Neutrophil-derived Microparticles Research Articles

SP0025 Neutrophil microparticles - what do they do?

Microparticles (MPs) are small vesicles shed from the plasma membrane that retain unique markers and mediators of their parent cell, enabling them to exert independent functions. Elevated levels of circulating...

200 NEUTROPHIL-DERIVED MICROPARTICLES MODULATE MONOCYTE MIGRATION IN AN ENDOTHELIAL DEPENDENT MANNER

<h3>Introduction</h3> Neutrophil depletion can delay atherogenesis and conversely increasing circulating neutrophils enhances plaque progression in mice. Lack of evidence for the presence of neutrophils in atherosclerotic plaques makes their role in disease progression less clear. Neutrophils may have a greater role in facilitating the movement of other cells into the vessel wall, possibly through the release of microparticles. The ability of microparticles to deliver cargo to target cells may be an explanation for neutrophil-specific markers being co-localised with endothelial cells in atherosclerotic plaques, despite the absence of neutrophils themselves. <h3>Methods</h3> Neutrophils were incubated with PBS, fMLP (10–5 M), and acetylated LDL (AcLDL, 20 µg/ml) to stimulate microparticle formation. To remove residual fMLP, microparticle suspensions were dialysed. Microparticles were characterised and quantified using a standardised flow cytometry method. Their adhesion to human coronary artery endothelial cells (hCAEC) after incubation with anti-ICAM-1 or isotype control was quantified using flow cytometry. Internalisation was visualised using confocal microscopy. Microparticle binding to and activation of monocytes was also investigated using flow cytometry. Monocytes migration to CCL-2 in the presence of neutrophil-derived microparticles with and without hCAEC was also investigated. <h3>Results</h3> Neutrophil-derived microparticles adhered to hCAEC in an ICAM-1 dependent manner and were internalised. Microparticles bound to monocytes regardless of stimuli, but their adhesion was lower than observed with hCAEC. Only microparticles from neutrophils stimulated with fMLP induced monocyte L-selectin shedding and significantly enhanced monocyte-hCAEC adhesion. Microparticles increased monocyte migration to CCL2 in an endothelial cell and CD18-dependent manner. <h3>Conclusions</h3> Microparticles bind to ICAM-1 on the surface of hCAEC and are internalised. Monocyte migration is enhanced by neutrophil-derived microparticles only in the presence of endothelial cells. In conclusion, neutrophil-derived microparticles may play a role in activating hCAEC, potentially by their uptake, and thus amplify inflammatory responses at sites of atherosclerotic lesion development.

Pro-inflammatory effect of cystic fibrosis sputum microparticles in the murine lung

The role of microparticles (MPs) in the inflammatory process of cystic fibrosis (CF) airways is not known. Here, we have studied the pro-inflammatory potential of CF MPs in a model of acute lung injury. Swiss mice were subjected to intratracheal administration of MPs obtained from CF and primary ciliary diskinesia (PCD) patients. Histopathology, total and differential cell counts in bronchoalveolar lavage fluid were used to evaluate the inflammatory reaction in the lung. Lipopolysaccharide (LPS)-like activity of MPs was studied by Limulus amebocyte lysate assay. MPs obtained from acute CF patients determined peribronchial and perivascular inflammatory infiltrates similar to those elicited by LPS. This inflammation was granulocyte-dominated and higher than that determined by MPs obtained from stable CF, whereas PCD MPs caused a macrophage-dominated inflammation. While LPS-activity was not found in circulating blood MPs prepared from CF patients, it was present in MPs obtained from CF sputum and sputum CD66b(+) neutrophils. Finally, LPS-like activity was only detected in circulating MPs after incubation with LPS as well as in MPs obtained from LPS-stimulated neutrophils obtained from healthy donors. These data suggest that the pro-inflammatory potential of neutrophil-derived MPs in the CF airways may be subsequent to the binding of shedded LPS.

