isolation-characterization-neutrophil-derived-microparticles-for

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Northwestern University

P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) play important roles in mediating the inflammatory cascade. Selectin kinetics, together with neutrophil hydrodynamics, regulate the fundamental adhesion cascade of cell tethering and rolling on the endothelium. The current study uses the Multiscale Adhesive Dynamics computational model to simulate, for the first time, the tethering and rolling behavior of pseudopod-containing neutrophils as mediated by P-selectin/PSGL-1 bonds. This paper looks at the effect of including P-selectin/PSGL-1 adhesion kinetics. The parameters examined included the shear rate, adhesion on-rate, initial neutrophil position, and receptor number sensitivity. The outcomes analyzed included types of adhesive behavior observed, tether rolling distance and time, number of bonds formed during an adhesive event, contact area, and contact time. In contrast to the hydrodynamic model, P-selectin/PSGL-1 binding slows the neutrophil's translation in the direction of flow and causes the neutrophil to swing around perpendicular to flow. Several behaviors were observed during the simulations, including tethering without firm adhesion, tethering with downstream firm adhesion, and firm adhesion upon first contact with the endothelium. These behaviors were qualitatively consistent with in vivo data of murine neutrophils with pseudopods. In the simulations, increasing shear rate, receptor count, and bond formation rate increased the incidence of firm adhesion upon first contact with the endothelium. Tethering was conserved across a range of physiological shear rates and was resistant to fluctuations in the number of surface PSGL-1 molecules. In simulations where bonding occurred, interaction with the side of the pseudopod, rather than the tip, afforded more surface area and greater contact time with the endothelial wall.

Simulation and Analysis of Tethering Behavior of Neutrophils with Pseudopods.

Mobilization of the innate immune response to clear and metabolize necrotic and apoptotic cardiomyocytes is a prerequisite to heart repair after cardiac injury. Suboptimal kinetics of dying myocyte clearance leads to secondary necrosis, and in the case of the heart, increased potential for collateral loss of neighboring non-regenerative myocytes. Despite the importance of myocyte phagocytic clearance during heart repair, surprisingly little is known about its underlying cell and molecular biology.

Cardiomyocytes induce macrophage receptor shedding to suppress phagocytosis.

Polymorphonuclear leukocytes (PMNs) are innate immune cells whose principal function is to migrate from the blood to sites of inflammation, where they exert crucial anti-infectious and immunomodulatory effects. However, dysregulated migration of PMNs into mucosal epithelial tissues is characteristic of chronic inflammatory disorders, including inflammatory bowel disease. Carbohydrate-mediated binding interactions between PMN Lewis glycans and endothelial glycan-binding proteins are critical for initial migration of PMN out of the vasculature. However, the role of Lewis glycans during transepithelial migration (TEM) has not been well characterized. Herein, we show that antibody blockade of Lewis X (Le(x)) displayed as terminal glycan residues on the PMN surface blocks chemotaxis and TEM while enhancing PMN-adhesive interactions with intestinal epithelia. Unexpectedly, targeting of subterminal Le(x) residues within glycan chains had no effect on PMN migration or adhesive interactions. There was increased surface expression of Le(x) on PMN after TEM, and blockade of terminal Le(x) regulated post-migratory PMN functions, increasing PMN phagocytosis and the surface mobilization of azurophilic (CD63, myeloperoxidase, and neutrophil elastase) and specific (CD66b and lactoferrin) granule markers. These findings suggest that terminal Le(x) represents a potential target for regulating PMN trafficking and function in inflamed mucosa. Furthermore, given its abundant expression on migrating PMN, Le(x) may be a rational target for modulating inflammation in diseases where dysregulated PMN influx is associated with host tissue damage.

Targeting of Neutrophil Lewis X Blocks Transepithelial Migration and Increases Phagocytosis and Degranulation.

Intestinal wounds often occur during inflammatory and ischemic disorders of the gut. To repair damage, intestinal epithelial cells must rapidly spread and migrate to cover exposed lamina propria, events that involve redox signaling. Wounds are subject to extensive redox alterations, particularly resulting from H2O2 produced in the adjacent tissue by both the epithelium and emigrating leukocytes. The mechanisms governing these processes are not fully understood, particularly at the level of protein signaling. Crk-associated substrate, or Cas, is an important signaling protein known to modulate focal adhesion and actin cytoskeletal dynamics, whose association with Crk is regulated by Abl kinase, a ubiquitously expressed tyrosine kinase. We sought to evaluate the role of Abl regulation of Cas at the level of cell spreading and migration during wound closure. As a model, we used intestinal epithelial cells exposed to H2O2 or scratch wounded to assess the Abl-Cas signaling pathway. We characterized the localization of phosphorylated Cas in mouse colonic epithelium under baseline conditions and after biopsy wounding the mucosa. Analysis of actin and focal adhesion dynamics by microscopy or biochemical analysis after manipulating Abl kinase revealed that Abl controls redox-dependent Cas phosphorylation and localization to influence cell spreading and migration. Collectively, our data shed new light on redox-sensitive protein signaling modules controlling intestinal wound healing.

