Ang-2/1 and polymorphonuclear neutrophil transmigration. Tranexamic acid if administered in patients with T/HS should be administered "early"; this includes in the prehospital setting.

Frontline Science: Activation of metabolic nuclear receptors restores periodontal tissue homeostasis in mice with leukocyte adhesion deficiency-1.

β2 Integrins mediate neutrophil-endothelial adhesion and recruitment of neutrophils to sites of inflammation. The diminished expression of β2 integrins in patients with mutations in the ITGB2 (CD18) gene (leukocyte adhesion deficiency-Type 1; LAD1) results in few or no neutrophils in peripheral tissues. In the periodontium, neutrophil paucity is associated with up-regulation of IL-23 and IL-17, which drive inflammatory bone loss. Using a relevant mouse model, we investigated whether diminished efferocytosis (owing to neutrophil scarcity) is associated with LAD1 periodontitis pathogenesis and aimed to develop approaches to restore the missing efferocytosis signals. We first showed that CD18-/- mice phenocopied human LAD1 in terms of IL-23/IL-17-driven inflammatory bone loss. Ab-mediated blockade of c-Mer tyrosine kinase (Mer), a major efferocytic receptor, mimicked LAD1-associated up-regulation of gingival IL-23 and IL-17 mRNA expression in wild-type (WT) mice. Consistently, soluble Mer-Fc reversed the inhibitory effect of efferocytosis on IL-23 expression in LPS-activated Mϕs. Adoptive transfer of WT neutrophils to CD18-/- mice down-regulated IL-23 and IL-17 expression to normal levels, but not when CD18-/- mice were treated with blocking anti-Mer Ab. Synthetic agonist-induced activation of liver X receptors (LXR) and peroxisome proliferator-activated receptors (PPAR), which link efferocytosis to generation of homeostatic signals, inhibited the expression of IL-23 and IL-17 and favorably affected the bone levels of CD18-/- mice. Therefore, our data link diminished efferocytosis-associated signaling due to impaired neutrophil recruitment to dysregulation of the IL-23-IL-17 axis and, moreover, suggest LXR and PPAR as potential therapeutic targets for treating LAD1 periodontitis.

Kinin B1 Receptor Is Important in the Pathogenesis of Myeloperoxidase-Specific ANCA GN.

Myeloperoxidase-specific ANCA (MPO-ANCA) are implicated in the pathogenesis of vasculitis and GN. Kinins play a major role during acute inflammation by regulating vasodilatation and vascular permeability and by modulating adhesion and migration of leukocytes. Kinin system activation occurs in patients with ANCA vasculitis. Previous studies in animal models of GN and sclerosing kidney diseases have demonstrated protective effects of bradykinin receptor 1 (B1R) blockade via interference with myeloid cell trafficking. To investigate the role of B1R in a murine model of MPO-ANCA GN, we evaluated effects of B1R genetic ablation and pharmacologic blockade. We used bone marrow chimeric mice to determine the role of B1R in bone marrow-derived cells (leukocytes) versus nonbone marrow-derived cells. We elucidated mechanisms of B1R effects using in vitro assays for MPO-ANCA-induced neutrophil activation, endothelial adherence, endothelial transmigration, and neutrophil adhesion molecule surface display. B1R deficiency or blockade prevented or markedly reduced ANCA-induced glomerular crescents, necrosis, and leukocyte influx in mice. B1R was not required for in vitro MPO-ANCA-induced neutrophil activation. Leukocyte B1R deficiency, but not endothelial B1R deficiency, decreased glomerular neutrophil infiltration induced by MPO-ANCA in vivo. B1R enhanced ANCA-induced neutrophil endothelial adhesion and transmigration in vitro. ANCA-activated neutrophils exhibited changes in Mac-1 and LFA-1, important regulators of neutrophil endothelial adhesion and transmigration: ANCA-activated neutrophils increased surface expression of Mac-1 and increased shedding of LFA-1, whereas B1R blockade reduced these effects. The leukocyte B1R plays a critical role in the pathogenesis of MPO-ANCA-induced GN in a mouse model by modulating neutrophil-endothelial interaction. B1R blockade may have potential as a therapy for ANCA GN and vasculitis.