Neutrophil‐Derived Microparticles as Novel Effectors in Joint Disease

Microparticles (MPs) are small membrane‐bound vesicles released by cells upon activation. Numerous reports have detected MPs in the synovial fluid of Rheumatoid Arthritis (RA) patients. Whilst Tcell‐ derived MPs activate synoviocytes, recent reports have shed light on the possible anti‐inflammatory phenotype of neutrophilderived MPs, challenging the linear model of inflammation. The aim of the study is to unveil the role of neutrophil‐derived MPs in RA, particularly in the context of cartilage biology. Microparticles generated from human neutrophils were characterised by flow cytometry and tested for chondroprotective efficacy in a C28/I2 micromass model of cartilage turnover. These experiments revealed protective actions. Investigation in whole cartilage explants indicated chondroprotection (demonstrated by safranin O staining) as studied over a seven‐day period. Microparticle entry into cartilage was also investigated using confocal microscopy. In conclusion, we report novel biological functions of neutrophil MPs with potential relevance to cartilage biology and arthritis.Supported by Oliver Bird Studentship Scheme QMUL/KCL

Extravasating Neutrophil-derived Microparticles Preserve Vascular Barrier Function in Inflamed Tissue

Emerging evidence suggests that gap formation and opening of the endothelial junctions during leukocyte extravasation is actively controlled to maintain the integrity of the vascular barrier. While the role for endothelial cells to this process has been well defined, it is not clear whether leukocytes are also actively contributing to endothelial barrier function. We have recently showed that extravasating leukocytes deposit microparticles on the subendothelium during the late stages of extravasation, which is LFA-1 dependent. Using multiphotonintravital microscopy (MP-IVM) of mouse cremaster muscle vessels in the current work, we show that microparticle formation and deposition maintains the integrity of the microvascular barrier during leukocyte extravasation. Inhibition of neutrophil-derived microparticle formation resulted in dramatically increased vascular leakage. These findings suggest that deposition of microparticles during neutrophil extravasation is essential for maintaining endothelial barrier function and may result in temporal difference between neutrophil extravasation and an increase in vascular leakage.

10 Regulation of Monocyte-Endothelial Cell Interactions by Neutrophil-Derived Microparticles

Rationale Monocytes play a major role in atherosclerosis progression through migration into the arterial wall. Neutrophil involvement in atherosclerosis was previously only thought to be via enzymatic weakening of the fibrous cap. However, neutrophil depletion can delay atherogenesis and conversely increasing circulating neutrophil enhance plaque progression in mice. Lack of evidence for the presence of neutrophils in atherosclerotic plaques makes their role in disease progression less clear. We have found neutrophil-derived microparticles increase migration of neutrophils. Our hypothesis: neutrophil-derived microparticles increase endothelial cell-monocyte interactions and facilitate monocyte transendothelial migration. Methodology Neutrophils were incubated with various agents to stimulate microparticle formation. Microparticles were characterised and quantified using a novel, standardised flow cytometry method and electron microscopy. Monocytes were migrated to MCP-1 across CAEC incubated with neutrophil-derived microparticles using a transendothelial migration assay. Cytokine release and adhesion molecule expression by hCAEC was investigated using a cytometric bead array and fluorescent antibody binding respectively. Microparticle adhesion to hCAECs was investigated under static and flow conditions. Findings Neutrophil-derived microparticles were between 0.5 and 0.9µm. The most abundant surface markers were Annexin V with 20-30% positive microparticles, CD66c (10–15%) and CD18 (5–15%). Low levels of CD11b, CXCR2, and L-selectin were observed. More microparticles were produced in response to fMLP and aLDL compared to PBS. Microparticles bind to and induce MCP-1 release from hCAEC. Monocyte migration increased upon hCAEC incubation with neutrophil-derived microparticles and the extent of the increase was dependent on the stimulus used with aLDL>fMLP>PBS. This highlights a role for microparticles in endothelial activation.

Circulating microparticles in severe preeclampsia

The present study aimed to evaluate microparticles (MPs) from different sources in women with severe preeclampsia (PE) compared with normotensive pregnant women and non-pregnant women. This case–control study evaluated 28 pregnant women with severe PE, 30 normotensive pregnant women, and 29 non-pregnant women. MPs from neutrophils, endothelial cells, monocytes, platelets, leukocytes, erythrocytes, and syncytiotrophoblast were evaluated using flow cytometry. A higher total number of MPs were observed in women with severe PE compared with normotensive pregnant women and non-pregnant women (P=0.004 and P=0.001, respectively). MPs derived from erythrocytes were increased in women with severe PE compared with normotensive pregnant women (P=0.002). A trend towards association was observed between platelet count and the number of MPs derived from platelets (P=0.09) in severe PE group. A positive correlation was also found between the number of endothelial cell-derived MPs and the number of platelet-derived MPs, leukocyte-derived MPs, neutrophil-derived MPs, and lymphocyte-derived MPs (P<0.05) in severe PE pregnant women. MP counts can be increased in severe PE, and erythrocyte and endothelial cell-derived MPs seem to be associated to severe PE.