Redox control of Cas phosphorylation requires Abl kinase in regulation of intestinal epithelial cell spreading and migration.

Neutrophil [polymorphonuclear leukocyte (PMN)] transepithelial migration (TEM) is a hallmark of inflammatory mucosal disorders. PMN TEM is associated with epithelial injury; however, mechanisms involved in this process are not well defined. The current work describes a new mechanism whereby deposition of PMN membrane-derived microparticles (PMN-MPs) onto intestinal epithelial cells (IECs) during TEM leads to loss of epithelial cadherins, thus promoting epithelial injury and increased PMN recruitment. PMN-MPs secreted by activated PMNs during TEM displayed a high level of enzymatically active matrix metalloproteinase 9 (MMP-9), and were capable of mediating potent effects on IEC integrity. Isolated PMN-MPs efficiently bound to IEC monolayers and induced cleavage of desmoglein-2 (DSG-2) but not E-cadherin, leading to disruption of IEC intercellular adhesions. Furthermore, PMN-MP binding to intestinal epithelium in vitro in transwell assays and in vivo in ligated intestinal loop preparations facilitated increases in PMN TEM. These effects were MMP-9 dependent and were reversed in the presence of specific pharmacological inhibitors. Finally, we demonstrated that IEC Dsg-2 serves as a barrier for migrating PMNs, and its removal by PMN-MP-associated MMP-9 facilitates PMN trafficking across epithelial layers. Our findings thus implicate PMN-MPs in PMN-mediated inflammation and epithelial damage, as observed in inflammatory disorders of mucosal surfaces.-Butin-Israeli, V., Houser, M. C., Feng, M., Thorp, E. B., Nusrat, A., Parkos, C. A, Sumagin, R. Deposition of microparticles by neutrophils onto inflamed epithelium: a new mechanism to disrupt epithelial intercellular adhesions and promote transepithelial migration.

Deposition of microparticles by neutrophils onto inflamed epithelium: a new mechanism to disrupt epithelial intercellular adhesions and promote transepithelial migration.

Neutrophil (PMN) infiltration of the intestinal mucosa often leads to severe epithelial injury; however, how this process occurs is unclear. This article describes a novel mechanism whereby membrane-derived microparticles released by tissue infiltrating PMNs (PMN-MPs) serve as shuttles to protect and deliver active mediators to locally modulate cellular function during inflammation. Specifically, myeloperoxidase (MPO), which is abundantly expressed in PMN azurophilic granules and is used for microbial killing, was found to be mobilized to the PMN surface and subsequently released in association with PMN-MPs upon PMN activation and binding to intestinal epithelial cells (IECs). The enzymatic activity of PMN-MP-associated MPO was enhanced compared with soluble protein, leading to potent inhibition of wound closure following PMN-MP binding to IECs. Importantly, localized microinjection of PMN-MPs into wounded colonic mucosa was sufficient to impair epithelial wound healing in vivo. PMN-MP/MPO-dependent inhibition of IEC wound healing was due to impaired IEC migration and proliferation, resulting from impeded actin dynamics, cell spreading, and cell cycle arrest. Thus, our findings provide new insight into mechanisms governing PMN-induced tissue injury and implicate PMN-MPs and MPO as important regulators of cellular function.

Neutrophil Microparticles Deliver Active Myeloperoxidase to Injured Mucosa To Inhibit Epithelial Wound Healing.

PMN-expressed fucosylated glycans from the Lewis glycan family, including Lewis-x (Le) and sialyl Lewis-x (sLe), have previously been implicated in the regulation of important PMN functions, including selectin-mediated trafficking across vascular endothelium. Although glycans, such as Leand sLe, which are based on the type 2 sequence (Galβ1-4GlcNAc-R), are abundant on PMNs, the presence of type 1 Galβ1-3GlcNAc-R glycans required for PMN expression of the closely related stereoisomer of Le, termed Lewis-A (Le), has not, to our knowledge, been reported. Here, we show that Leis abundantly expressed by human PMNs and functionally regulates PMN migration. Using mAbs whose precise epitopes were determined using glycan array technology, Lefunction was probed using Le-selective mAbs and lectins, revealing increased PMN transmigration across model intestinal epithelia, which was independent of epithelial-expressed LeAnalyses of glycan synthetic machinery in PMNs revealed expression of β1-3 galactosyltransferase and α1-4 fucosyltransferase, which are required for Lesynthesis. Specificity of functional effects observed after ligation of Lewas confirmed by failure of anti-LemAbs to enhance migration using PMNs from individuals deficient in α1-4 fucosylation. These results demonstrate that Leis expressed on human PMNs, and its specific engagement enhances PMN migration responses. We propose that PMN Lerepresents a new target for modulating inflammation and regulating intestinal, innate immunity.