Neutrophil accumulation and NET release contribute to thrombosis in HIT.

Heparin-induced thrombocytopenia (HIT) is an immune-mediated thrombocytopenic disorder associated with a severe prothrombotic state. We investigated whether neutrophils and neutrophil extracellular traps (NETs) contribute to the development of thrombosis in HIT. Using an endothelialized microfluidic system and a murine passive immunization model, we show that HIT induction leads to increased neutrophil adherence to venous endothelium. In HIT mice, endothelial adherence is enhanced immediately downstream of nascent venous thrombi, after which neutrophils undergo retrograde migration via a CXCR2-dependent mechanism to accumulate into the thrombi. Using a microfluidic system, we found that PF4 binds to NETs, leading them to become compact and DNase resistant. PF4-NET complexes selectively bind HIT antibodies, which further protect them from nuclease digestion. In HIT mice, inhibition of NET formation through Padi4 gene disruption or DNase treatment limited venous thrombus size. PAD4 inactivation did affect arterial thrombi or severity of thrombocytopenia in HIT. Thus, neutrophil activation contributes to the development of venous thrombosis in HIT by enhancing neutrophil-endothelial adhesion and neutrophil clot infiltration, where incorporated PF4-NET-HIT antibody complexes lead to thrombosis propagation. Inhibition of neutrophil endothelial adhesion, prevention of neutrophil chemokine-dependent recruitment of neutrophils to thrombi, or suppression of NET release should be explored as strategies to prevent venous thrombosis in HIT.

Mutations in Lyn Kinase Causes Changes in Neutrophil Function and Migration

The Src Family Kinase (SFK) Lyn plays important regulatory roles in immune cells including neutrophils. As a signaling protein, Lyn has been shown to regulate neutrophil migration and endothelial adhesion, contributing to inflammation. Lyn contains a tyrosine kinase domain as well as SH2 and SH3 domains allowing for adaptor functions. A set of patients with systemic neutrophil‐induced inflammation have been shown to have de novo germline mutations in Lyn, Y508* and Y508F, affecting the negative regulatory tyrosine residue of Lyn. The purpose of this study is to evaluate the physiological, cellular, and biochemical effects of this mutation, and further understand the role of Lyn Kinase in neutrophil function. Patients were found to have a gain‐of‐function mutation and presented with skin vasculitis that improved with administration of the SFK inhibitor, dasatinib. We generated stable expression lines of WT, Y508*, and Y508F pMX‐Lyn‐GFP in neutrophil‐like PLB‐985 cells. Kinase activity assays show increased phosphorylation of SFK substrate, SAM68, and overall tyrosine phosphorylation with both Y508*and Y508F expression compared to WT, indicating higher kinase activity in the mutants. Further experiments using kinase‐dead mutants indicate that the higher kinase activity is caused by increases in Lyn's kinase activity and not its adaptor function. In addition, we evaluated the chemotaxis of PLB‐985 cells towards the chemoattractant fmlp using transwell migration assays and microfluidic devices that allow for the generation of chemoattractant gradients. Y508* and Y508F PLB‐985 cells were found to have significantly increased chemotaxis towards 10 mM fmlp (p<0.05) than WT Lyn. Our data supports the role of Lyn as a positive regulator of migration, demonstrating that disruption in the regulatory domain of Lyn can cause cellular and physiological changes. Furthermore, it highlights the role of the de novo germline mutations in Lyn as a cause of human inflammatory disease.Support or Funding InformationSupported by the Hilldale Undergraduate Research FellowshipThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Post‐septic Down‐regulation of Sulfatase‐1 Suppresses Pulmonary Endothelial ICAM‐1 Expression