Human microparticles generated during sepsis in patients with critical illness are neutrophil-derived and modulate the immune response

Microparticles (MPs) are 0.3 μm to 1.0 μm vesicles generated after cell activation or apoptosis that may play a role in the pathophysiology of sepsis. We sought to elucidate the role of MPs in patients with critical illness and hypothesized that MPs are generated at the site of inflammation and can modulate the immune response. Surgical patients with critical illness with ongoing sepsis were enrolled from the intensive care unit of an urban, Level I trauma center from March to June 2011. Abdominal washings and bronchoalveolar lavage fluid were collected from sites of inflammation. MPs were isolated using differential centrifugation, then characterized by flow cytometry. Immunologic assays were conducted by incubating neutrophil-derived MPs (NDMPs) with a human monocytic cell line (THP-1). A p value ≤0.05 was considered significant. MPs were absent in noninflamed foci in patients, whereas NDMPs were present in locations of inflammation. NDMPs were added to cultured THP-1 cells to quantify immunomodulatory effects. THP-1 cells were able to phagocytose NDMPs. Cells that ingested NDMPs demonstrated increased activation. In contrast, bystander THP-1 cells without ingested NDMPs demonstrated decreased activation. NDMPs are generated at the site of inflammation in patients with critical illness during sepsis. They have a divergent effect on the immune response by activating phagocytic cells and deactivating bystander cells. NDMPs may play an important role in regulating the inflammatory response to sepsis in patients with critical illness.

Cutting Edge: Humanized Nano-Proresolving Medicines Mimic Inflammation-Resolution and Enhance Wound Healing

Endogenous microparticles (MPs) were systematically profiled during the time course of self-limited inflammation. Precursors for specialized proresolving lipid mediators were identified in MPs from inflammatory exudates using liquid chromatography tandem mass spectrometry-based metabolomics. Hence, we postulated that formation of anti-inflammatory and proresolving lipid mediators could underlie beneficial effects attributed to MPs and that this process could serve as a basis for biomimicry. Using human neutrophil-derived MPs, we constructed novel nanoparticles (NPs) containing aspirin-triggered resolvin D1 or a lipoxin A(4) analog. Enriched NPs dramatically reduced polymorphonuclear cell influx in murine peritonitis, shortened resolution intervals, and exhibited proresolving actions accelerating keratinocyte healing. The enriched NPs protected against inflammation in the temporomandibular joint. These findings indicate that humanized NPs, termed nano-proresolving medicines, are mimetics of endogenous resolving mechanisms, possess potent beneficial bioactions, can reduce nanotoxicity, and offer new therapeutic approaches.

Interval and Continuous Exercise Regimens Suppress Hypoxia‐induced Thrombin Generation by Decreased Release of Procoagulant‐rich Microparticles from Neutrophils

The release of procoagulant-rich microparticles from neutrophils accelerates the pathogenesis of inflammatory thrombosis. This study explicates the manner in which interval and continuous exercise regimens affect neutrophil-derived microparticles (NDMP) release and NDMP-mediated thrombin generation (TG). Twenty sedentary males were randomly divided into moderate continuous exercise training group (MCT; 60% VO2max, n=10) and aerobic interval exercise training group (AIT; 3-minute intervals at 80% and 40% of VO2max, n=10) for 30 minutes per day, 5 days per week for 5 weeks. The NDMP characteristics and dynamic TG parameters were measured by two-color flow cytometry and calibrated, automatic thrombinography, respectively. Acute 12%O2 exercise increased release of micrparticles from neutrophils and up-regulated expression of phosphatidylserine (PS) on NDMP, which responses were accompanied by elevated thrombin peak height and increased TG rate in NDMP-rich plasma. However, both MCT and AIT for 5 weeks decreased levels of tissue factor-rich and PS-exposed microparticles released from neutrophils and depressed NDMP-mediated dynamic TG in plasma at rest and following the 12%O2 exercise. Therefore, we conclude that both MCT and AIT ameliorates NDMP-induced TG by down-regulated expressions of pro-coagulant factors on NDMP at performing 12%O2 exercise, which may, in turn, decrease the risk of inflammatory thrombosis evoked by hypoxic stress.