Expression of Lewis-a glycans on polymorphonuclear leukocytes augments function by increasing transmigration.

V617F driver mutation of JAK2 is the leading cause of the Philadelphia-chromosome-negative myeloproliferative neoplasms (MPNs). Although thrombosis is a leading cause of mortality and morbidity in MPNs, the mechanisms underlying their pathogenesis are unclear. Here, we identified pleckstrin-2 (Plek2) as a downstream target of the JAK2/STAT5 pathway in erythroid and myeloid cells, and showed that it is upregulated in a JAK2V617F-positive MPN mouse model and in patients with MPNs. Loss of Plek2 ameliorated JAK2V617F-induced myeloproliferative phenotypes including erythrocytosis, neutrophilia, thrombocytosis, and splenomegaly, thereby reverting the widespread vascular occlusions and lethality in JAK2V617F-knockin mice. Additionally, we demonstrated that a reduction in red blood cell mass was the main contributing factor in the reversion of vascular occlusions. Thus, our study identifies Plek2 as an effector of the JAK2/STAT5 pathway and a key factor in the pathogenesis of JAK2V617F-induced MPNs, pointing to Plek2 as a viable target for the treatment of MPNs.

Loss of pleckstrin-2 reverts lethality and vascular occlusions in JAK2V617F-positive myeloproliferative neoplasms.

Formin protein mDia1 is involved in actin polymerization and plays important roles in the migration and adhesion of hematopoietic cells. The mDia1 encoding gene is located on chromosome 5q, which is commonly deleted in patients with del(5q) myelodysplastic syndromes (MDSs). We previously reported that mice with mDia1 deficiency developed neutropenia that closely mimics MDS. However, the mechanism of neutropenia in these mice and patients with del(5q) MDS remains incompletely defined. Here, we reveal that mDia1 knockout mice show cell-autonomously increased CD11b expression on neutrophils in the peripheral blood and bone marrow. The level of CD11b was also higher in patients with del(5q) MDS compared with normal individuals. Mechanistically, loss of mDia1 significantly attenuated the endocytosis of CD11b on neutrophils, which led to an increased number of neutrophils adhering to the blood vessels in mDia1 knockout mice. Administration of CD11b antibody to mDia1 knockout mice reduced the adhesion of neutrophils to the vessels and rescued neutropenia. Our study reveals the role of mDia1 deficiency in the upregulation of CD11b on neutrophils, which leads to their increased binding to the blood vessels. These results may provide important clues for the pathogenesis of neutropenia in patients with del(5q) MDS.

Loss of mDia1 causes neutropenia via attenuated CD11b endocytosis and increased neutrophil adhesion to the endothelium.

Tightly controlled communication among the various resident and recruited cells in the intestinal tissue is critical for maintaining tissue homeostasis, re-establishment of the barrier function and healing responses following injury. Emerging evidence convincingly implicates extracellular vesicles (EVs) in facilitating this important cell-to-cell crosstalk by transporting bioactive effectors and genetic information in healthy tissue and disease. While many aspects of EV biology, including release mechanisms, cargo packaging, and uptake by target cells are still not completely understood, EVs contribution to cellular signaling and function is apparent. Moreover, EV research has already sparked a clinical interest, as a potential diagnostic, prognostic and therapeutic tool. The current review will discuss the function of EVs originating from innate immune cells, namely, neutrophils, monocytes and macrophages, as well as intestinal epithelial cells in healthy tissue and inflammatory disorders of the intestinal tract. Our discussion will specifically emphasize the contribution of EVs to the regulation of vascular and epithelial barrier function in inflamed intestines, wound healing, as well as trafficking and activity of resident and recruited immune cells.

Ronen Sumagin

Department of Pathology,
Feinberg School of Medicine,
Department of Pathology, Feinberg School of Medicine

Northwestern University

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Ronen Sumagin

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pathology,Feinberg School of Medicine,Department of Pathology, Feinberg School of Medicine

Northwestern University

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Department of Pathology,
Feinberg School of Medicine,
Department of Pathology, Feinberg School of Medicine