RationalePost‐septic compensatory anti‐inflammatory syndrome (CARS) is a state of immunoparalysis that leaves patients susceptible to secondary infections. Interestingly, CARS coincides with post‐septic reconstitution of the pulmonary endothelial glycocalyx, a heparan sulfate (HS)‐enriched layer that regulates endothelial‐neutrophil adhesion and is degraded during early sepsis. CARS is generally described as a consequence of impaired leukocyte function, with little understanding of the role of the post‐septic endothelium. We hypothesized that the post‐septic pulmonary endothelial glycocalyx is characterized by changes in HS sulfation (via changes in sulfatase expression), leading to impaired endothelial inflammatory responses.MethodsWe induced sepsis in male C57BL/6J mice with cecal ligation and puncture (CLP). To recapitulate CARS, post‐septic mice (3 days after CLP) animals were treated with intratracheal lipopolysaccharide (LPS), and the degree of inflammation was assessed thereafter by bronchoalveolar lavage (BAL) cell count and protein concentration. To determine changes in post‐septic pulmonary endothelial gene expression during CARS, we flow‐sorted pulmonary endothelial cells and performed RNA microarray and confirmatory qRT‐PCR. To determine post‐septic changes in HS sulfation, we performed mass spectrometry of HS isolated from either the pulmonary endothelial glycocalyx or the plasma of post‐CLP or post‐sham mice. We determined the impact of altered endothelial HS sulfation on CARS by either (a) degrading HS from the endothelial glycocalyx by intravenous heparinase I or (b) administering exogenous sulfated HS intravenously prior to intratracheal LPS. We specifically measured the effect of sulfatase‐1 (sulf‐1, an extracellular enzyme that specifically cleaves 6‐O sulfate of HS) on endothelial cell ICAM‐1 expression using siRNA.ResultsIn comparison to sham mice, post‐septic mice showed decreased cellular and protein infiltration in BAL fluid 48 hrs after intratracheal LPS (n=5–6, P < 0.05). Microarray and qRT‐PCR analyses of flow‐sorted endothelial cells (harvested 48 hrs after CLP or sham) detected downregulation of sulf‐1 in CLP mice (n=3–4, P < 0.05). While these changes correlated with increased 6‐O sulfation in the endothelial glycocalyx and circulating HS, experimental modulation of these HS compartments did not influence CARS. However, in vitro, transfection of siRNA targeted to sulf‐1 resulted in 90% reduction of sulf‐1 mRNA and down‐regulation of ICAM‐1 by 57–70% (n=2). These in vitro changes surprisingly occurred independently of HS 6‐O sulfation.ConclusionsConcurrently with CARS, post‐septic pulmonary endothelial cells undergo downregulation of sulf‐1, causing loss of endothelial ICAM‐1 expression. Surprisingly, this impaired ICAM‐1 expression occurred in the absence of changes in HS 6‐O sulfation.Support or Funding InformationDepartment of Defense PR150655 (to EPS)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fasudil inhibits neutrophil-endothelial cell interactions by regulating the expressions of GRP78 and BMPR2

Regulation of leukocyte-endothelial cell interactions and of vascular permeability plays a critical role in the maintenance of functional pulmonary microvascular barriers. Little is yet known about the effect of the Rho-associated protein kinase (ROCK) inhibitor fasudil on leukocyte-endothelial cell interactions or the underlying mechanism. In the present study, as evaluated using co-culture systems of neutrophils and human pulmonary microvascular endothelial cells (HPMECs), fasudil dose-dependently suppressed neutrophil chemotaxis by decreasing the production of chemotactic factors in lipopolysaccharide (LPS)-treated HPMECs. The inhibitory role of fasudil in neutrophil chemotaxis was mediated by down-regulation of the chaperone glucose-regulated protein 78 (GRP78), since the inhibition was abolished by 4-phenyl butyric acid (a chemical chaperone mimicking GRP78). In addition, fasudil inhibited LPS-induced neutrophil-endothelial adhesion by reducing the expression of intercellular adhesion molecule (ICAM)-1. By use of lentiviral transfection in HPMECs, bone morphogenic protein receptor 2 (BMPR2) overexpression suppressed the LPS-induced increase of both ICAM-1 expression and neutrophil-endothelial adhesion, whereas knocking down BMPR2 abolished the inhibitory role of fasudil in both ICAM-1 expression and neutrophil-endothelial adhesion. Moreover, fasudil alleviated LPS-induced hyperpermeability of HPMEC monolayers by leading to the recovery of intercellular junctions, thereafter reduced neutrophil transendothelial cell migration. Therefore, fasudil inhibited leukocyte-endothelial cell interactions and vascular hyperpermeability through modulation of GRP78 and BMPR2 expression, suggesting a potential role for ROCK as a switch for inhibiting leukocyte-endothelial cell interactions.