A new biological marker isolated and characterizated in sputum from lung pathology patients: microparticles

Sputum is recognized as a very useful sampling method in Cystic Fibrosis (CF) for both research and clinical use aiding both the diagnosis and monitoring of lung disease inflammatory status. A great advantage of the technique is that it enables sampling of the airways in a non-invasive manner. We investigated on the presence and phenotype of microparticles (MPs) in the sputum, of acute and intermittent CF and Primary Ciliary Dyskinesia (PCD) adult patients. MPs are membrane vesicles that are released during cell activation and apoptosis. MPs have different biological effects depending on the cell from they originate. CF lung disease is characterized by massive neutrophil influx in the airways, their activation and eventually apoptosis. Spontaneous sputum, obtained from 21 CF patients (10 acute and 11 intermittent) and 7 patients with PCD, was liquefied with Sputasol. MPs were counted, visualized by electron microscopy, identified in the supernatants of treated sputum by cytofluorimetry and immunodecoration for leukocyte (CD11a), granulocyte (CD66b), and monocyte-macrophage (CD11b) antigens. Electron microscopy revealed that sputum MPs were in the 100-500 nm range and did not contain bacteria. CF sputa contained higher number of MPs in comparison with PCD sputa. CD66b and CD11a, but not CD11b, levels in MPs were significantly higher in CF than in PCD. Acute and intermittent patients presented significantly higher levels of CD11aand CD66b-expressing MPs respect to PCD patients. MPs are detectable in sputa obtained from CF patients and are predominantly of granulocyte origin. These findings are consistent with massive influx of neutrophils into CF airways and their accumulation on the surface of the airway epithelium. In this environment, neutrophils are activated by bacterial products, pro-inflammatory cytokines, and chemokines. Neutrophils undergo apoptosis, as normally happens in acute inflammation, but also postapoptotic necrosis, releasing toxic enzymes and oxygen radicals. MPs in CF sputa likely reflect both activation and apoptosis of neutrophils. The significantly less presence of granulocyte-derived MPs in PCD respect to CF patients, may be due to the fact that lung disease in PCD, although similar, is delayed in time as compared with CF. In CF airways, neutrophil-derived MPs could contribute to self-perpetuating inflammatory cycle, and may account for the exaggerated pro-inflammatory response of cells in CF patients. The MPs isolated from sputum could be a new non invasive methods and opens a new opportunity for the study of lung disease.

Expression, activation, and function of integrin αMβ2 (Mac-1) on neutrophil-derived microparticles

AbstractLeukocyte-derived microparticles (MPs) are markers of cardiovascular diseases and contribute to pathogenesis by their interaction with various cell types. The presence and activation state of a multifunctional leukocyte receptor, integrin αMβ2 (CD11b/18), on MPs derived from human neutrophils (PMNs) were examined. αMβ2 expression was significantly enhanced on MPs derived from stimulated compared with resting PMNs. Furthermore, αMβ2 on MPs from stimulated but not resting PMNs was in an activated conformation because it was capable of binding activation-specific monoclonal antibodies (CBRM1/5 and mAb24) and soluble fibrinogen. MPs expressing active αMβ2 interacted with and were potent activators of resting platelets as assessed by induction of P-selectin expression and activation of αIIbβ3. With the use of function-blocking antibodies and MPs obtained from αM−/−-deficient mice, we found that engagement of GPIbα on platelets by αMβ2 on MPs plays a pivotal role in MP binding. Platelet activation by MPs occurs by a pathway dependent on Akt phosphorylation. PSGL-1/P-selectin interaction also is involved in the conjugation of MPs to platelets, and the combination of blocking reagents to both αMβ2/GPIbα and to PSGL-1/P-selectin completely abrogates MP-induced platelet activation. Thus, cooperation of these 2 receptor/counterreceptor systems regulates the prothrombotic properties of PMN-derived MPs.

Annexin 1 mediates the rapid anti-inflammatory effects of neutrophil-derived microparticles

AbstractPolymorphonuclear leukocyte (PMN)–derived microparticles display inhibitory properties on target cells as assessed in vitro; since PMNs contain abundant amounts of the endogenous anti-inflammatory protein annexin 1 (AnxA1), we tested here whether biologically active AnxA1 could be present in PMN-derived microparticles. PMN adhesion to human umbilical vein endothelial cell (HUVEC) monolayers led to the generation of microparticles that contained AnxA1, as detected by Western blotting, flow cytometry, and mass spectrometry analyses. Addition of these microparticles to recipient PMNs prior to flow over HUVEC monolayers significantly inhibited cell adhesion, an effect abrogated by a neutralizing anti-AnxA1 antibody, or an antibody raised against the AnxA1 receptor, that is termed lipoxin A4 receptor or ALX. Intravenous delivery of human PMN–derived microparticles markedly inhibited PMN recruitment to an air pouch inflamed with IL-1β. This anti-inflammatory effect was also dependent on endogenous AnxA1, since injection of microparticles produced from wild-type PMNs (bone marrow derived), but not from AnxA1-null PMNs, inhibited IL-1β–induced leukocyte trafficking. In conclusion, PMN-derived microparticles contain functionally active AnxA1 that confers them anti-inflammatory properties; generation of these microparticles in the microcirculation could promote inflammatory resolution by time-dependent dampening of cell recruitment.