Hyperbaric Oxygen Inhibits Reperfusion-Induced Neutrophil Polarization and Adhesion Via Plasmin-Mediated VEGF Release.

Background:Ischemia-reperfusion (IR) injury is seen in many settings such as free flap salvage and limb replantation/revascularization. The consequences—partial/total flap loss, functional muscle loss, or amputation—can be devastating. Of the treatment options available for IR injury, hyperbaric oxygen (HBO) is the most beneficial. HBO inhibits neutrophil-endothelial adhesion through interference of CD18 neutrophil polarization in IR, a process mediated by nitric oxide. The purposes of this study were to examine the involvement of vascular endothelial growth factor (VEGF) in the beneficial HBO effect on CD18 polarization and neutrophil adhesion and investigate the effect of plasmin on VEGF expression in skeletal muscle following IR injury.Methods:A rat gracilis muscle model of IR injury was used to evaluate the effect of VEGF in IR, with and without HBO, on neutrophil CD18 polarization and adhesion in vivo and ex vivo. Furthermore, we investigated the effects that plasmin has on VEGF expression in gracilis muscle and pulmonary tissue by blocking its activation with alpha-2-antiplasmin.Results:HBO treatment following IR injury significantly decreased neutrophil polarization and adhesion ex vivo compared with the IR group. Anti-VEGF reversed the beneficial HBO effect after IR with polarization and adhesion. In vivo adhesion was also increased by anti-VEGF. HBO treatment of IR significantly increased the VEGF protein in both gracilis and pulmonary vasculature. Alpha-2-antiplasmin significantly reversed the HBO-induced increase of VEGF in gracilis muscle.Conclusions:These results suggest that HBO inhibits CD18 polarization and neutrophil adhesion in IR injury through a VEGF-mediated pathway involving the extracellular matrix plasminogen system.

Sialic acid and Galectins mediate Xenogenic Neutrophil-Endothelial Adhesion

Abstract Sialic acids (Sias) play multifarious roles in immunity, and likely contribute to inflammation seen in xenograft failure. The ‘net’ cellular sialyation state depends on many things including transient loss of Sia moieties with exposure of subterminal galactosyl structures (mediated by sialyltransferases and sialidases [neuraminidases]), which can function as receptors for extracellular lectins, such as galectins. Here, we report the role of Sias in modulating adhesion of human neutrophils (NΦ’s) to pig endothelial cells. Human (hAECs) and pig (pAECs) aortic endothelial cells were pretreated with Clostridium perfringens neuraminidase (NA, to remove Sias), human tumor necrosis factor (hTNF, 25 ug/ml), or both. CalceinAM-labelled human NΦ’s were treated with rhIL-8, NA, or N-acetyllactosamine (LacNAc, a galectin inhibitor). NΦ adhesion was measured and expressed as percent adhesion. NA treatment of AECs increased NΦ adhesion to pig (36.3% vs 22.4%) more than to human (24.2% vs 20.7%) AECs. NΦ adhesion was similarly increased by TNF activation of AECs in static and flow conditions. hIL-8 dramatically increased NΦ adhesion to pAECs (76.5%, p<.001) and hAECs (45.3%, p<.001), with the highest adhesion observed with NA-treated pAECs and hIL-8-activated NΦ’s. Pre-exposure of the NΦ’s to LacNAc prevented their galectin-mediated adhesion to galactose moieties exposed on the NA-treated pAECs (13.7%, p<.0001). In summary, AEC de-sialylation promotes xenogenic NΦ adhesion, an effect completely inhibited by pre-incubation of Nf’s with LacNAc. These results suggest the inhibition of sialidase activity or galectin binding as promising therapeutic approaches to limit inflammation in xenogeneic organ injury.

Gastric injury induced by Non-steroidal Anti-inflammatory Drugs and therapeutic strategies for prevention

The toxicity of gastrointestinal (GI) tract associated with the use of nonsteroidal anti-inflammatory drugs (NSAIDs) is an important medical and socio-economic problem. The coprescription of traditional gastroprotective drugs such as proton pump inhibitors (PPIs) and H2-receptor antagonist, to reduce gastric acid secretion are the common clinical therapeutically approach. However, these strategies are not effectively to reduce gastrointestinal adverse events of NSAIDs and recently have demonstrated their low safety in long-term use. In the search for new therapeutically approach, the use of selective COX-2 inhibitors and the use of prostaglandin analogue in order to maintain the local prostaglandins was considered. Unfortunately, their adverse events in cardiovascular or renal system provoked that their therapeutic use were discarded. Therefore, the search for new therapeutic strategies has been promoted. The current experimental and preclinical approaches includes very promising alternatives such as H2S-NSAIDs, NO-NSAIDs and the possible use of Docosahexaenoic acid (DHA), an Omega-3 Fatty acid. In this regard, DHA is focused in face the prostaglandin independent mechanism of NSAID-induce gastric injury such as increment of proinflammatory molecules expression and neutrophil–endothelial adherence. However, large studies of these therapeutically approaches are still needed.

Ischemic preconditioning ameliorates intestinal injury induced by ischemia-reperfusion in rats.

To evaluate preventative effects of ischemic preconditioning (IP) in a rat model of intestinal injury induced by ischemia-reperfusion (IR). Male Sprague-Dawley rats (250-300 g) were fasted for 24 h with free access to water prior to the operation. Eighteen rats were randomly divided into three experimental groups: S group (n = 6), rats were subjected to isolation of the superior mesenteric artery (SMA) for 40 min, then the abdomen was closed; IR group (n = 6), rats were subjected to clamping the SMA 40 min, and the abdomen was closed followed by a 4-h reperfusion; IP group (n = 6) rats underwent three cycles of 5 min ischemia and 5 min reperfusion, then clamping of the SMA for 40 min, then the abdomen was closed and a 4-h reperfusion followed. All animals were euthanized by barbiturate overdose (150 mg/kg pentobarbital sodium, i.v.) for tissue collection, and the SMA was isolated via median abdominal incision. Intestinal histologic injury was observed. Malondialdehyde (MDA), myeloperoxidase (MPO) and tumor necrosis factor (TNF)-α concentrations in intestinal tissue were measured. Intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression, as well as nuclear factor (NF)-κB activity and expression in intestinal tissue were also determined. Compared with the IR group, IP reduced IR-induced histologic injury of the intestine in rats (2.00 ± 0.71 vs 3.60 ± 0.84, P < 0.05). IP significantly inhibited the increase in MDA content (5.6 ± 0.15 μmol/L vs 6.84 ± 0.18 μmol/L, P < 0.01), MPO activity (0.13 ± 0.01 U/L vs 0.24 ± 0.01 U/L, P < 0.01), and TNF-α levels (7.79 ± 2.35 pg/mL vs 10.87 ± 2.48 pg/mL, P < 0.05) in the intestinal tissue of rats. IP also markedly ameliorated the increase in ICAM-1 (204.67 ± 53.27 vs 353.33 ± 45.19, P < 0.05) and VCAM-1 (256.67 ± 58.59 vs 377.33 ± 41.42, P < 0.05) protein expression in the intestinal tissues. Additionally, IP remarkably decreased NF-κB activity (0.48 ± 0.16 vs 0.76 ± 0.22, P < 0.05) and protein expression (320.23 ± 38.16 vs 520.76 ± 40.53, P < 0.01) in rat intestinal tissue. IP may protect against IR-induced intestinal injury by attenuation of the neutrophil-endothelial adhesion cascade via reducing ICAM-1 and VCAM-1 expression and TNF-α-induced NF-κB signaling pathway activity.