Nitric Oxide Regulates Neutrophil Migration through Microparticle Formation

The role of nitric oxide (NO) in regulating neutrophil migration has been investigated. Human neutrophil migration to interleukin (IL)-8 (1 nmol/L) was measured after a 1-hour incubation using a 96-well chemotaxis plate assay. The NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME) significantly (P < 0.001) enhanced IL-8-induced migration by up to 45%. Anti-CD18 significantly (P < 0.001) inhibited both IL-8-induced and L-NAME enhanced migration. Antibodies to L-selectin or PSGL-1 had no effect on IL-8-induced migration but prevented the increased migration to IL-8 induced by L-NAME. L-NAME induced generation of neutrophil-derived microparticles that was significantly (P < 0.01) greater than untreated neutrophils or D-NAME. This microparticle formation was dependent on calpain activity and superoxide production. Only microparticles from L-NAME and not untreated or D-NAME-treated neutrophils induced a significant (P < 0.01) increase in IL-8-induced migration and transendothelial migration. Pretreatment of microparticles with antibodies to L-selectin (DREG-200) or PSGL-1 (PL-1) significantly (P < 0.001) inhibited this effect. The ability of L-NAME-induced microparticles to enhance migration was found to be dependent on the number of microparticles produced and not an increase in microparticle surface L-selectin or PSGL-1 expression. These data show that NO can modulate neutrophil migration by regulating microparticle formation.

Circulating Microparticles in Lebanese Patients with beta-Thalassemia Intermedia.

Introduction: Beta-thalassemia is a genetic disease with a prevalence that varies in the world reaching 3% in Lebanon. One-third of these patients have beta-thalassemia intermedia. Patients with thalassemia intermedia have complications not present in those with thalassemia major, mainly leg ulcers, extramedullary hematopoiesis and thrombosis. The beta-thalassemia intermedia patients not receiving transfusions and who are splectomized, are at higher risk of thrombosis as well as the other complications. Microparticles (MP) are shed submicrometric plasma membrane fragments (~0.1–1μm) harboring negatively charged phosphatidylserine (PS) in their extracellular membrane leaflet. They mainly stem from apoptotic or activated cells, and generally present a procoagulant potential. Increased levels of MP were described in disorders with major vascular and thrombotic symptoms such as myocardial infarction, diabetes, renal failure or sickle cell disease.Methods: In this study we determined circulating MP levels in platelet-free plasma samples of patients and healthy donors using a solid-phase capture assay on insolubilized streptavidin. The presence of circulating MP was first evaluated using annexin V-coupled biotin that binds the negatively charged PS in MP. Independent sample t-test was conducted to examine significant difference between patients and controls. Thirty-two patients with beta-thalassemia intermedia and 17 healthy controls were included. P-values less than 0.05 were considered to be statistically significant.Results: Fifty percent of the patients and 81.3% of the control group were males. The average age of the patients was 26.5 ± 9.5 years. Around 77% of the patients had splenectomy. Shed membrane MP were elevated to near statistical significance in platelet-free plasma of patients with beta-thalassemia intermedia but not in control individuals (7.3 ± 5.7 vs 4.7 ± 2.9 nmol/L PS equivalent, p=0.09). The cell origin of MP was further investigated after capture onto specific biotinylated antibodies used instead of annexin V. MP of red blood cell origins were elevated, as expected in patients with beta-thalassemia intermedia vs controls (6.05 ± 3.8 vs 2.54 ± 3.8 nmol/L PS equivalent, p<0.01 for patients and controls, respectively). Interestingly, both platelet and endothelial-derived MP were elevated in patients vs controls (of platelet origin: 3.02 ± 2.06 vs 1.91 ± 1.15 nmol/L PS equivalent, p=0.05; and of endothelial origin: 1.58 ± 0.87 vs 0.41 ± 0.3 nmol/L PS equivalent, p<0.01). Leukocyte and neutrophil-derived MP were also elevated in patients vs control (p=<0.01).Conclusion: These results indicate that circulating membrane MP are increased in beta-thalassemia intermedia and testify to increased platelet, endothelium and leukocyte activation, in probable association with the increased thrombotic risk. Further investigations are required to assess the significance of these enhanced markers of cell activation, with respect to a possible deleterious potential towards vascular or blood cell functions.