PWE-143 Abnormal Platelets And The Formation Of Activated Neutrophil-platelet Complexes Following Platelet Administration Induces Neutrophil Activation And Release Of Reactive Oxygen Species In Liver Cirrhosis

IntroductionThe susceptibility to developing infection is well recognised in cirrhosis and circulating neutrophil dysfunction including excessive production of reactive oxygen species (ROS) is a major contributor to innate immune paresis....

Microparticle alpha‐2‐macroglobulin enhances pro‐resolving responses and promotes survival in sepsis

Incorporation of locally produced signaling molecules into cell-derived vesicles may serve as an endogenous mediator delivery system. We recently reported that levels alpha-2-macroglobulin (A2MG)-containing microparticles are elevated in plasma from patients with sepsis. Herein, we investigated the immunomodulatory actions of A2MG containing microparticles during sepsis. Administration of A2MG-enriched (A2MG-E)-microparticles to mice with microbial sepsis protected against hypothermia, reduced bacterial titers, elevated immunoresolvent lipid mediator levels in inflammatory exudates and reduced systemic inflammation. A2MG-E microparticles also enhanced survival in murine sepsis, an action lost in mice transfected with siRNA for LRP1, a putative A2MG receptor. In vitro, A2MG was functionally transferred onto endothelial cell plasma membranes from microparticles, augmenting neutrophil–endothelial adhesion. A2MG also modulated human leukocyte responses: enhanced bacterial phagocytosis, reactive oxygen species production, cathelicidin release, prevented endotoxin induced CXCR2 downregulation and preserved neutrophil chemotaxis in the presence of LPS. A significant association was also found between elevated plasma levels of A2MG-containing microparticles and survival in human sepsis patients. Taken together, these results identify A2MG enrichment in microparticles as an important host protective mechanism in sepsis.

Cardiovascular protection of magnolol: cell-type specificity and dose-related effects

Magnolia officinalis has been widely used in traditional Chinese medicine. Magnolol, an active component isolated from Magnolia officinalis, is known to be a cardiovascular protector since 1994. The multiplex mechanisms of magnolol on cardiovascular protection depends on cell types and dosages, and will be reviewed and discussed in this article. Magnolol under low and moderate dosage possesses the ability to protect heart from ischemic/reperfusion injury, reduces atherosclerotic change, protects endothelial cell against apoptosis and inhibits neutrophil-endothelial adhesion. The moderate to high concentration of magnolol mainly acts on smooth muscle cells and platelets. Magnolol induces apoptosis in vascular smooth muscle cells at moderate concentration and inhibits proliferation at moderate and high concentration. High concentration of magnolol also abrogates platelet activation, aggregation and thrombus formation. Magnolol also serves as an smooth muscle relaxant only upon the high concentration. Oral intake of magnolol to reach the therapeutic level for cardiovascular protection is applicable, thus makes magnolol an agent of great potential for preventing cardiovascular diseases in high-risk patients.

On, Around, and Through: Neutrophil-Endothelial Interactions in Innate Immunity

This manuscript will review our current understanding of neutrophilic polymorphonuclear leukocyte (neutrophil) interactions with the endothelium during immune and inflammatory responses, focusing on the molecular mechanisms regulating neutrophil adhesion to and migration through the endothelium in response to infection or tissue injury. This is a complex and dynamic area of research and one that has been the topic of several recent comprehensive reviews to which the interested reader is referred (64, 118, 131). By design, this review will begin with a brief review of some basic aspects of neutrophil biology and endothelial adhesion to provide a foundation. The remainder of the review will focus on selected areas of this complex field, specifically the role of the endothelial glycocalyx in regulating neutrophil adhesion and the mechanisms and consequences of migration of neutrophils between (paracellular) and through (transcellular) endothelial cells during egress from the vasculature.

Advanced glycation end products in diabetic patients with optimized glycaemic control and their effects on endothelial reactivity: possible implications in venous graft failure

Diabetic patients exhibit an increased risk of saphenous graft occlusion after coronary bypass. Advanced glycation end products (AGEs) are ubiquitous signalling proteins that are associated with vascular and neurological complication of diabetes. The aim of this study is to verify whether AGE levels may promote endothelial cell alterations responsible for vein graft failure. Segments of saphenous vein were obtained from both normal people and diabetic patients (HbA(1c) < 6.0%) at the time of coronary surgery. Cultured endothelial cells were incubated in the absence/presence of AGEs (2 and 20 microM), and mRNA and protein for both receptor of AGEs (RAGE) and peroxisome proliferator-activated receptors-gamma (PPAR-gamma) were analysed by real-time polymerised chain reaction (PCR) and Western blot analysis. In the same fashion, the cell release of reactive oxygen species (ROS) was estimated in the absence/presence of AGEs by spectrofluorimetric analysis. Finally, neutrophil-endothelial adhesion was evaluated in saphenous vein segments with and without the addition of AGEs. AGEs activated in a dose-dependent manner the expression of RAGE and inhibited PPAR-gamma expression in endothelial cells as testified by both reverse transcription-PCR (RT-PCR) and Western blot analysis. Stimulation of cultured endothelial cells with AGEs significantly enhanced intracellular ROS formation in a dose-dependent manner. Finally, neutrophil-endothelial adhesion was significantly increased after incubation of control veins with AGEs. These findings indicate that even in diabetic patients with HbA(1c) < 6.0%, elevated serum levels of AGE determine a sort of a pro-thrombotic state, providing a common mechanism that could explain the increased rate of vein graft occlusion in this population.

Actin S-Nitrosylation Inhibits Neutrophil β2 Integrin Function

The focus of this work was to elucidate the mechanism for inhibition of neutrophil beta(2) integrin adhesion molecules by hyperoxia. Results demonstrate that exposure to high oxygen partial pressures increases synthesis of reactive species derived from type 2 nitric-oxide synthase and myeloperoxidase, leading to excessive S-nitrosylation of beta-actin and possibly profilin. Hyperoxia causes S-nitrosylation of the four cysteine moieties closest to the carboxyl-terminal end of actin, which results in formation of short actin filaments. This alters actin polymerization, network formation, and intracellular distribution, as well as inhibits beta(2) integrin clustering. If neutrophils are exposed to ultraviolet light to reverse S-nitrosylation, or are incubated with N-formyl-methionyl-leucine-phenylalanine to trigger "inside-out" activation, the effects of hyperoxia are reversed. We conclude that cytoskeletal changes triggered by hyperoxia inhibit beta(2) integrin-dependent neutrophil adhesion.

Neutrophil-derived Reactive Oxygen Species Can Modulate Neutrophil Adhesion to Endothelial Cells in Preeclampsia

Neutrophil activation has been implicated in the pathophysiology of preeclampsia. The aim of this study was to investigate whether neutrophil-derived reactive oxygen species (ROS) modulate adhesion to endothelial cells in preeclampsia. We first assessed neutrophil superoxide production and neutrophil-endothelial cell adhesion in normal nonpregnant women (n = 8), normal pregnant women (n = 10), and preeclamptic pregnant women (n = 8). We then examined the effects of neutrophil-derived ROS on neutrophil-endothelial adhesion. The release of neutrophil superoxide was measured using cytochrome C reduction. N-formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated superoxide production by neutrophils was significantly increased in women with preeclampsia when compared with the other two groups. Neutrophils from women with preeclampsia were more likely to adhere to endothelial cells, than were those from the other two groups (mean adhesion rate +/- s.d. (%); 20.6 +/- 2.7 in preeclampsia, 10.2 +/- 1.2 in normal pregnancy, 11.0 +/- 0.9 in normal nonpregnancy, P < 0.01). Superoxide dismutase (SOD), which dismutates the excess superoxide to hydrogen peroxide, did not affect neutrophil-endothelial adhesion. In contrast, catalase, which catalyzes the conversion of hydrogen peroxide to oxygen and water, inhibited neutrophil-endothelial adhesion in the preeclamptic group (8.1 +/- 0.5%, P < 0.01). Neutrophils from women with preeclampsia demonstrate increased CD11b expression and adhesion to endothelial cells. This is likely caused by elevations in superoxide and its derivative, hydrogen peroxide.